Handbook of DNA Profiling 9789811643170, 9789811643187, 9789811643194, 9811643172

Table of contents :
Preface
Contents
About the Editors
Contributors
Part I: Principles of Forensic DNA Profiling
1 Forensic DNA Investigation
Definitions
Introduction
Value of Forensic DNA Investigations
Evidential Value
Forensic DNA Evidence
Forensic DNA Analysis
Forensic DNA Databases
Use of Forensic Lead Investigative Information
Familial DNA Searches
Modus Operandi and Signature of Serial Offenders
Offender Profiling
Best Practices Using Forensic Investigative Leads as Effective Investigative Tool
Step 1: Notification
Step 2: The Investigation
Appeal to Victims to Come Forward
Search for Similar Cases
Revisit the Crime Scenes
Exhibits Material
Reinterview Victims
Cellular Phones
Step 3: After the Arrest
Preparation for the Trial
Sentencing
Reasons for Unsuccessful Investigations of Serial Casework
Serial Related Cases Not Consolidated
Serial Casework Investigated by Inexperienced Detectives
Cases Closed as Undetected Are Not Reopened
Informal Identity Parades
Pretrial Preparation
Erroneous Forensic DNA Findings
Conclusion
References
2 Introduction to Forensic DNA Typing and Current Trends
From Sampling to Identification
Improved Software Tools
Massively Parallel Sequencing/Next-Generation Sequencing: Advanced Human Identification
The NGS Technology
Comparing CE and NGS
Forensic Applications of NGS Technology
STR and SNP Sequencing
Mitochondrial DNA Sequencing in Forensics Using NGS
DNA Intelligence
Forensic DNA Phenotyping (FDP): Visible Phenotype Estimation
Ancestry Informative Markers
Degraded Samples
Identifying Monozygotic Twins
Emerging Applications of NGS
Utility of NGS/MPS over CE
Forensic Genetic Genealogy: The New Means of Genetic Identification
The Ekeby Man Case
The Golden State Killer
The Rapid DNA Instrument
Conclusion
References
3 Biological Sources of DNA: The Target Materials for Forensic DNA Typing
Introduction
Blood
Semen
Vaginal Secretion
Oral Fluids
Sweat
Urine
Fecal Matter
References
4 Collection, Preservation, and Transportation of Biological Evidences
Introduction
Biological Evidence
DNA Content in Biological Samples
Collection of Biological Samples
Methodology for the Collection and Preservation of Biological Evidence
Scraping Method
Double Swabbing Method
Cutting Method
Tape Lifting Method
Picking Method
Collection, Preservation, Transportation, and Storage Procedures for Commonly Encountered Biological Evidences at the Crime Sc...
Liquid Blood
Liquid Blood and Wet Bloodstains from Crime Scene
Dried Blood Stains
Semen and Seminal Stains and Evidences from Sexual Assault Cases
Soft Tissues and Organs
Teeth and Bone
Hair
Touch DNA Samples
Chain of Custody of Forensic Samples
References
5 Forensic DNA: From New Approaches for the Bio-stain Identification to the Evaluation of the Genetics Evidence in Courtroom
Introduction About the Bio-Stain Identification
New Approaches for the Bio-Stain Identification
Analysis Techniques in Forensic Genetics and New Technologies
Genetic Traces in a Fire Scene
Genetic Traces in a Fire Scenario
The Biological Traces Evidence
From Scientific Proof to ``Beyond Reasonable Doubt´´
DNA and Fire
Final Remarks
The New Popperian Epistemology of the Criminal Process: Strong Scientific Evidence and Reduction of the Clues to Conjectures
The Revolution of the Justice Method: Popper´s Way for Strong Proofs
The Neutrality of the Researcher and the Rigorous Acquisition of the Proofs
References
6 Tools and Techniques Used in Forensic DNA Typing
Introduction
Basic Principles of DNA
DNA Typing Methods
Blood Group Testing
Forensic Protein Profiling
RFLP-Based DNA Testing
PCR-Based Tests
Capillary Electrophoresis
Y Chromosome DNA Testing
STR Typing
Mitochondrial DNA
X Chromosome Analysis
Microbial and Animal Forensics
Next-Generation Sequencing
Conclusion
References
7 Evaluation of the Autosomal STR Markers and Kits
Introduction
Autosomal STR Marker
Nomenclature for DNA Markers
Types of Autosomal STR Marker
CODIS Markers
Autosomal STR Multiplex Kits
Earlier STR Multiplex Kits
Identifiler Kit
``AmpFLSTR Identifiler Direct´´
``AmpFLSTR Identifiler Plus´´
Powerplex 16HS Kit
New Generation Kit
Global Filer Kit
Verifiler Kit
Fusion 6C Kit
Forensic Relevance of Amelogenin Marker
Forensic Validation of Autosomal STR Kits
Usefulness of Autosomal STR Kits in Forensic Practice
Conclusion
References
8 Forensic Human Y-Chromosome Markers: Principles and Applications
Introduction
Y-Chromosome Structure
Markers on Y Chromosome and Polymorphisms
Y-STRs
Rapidly Mutating Y-STRs
Mini Y-STRs
Inheritance Pattern of Y-STR Markers
Y-SNPs
Y-STR Kits
Interpretation of Y-STR Results
No Interpretable Results
Inconclusive Results
Exclusion
Inclusion/Match
Mixture Interpretation Via Y-STR Analysis
Forensic Use of Y-Chromosome Testing
Sexual Assault Investigation
Sex Determination
Paternity Testing
Disaster Victim Identification
Historical Investigations
Y-STR Haplotype Databases
Familial Searching
Haplogroup Prediction
Conclusion
References
9 Haplodiploid Markers and Their Forensic Relevance
Introduction
Theoretical and Statistical Analyses
Single Locus
Haplotype
Association at Gametogenesis Level: Linkage
Association at the Population Level
Applications
Individual Profiling
Genetic Relatedness
Conclusions
Conflict of Interest
References
10 Single-Nucleotide Polymorphism
Introduction
SNPs and Their Attributes
Advantages of SNP
Categories of SNP Markers
Identity-Testing SNPs
Lineage SNPs
Ancestry Informative Markers
Phenotype Informative SNPs
Techniques of SNP Analysis
Molecular Beacons (MB)
Microarrays/DNA Chips
SNaPshot Multiplexing Method
Mass Spectrometry
Forensic Application of SNP Profiling
Human Identification (HID) from Skeletal Remains
Paternity Testing and Kinship Analysis
Phenotypic Information of a Missing Suspect
Constraints of SNP
SNP Information Databases
Conclusion
References
11 Ethical Governance of Forensic DNA Databases in Southeast Asia
Introduction
Research Involving Human DNA Databases
Ethical Elements Applied to Genetic Research and DNA Databasing
Ethical Framework for Multiple Stakeholder Responsibility
Research on Reference Databases on Forensically Relevant DNA Markers in Southeast Asia
Brunei Darussalam
Cambodia
Indonesia
Laos
Malaysia
Myanmar
Philippines
Singapore
Thailand
Vietnam
Survey of Publications on Forensic DNA databases in SE Asia
Conclusions and Recommendations
References
12 Using Laboratory Validation to Identify and Establish Limits to the Reliability of Probabilistic Genotyping Systems
Introduction
Standards and Guidelines Governing Internal Validation
Published Guidance Documents
Validation Workshops and Technical Merit Evaluation by NIST Scientists
Key Principles
Pushing the System Until It Fails
Dimensions That Must Be Evaluated Individually and Collectively
Suboptimal Amounts of Template DNA
Variation in Contributor Ratios
A Large and/or Unknown Number of Contributors (NOC) to a Sample
Allele Sharing Between Contributors to a Sample, and with Noncontributors
Degradation/Inhibition and Differential Degradation
Additional Factors That Complicate the Interpretation of Mixed DNA Samples
Key Principles
Using and Sharing Validation Data and Results
Incorporating Validation Results into Standard Operating Procedures
Transparency of Validation Data for Independent Review
Key Principles
Conclusions
References
13 Potential of DNA Technique-Based Body Fluid Identification
Introduction
Biological Background of Molecular Markers
mRNA Profiling
miRNAs Profiling
Epigenetics
DNA Methylation Profiling Method
DNA-Compatible Cell-Specific Identification
Conclusion
References
14 Overview of Familial DNA and Forensic Phenotyping
The Basics of DNA
The Building Blocks for the Human Body
The Use of DNA in Forensics
DNA Fingerprinting
Limitations of DNA Evidence
The Right to Collect DNA from Suspects
Recent DNA Controversies
Familial DNA
DNA Phenotyping
Racial Profiling
Right to Privacy
Legislative Response
Conclusion
References
15 A Glimpse of Famous Cases in History Solved by DNA Typing
Introduction
Famous Cases in History Solved by Human DNA Analysis
Colin Pitchfork Case
Tommie Lee Andrews Case
Joseph Castro Case
O. J. Simpson Murder Case
Craig Harman Case
Identification of 9/11 Victims
Famous Cases in History Solved by Nonhuman DNA Analysis
Snowball Case
The Bogan Case
Cold Cases Solved by DNA Analysis
The Boston Strangler Case
Murder and Sexual Assault of Girl in 1988
Arrest of Nebraska Sex Offender in 1983 Slaying of UNO Student
DNA Examination of Unsolved Female Homicide Cases During 1990-1999
Jeffrey Gafoor Case
Exoneration of Convicted Individuals Through DNA Analysis
Gary Dotson´s Case
Kirk Bloodsworth´s Case
Post-Conviction DNA Testing
Michael Shirley´s Case
Promise of NGS Technology in Solving Forensic Cases
Conclusive Remarks
References
16 Role of the Molecular Anthropologist in the Forensic Context
Introduction
The Identification Process
DNA from Skeletal Remains
DNA Damage
Contaminating DNA
PCR Inhibitors
Role of Forensic Molecular Anthropology
The Choice of Skeletal Elements and Their Sampling
dna Analysis for Human Identification
Molecular Information When No Reference Data from Relatives Are Available
Conclusion
References
Part II: Usefulness of Various Techniques of DNA Profiling in Solving Cases
17 Short Tandem Repeat Mutations in Paternity Analysis
Introduction
Types of Samples Received for Parentage Testing
Markers Used for DNA Fingerprinting
Short Tandem Repeat Markers
Autosomal and Y Chromosomal Short Tandem Repeats
Autosomal STRs
Y Chromosomal STRs
Single Nucleotide Polymorphism (SNP) Markers
Autosomal SNPs
Mitochondrial DNA Markers
Autosomal STRs in Human Identification and Parentage Testing
Utility of Other Autosomal and Lineage Markers in Parentage Testing
STR Typing Methodology
Statistical Analysis of the Paternity Test Results
STR Inconsistencies/Mismatches in Parentage Testing
Calculation of Mutation Rate
Avuncular Index
Factors Influencing Short Tandem Repeat Mutations
Molecular Basis of STR Inconsistencies
Primer Binding Site Mutations
Strand Slippage Mutation
Uniparental Disomy
Anomalous Band Patterns
Complementary Markers to Study Inconsistencies in Parentage Testing
Next Generation Sequencing
Case Study
Background
Methods
Results
Statistical Analysis of the DNA Typing Results
Discussion
Conclusions
References
18 Utility and Applications of Lineage Markers: Mitochondrial DNA and Y Chromosome
Introduction
Mitochondrial DNA
What Is the Mitochondrial DNA?
Localization, Origin, and Structure
mtDNA Characteristics
The High Number of Copies
Matrilineal Inheritance
Heteroplasmy
High Mutation Rate and Neutrality
Degradation Resistance
NUMTs (Nuclear Mitochondrial DNA Fragments)
Analysis Methodologies and Results Interpretation
Single-Nucleotide Polymorphisms (SNPs) and Other mtDNA Variations
Nomenclature: IUPAC-IUB Code and How to Use It
Results and Interpretation
Haplotype and Haplogroup Concepts
Haplotype Determination
Mitochondrial Reference Sequences (CRS, rCRS, RSRS)
Haplogroup Diagnosis
PhyloTree
Databases and Haplogroup Assignation Tools
Statistical Evaluation
Forensic Applications of mtDNA
Low-Template DNA Samples
When the Objective Is to Detect a Kinship Through the Maternal Lineage
To Approximate the Biogeographical Origin
New Perspectives: Twins Differentiation
Y Chromosome
Origin of the Y Chromosome
Structure of the Y Chromosome
Polymorphisms of the Y Chromosome
Y-STRs
Y Chromosome Microsatellite Mutation Rates
Interpretation of Y-STR Results in Forensic Analysis
Y-SNPs
Origin and Rates of Mutation in Y Chromosome Biallelic Markers
Applications of Y Chromosome
Forensic Analysis
Paternity Testing and Historical Investigations
Genealogical Studies
Biogeography Ancestry and Study of Migrations of Human
Final Conclusion
References
19 Usefulness of the X-Chromosome on Forensic Science
Introduction
The X-Chromosome Evolution
Structural Characteristics
Types of Markers and Their Usefulness on Forensic Science: X- STRs, X-InDels X-SNPs
Worldwide X-Chromosome Population Data (Population Databases)
The Statistical Approach: IBD, IBS, and Likelihood Ratio
X-chromosome and Kinship Analysis
Female-Female Duo (XFF)
Male-Male Duo (XMM)
Female-Male Duo (XFM)
Indistinguishable Genealogies
Classes Distinguishable by Analyzing X-Chromosome Markers
Incest Cases
X-Chromosome and Forensic Analysis of Low Template DNA
Conclusion
References
20 Using Mitochondrial DNA in Human Identification
Mitochondria: Structure, Physiology, and Functions
Mitochondrial Genome
Inheritance of Mitochondrial Genome
Application of Mitochondrial DNA Analysis in Forensic Caseworks
Human Identification from Degraded Compromised Samples
Age Estimation Using mtDNA Analysis
Genealogy Tracing
Mitochondrial DNA Analytical Methodologies in Forensic Caseworks
Data Interpretation
Cambridge Reference Sequence(s)
Nomenclature of mtDNA Sequence
mtDNA Sequencing Result Interpretation
Human Mitochondrial DNA Sequence Database
EMPOP: Innovative and Forensically Significant Human mtDNA Database
Modifications to the Current Methods of Mitochondrial DNA Analysis and Future Perspective
Screening Approaches for mtDNA Analysis
Currently Employed CE Technique Versus Next-Generation Sequencing Approaches
Hybridization Capture-Based Target Enrichment Coupled with Massive Parallel Sequencing
Conclusion
References
21 Applications of NGS in DNA Analysis
Introduction
A Brief History of DNA Typing in Forensic Science
Next-Generation Sequencing
Applications of NGS in DNA Profiling
Molecular Genetics and Diagnostics
Medical Microbiology
Forensic Microbiology and Biosurveillance
Environmental Microbiology
Forensic Biology
Plant Biology
Evolutionary Biology
Conclusion
References
22 Using Multiple Chromosomal Marker Analysis Tools, for DNA Profiling in Human Identification: New, Evolving and Productive A...
Introduction
The Evolution and Diversity STRs Markers for Human Identification
The Sex Chromosomal STRs in Human Genotyping
The Role of SNPs in Human Identification
Systems with STR Markers and Their Variations
Use of Combination Kits
Next Generation Sequencing-Based DNA Profiling Systems in Forensics
Conclusion
References
Part III: Varied Nature of Cases Solved by DNA Profiling
23 The Effect of Consanguineous Marriages in Solving DNA Cases
Introduction
Consanguineous Marriages
Homozygosity
Technical Updates
Conclusions
References
24 A Case Study on Murder Mystery Solved by DNA Typing
Introduction
Brief Case History
Description and Source of Samples
Procedure
Results and Observation
Conclusion
Precautions
References
25 Sexual Assault and Murder: When DNA Does Not Help Even Though It Is Present
Introduction
Case 1: Homicide
Case 2: Sexual Assault
Conclusion
References
26 DNA Profiling for Mass Disaster Victim Identification
Introduction
Background
Process
Sample Collection, Storage, and Transport
DNA Preprocessing, Extraction, and Quantification
PCR Amplification of STR Loci
Capillary Electrophoresis and DNA Profiling
Analysis
Case Reports
Case 1
Samples
DNA Extraction
DNA Profiling
Matching of DNA Profiles
Results
Case 2
Discussion
Conclusion
References
27 Potential Use of DNA Profiling in Solving Terrorism Cases
Introduction
Forensics DNA Intelligence
Forensic Investigative Team
Recovery of Touch DNA from DNA Contaminated with RDX-C4 Explosives
Technical Updates
Conclusions
Part One: Forensic DNA Intelligence and IEDs
Part Two: The Effect of RDX-C4 on the Recovery of Touch DNA
References
28 DNA Profiling in Forensic Odontology
Introduction
Identification Through Teeth
Applications of DNA in Forensic Odontology
Handling and Processing of Tooth Samples
Extraction of Dental DNA
Phenol-Chloroform/Organic Extraction Procedure
DNA Extraction of Teeth (Using Automate Express)
Recent Technologies in Human Identification
Analysis of Y Chromosome
Hatshepsut, Great Queen of Ancient Egypt
Identification of Carbonized Body Through Dental DNA
Conclusion
References
Part IV: DNA Analysis in Disease Diagnosis
29 Prenatal Diagnosis of Genetic Disorders by DNA Profiling
Introduction
Indications for Genetic Testing
Pre- and Post-test Genetic Counselling
Invasive Prenatal Diagnostic Procedures for Chromosomal or DNA Testing
Prenatal Cytogenetics and Its Limitations
Polymerase Chain Reaction (PCR)
Amplification-Refractory Mutation System (ARMS)-PCR
Applications of ARMS-PCR
Restriction Fragment Length Polymorphism (RFLP)
Sanger Sequencing
Applications of MLPA
Methylation-Specific MLPA (MS-MLPA)
Uses of MS-MLPA
Procedure
Noninvasive Prenatal Testing (NIPT) for Prenatal Cytogenetic Abnormalities
Illustrative Cases of Prenatal Diagnosis by Various Methods
Conclusions
References
30 CBU Posttransplant Chimerism Analysis Using ChimerMarker
Introduction
Definitions
Chimerism and Transplantation
HLA System
Umbilical Cord Blood (UCB)
Graft Versus Host Disease (GvHD)
Pre-analytic
Q-PCR
Short Tandem Repeats (STR)
Standardization
ChimerMarker
Case Report 256
Case Report 449
Cell Sorting
CBU Case Report
Case Report 269
Case Report 176
Conclusion
References
31 Diagnosis of Genetic Disorders by DNA Analysis
Introduction
DNA Testing for Classical Chromosomal Disorders
Common Microdeletion Syndromes
DiGeorge Syndrome/Velocardiofacial Syndrome (22q11.2 Deletion)
Prader-Willi Syndrome/Angelman Syndrome (15q11.2-q13 Deletion)
Subtelomeric Deletion/Duplication Syndromes
DNA Testing for Classical/Important Single-Gene Disorders
Beta-thalassemia
Alpha-thalassemia
Cystic Fibrosis
Congenital Adrenal Hyperplasia (CAH)
Spinal Muscular Atrophy
Duchenne Muscular Dystrophy (DMD)
Inborn Errors of Metabolism (IEMs)
Genodermatoses
Skeletal Dysplasias
Endocrine Disorders
Cardiac Disorders
Disorders of Sex Development (DSD)
Adult Onset/Late Onset Diseases
Newborn Screening
Premarital/Preconception/Prenatal/Preimplantation Genetic Screening/Diagnosis/Cancer Genetic Screening
Conclusion
References
Part V: Modifications in Routine Methodologies to Solve Challenging Cases
32 Challenges in DNA Extraction from Forensic Samples
Introduction
DNA Extraction History
Forensic Samples
DNA Extraction Methodologies
Organic Extraction
Chelex Extraction Method
Magnetic Beads Extraction Method
Spin Columns Extraction Method
Automated Extraction
Differential Extraction
Direct PCR
Storing Condition
Main Challenges
DNA Quantity and Quality
Contamination
Inhibitors
References
33 DNA Extraction in Human Bodies: From Fresh to Advanced Stages of Decomposition
Highlights
Introduction
DNA Extraction from Fresh Bodies
Overview
Biological Samples
DNA Extraction
Chelex
Chelex Protocol
Whatman FTA Paper
Organic Method
Organic Protocol
Solid-Phase
QIAamp DNA Investigator Kit (QIAGEN, Hilden, Germany) Protocol
Blood and Saliva
Buccal Swab
Urine
Hair
Purification and Elution (for All Samples)
DNA Extraction from Decomposed/Skeletonized Bodies
Overview
Biological Samples
DNA Extraction
Organic
Organic Protocol
Solid-Phase
QIAamp DNA Investigator Kit (QIAGEN, Hilden, Germany) Protocol
Silica-Coated Beads Protocol PrepFiler BTA Forensic DNA Extraction Kit (Thermofisher, Waltham, MA, USA)
Conclusions
References
34 DNA isolation from human remains
Introduction
Sample Pretreatment
Decontamination
Surface Washing
Sodium Hypochlorite Washing
Ethanol Washing
Acid Washing
Hydrogen Peroxide Washing
Enzymatic Predigestion
Milling and Sanding
Inner Part of the Compact Bone Sampling
Ultraviolet Irradiation
Combination of Techniques
Pulverization
Manual Mortar
Freezer Mill
TissueLyser
DNA Isolation
Manual DNA Isolation
Total Demineralization
Organic Extraction
Chelex Resin
Manual Purification of DNA Extracts
DNA Isolation with Commercial Kits
PrepFiler BTA Forensic DNA Extraction Kit
Bone DNA Extraction Kit
QIAamp DNA Investigator Kit
EZ1 DNA Investigator Kit
Cells and Tissue DNA Isolation Kit
Crime Prep Adem-Kits for Casework
Incorporation of Manual Pretreatment Protocols to Commercial Kits
Conclusions
References
35 Usefulness of Quantitative PCR in Forensic Genetics
Introduction
DNA Quantification
DNA Quantification Importance
DNA Quantification Methods
Quantitative PCR (qPCR) Fundamentals
Detection Methods
TaqMan Probes
SYBR Green qPCR Assays
Molecular Beacons
Calibration Curve and DNA Quantity Calculation
Quantitative PCR Instruments
qPCR Customization
Current Applications of qPCR in Forensic Genetics
Autosomal DNA Quantification
X and Y Chromosome Quantification
Mitochondrial DNA Quantification
Ancient DNA Studies
Non-human DNA Analysis
Single-Nucleotide Polymorphisms (SNPs)
Issues Associated with qPCR
Future Perspectives for qPCR in Forensic Genetics
Digital PCR (dPCR)
Conclusions
References
36 Logical Errors and Fallacies in DNA Evidence Interpretation
Introduction
Initial Assessment of DNA Evidence
The Probative Value of DNA Evidence
The Logic of Evidential Proof
Logical Fallacies and Reasoning Errors in DNA Evidence Interpretation in Reports and the Courtroom
The Prosecutor´s Fallacy
The Source Probability Error
The Ultimate Issue Error
The False-Positive Fallacy
How Not to Commit the Prosecution Fallacy
The Defendant´s Fallacy
The Uniqueness Fallacy
The Probability of another Match Error
The Numerical Conversion Error
Other Interpretation Difficulties
Relative Frequency of Occurrence
The ``Could be´´ or ``Could Have Come/Originated From´´ Approach
The ``Could Not Be Excluded´´ Approach
``Consistent with´´ Approach
The Association Fallacy
The Use of the Verbal Scale to Express the Strength of DNA Evidence
Statements about Identical Twins
Conclusions: Why Misinterpretation of Evidence Happens and What Can Be Done About It?
References
Part VI: Non-human Case Studies
37 Wildlife DNA Profiling and Its Forensic Relevance
Introduction
Identification of Species of Origin
Species Testing
Sequencing
Species Specific Primers
Common Locus Used for Species Identification
Cytochrome b (Cyt b)
Cytochrome Oxidase I (COI)
The Control Region (or D-Loop)
DNA Repositories
GenBank
EMBL
DDBJ
Identification of the Geographic Origin
Principle of Identification of the Geographic Origin
Methods of Determining Geographic Origin with DNA Markers
Lineage Markers
Microsatellites
Single Nucleotide Polymorphisms (SNPs)
Individual Identification
Individualization Testing
Short Tandem Repeats
Nonhuman Paternity Testing
Conclusion
References
38 Barcoding of Plant DNA and Its Forensic Relevance
Introduction
Forensic Botany and DNA Barcoding
Conclusion
References
39 DNA-Based Analysis of Plant Material in Forensic Investigations
Introduction
History of DNA-Based Analysis of Plant Evidence
Sample Types of Botanical Evidence, Collection, and DNA Isolation
Sample Types
Collection of Plant Materials and Soil
Isolation of DNA from Plant Materials
Applications of Analyzing Plant Material Associated with Forensic Evidence
Taxonomic Identification
Geographic Origin
Individualization
Case Studies
Applications of New Sequencing Technologies to Plant Evidence
Guidelines and Standards for DNA Analysis of Plant Evidence
Alternatives to DNA for Analyzing Plant Material Associated with Forensic Evidence
Conclusions
References
40 Molecular Markers and Genomics for Food and Beverages Characterization
Introduction
Epigenetic Influence of Food
Microbiome in Food
Tailoring DNA-Based Methods for Food Characterization
Overview of Molecular Methods for Food Authentication
Case Studies
Molecular Authentication Methods for Species Determination in Food
Molecular Authentication Methods for Variety or Breed Determination in Food, Condiments, and Beverages
The Growing Contribution of Bioinformatics in Food and Wine Traceability
Conclusion
References
41 Use of DNA Barcoding for Plant Species Identification
Introduction
Types of Cases
Transferred Trace Evidence in Physical and Sexual Assault Cases
Cases Related to Forbidden/Controlled Materials (Drugs and Psychoactive Substances)
Cases Related to Protected Plants Under Environment Laws
Cases Related to Poisoning
DNA Barcoding for Species Identification in Forensic Botany
matK (Maturase K)
Internal Transcribed Spacer (ITS)
Protocol for Plant Species Identification Using DNA Barcodes
Collection of Plant Samples for DNA Isolation
Preservations of Samples
Plant DNA Isolation
Organic Extraction Method
Cetryl Trimethylammonium Bromide (CTAB) Method
Quality and Quantity Assessment
PCR Amplification of the DNA Barcodes
Plant Genome Sequencing
Database
Conclusion
References
42 Molecular Techniques in Microbial Forensics
Introduction
Bioterrorism
Individualistic Microflora for Person Identification
Postmortem Examination
Detection of Body Fluids
Microflora of Soil and Water for Forensic Use
Epidemiology and Microbial Forensics
The Elements
Sample Collection
Detection Methods
Interpretation of Results
Conclusion
References
Part VII: Quality Control and Challenges in Forensic DNA Analysis
43 Touch DNA Analysis
Introduction
History of Touch DNA
Techniques for Collection of Touch DNA
Swab Technique
Cutting Out Technique
Scraping Technique
Tape Lift Technique
Techniques of Touch DNA Examination
Extraction and Isolation of Touch DNA
Evidentiary Value
Limitation
Case Study
A Valid Example: Charging the Waldo Attacker
Conclusion
References
44 The Use of Rapid DNA Technology in Forensic Science
Introduction
History of Forensic Rapid DNA
Instrumentation and Technology
Ease of Use
Portability
Uses and Applications
Booking Station
Crime Scene
Satellite DNA Laboratories
Disaster Victim Identification
Match Confirmation
Removing Bias in Mixture Interpretation
Retesting of Swabs
Comparison to Traditional DNA Methods
Sample Collection
Extraction
Quantification
PCR Amplification
Electrophoresis
Data Interpretation
Troubleshooting
Areas for Improvement and Future Development
Cost
Flexible Throughput
Size
Speed
Expanded Testing Capabilities
Conclusion
References
45 The Interpretation of Mixed DNA Samples
Introduction
Mixture and Complex DNA Profiles
Issues Surrounding the LCN Technique
Probabilistic Genotyping
The Application of Probabilistic Genotyping Methods During the Process
MPS Approaches to Forensic Genetics for DNA Mixture Deconvolution
Conclusion
References
46 Current Status of DNA Databases in the Forensic Field
Introduction
National DNA Databases
African Region
Middle East
European Region
Australasia
American Region
Asia
Ethical and Legal Standards
Major Ethical Concerns Include
Human Rights and Racial Contemplations
Privacy (Lack of Safeguard)
Familial Looking, Research Use, and Counter-Psychological Oppression
DNA Is Not Secure and Foolproof
Errors and Improper Utilization of Police Resource
Conclusion
References
47 InDel Loci in Forensic DNA Analysis
Introduction
Insertion
Deletion
InDel (Insertion/Deletion)
Use of Insertion/Deletion Polymorphism in Forensic Genetics
Commercial Kits Based on InDel Analysis
Investigator DIPplex Kit
InDelPlex INDEL Polymorphism Detection Kit
Mentype DIPplex PCR Amplification Kit
Use of Gonosomal InDel Loci in Forensic Genetics
Analysis of InDel Loci
Conclusion
References
48 The Role of DNA Profiling in Landscape of Human Migration
Introduction
Definitions and Numbers: The Magnitude of the Problem
Missing Persons and Migrations: A Difficult Crime to Solve
Human Trafficking: The Most Dangerous Scenario
The Role of Forensic Science: DNA Typing
The Advantages and Need for DNA Migrant Identification Databases
Robust, Reliable, Universal Technology
Power of Identification
Comparing Victim´s Data with Relatives´ Data
Possibility to Analyze All Kind of Biological Samples
Building, Updating, and Managing Genetic Databases
Missing Children: The DNA-PROKIDS Program
Organ Trafficking: The DNA-ProORGAN Program
Future Perspectives: Enlarging and Coordinating the Databases
Conclusion
References
49 Challenges in the DNA Analysis of Compromised Samples
Introduction
Inhibition of PCR
Inhibitory Mechanisms
Inhibition Factors
Degraded DNA
DNA Degradation Mechanisms
DNA Degradation Factors
Low Copy Number DNA
Problems with LCN DNA Analysis
DNA Contamination
DNA Contamination Sources
Laboratory Recommendations for the Analysis of Compromised Samples
Conclusions
References
50 Validating Forensic DNA Workflows
Introduction
What Is Validation?
Why Validate?
How to Validate?
Conclusion
References
51 Quality Control Measures in Short Tandem Repeat (STR) Analysis
Introduction
DNA Methods
Conclusions
References
52 DNA Phenotyping: The Technique of the Future
Inception of DNA Technology
Forensic DNA Phenotyping
Introduction
Science Behind DNA Phenotyping Technology
DNA-Based Prediction of Externally Visible Characteristics
Eye Color
Hair
Skin
DNA-Based Inference of Non-pigmented Traits
Face
Hair Structure
Hair Loss/Baldness
Age
Body Height/Stature
Currently Available Forensic DNA Phenotyping Test Systems
Legislations for Forensic DNA Phenotyping
Legal and Ethical Aspects of Forensic DNA Phenotyping
Case Studies: DNA Phenotyping
The Future of FDP
Conclusion
References
53 Swift DNA Technologies and Their Usefulness for Law Enforcement Agencies
Introduction
Growing Demand on Forensic Laboratories
Rapid DNA Testing as an Investigative Tool
Turnaround Time
Transportability
Contamination
Key Concerns
Privacy
Cognitive Challenges
Function Creep
Going Forward
Conclusion
References
54 Tracing of Human Migration and Diversity by Forensic DNA Analysis
Introduction
DNA as a Palimpsest of Human Migration
General Markers Used in DNA Profiling
Short Tandem Repeat (STR) Markers
Mitochondrial DNA (mtDNA)
Y Chromosome Marker
The Human Migration: Origins in Africa
Out of Africa and the Conjunction with Neanderthals
The Peopling of Europe
The Colonization of Asia and Oceania
Populating Americas
The Denisovans
Future Directions
References
Index

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Hirak Ranjan Dash Pankaj Shrivastava J. A. Lorente Editors

Handbook of DNA Profiling

Handbook of DNA Profiling

Hirak Ranjan Dash • Pankaj Shrivastava • J. A. Lorente Editors

Handbook of DNA Profiling With 173 Figures and 95 Tables

Editors Hirak Ranjan Dash School of Forensic Science National Forensic Sciences University Delhi Campus New Delhi, India

Pankaj Shrivastava DNA Fingerprinting Unit State Forensic Science Laboratory Sagar, Madhya Pradesh, India

J. A. Lorente Laboratory of Genetic Identification Department of Legal Medicine Toxicology and Physical Anthropology Faculty of Medicine University of Granada Granada, Spain GENYO Centre for Genomics and Oncological Research (Pfizer / University of Granada / Andalusian Regional Government) Granada, Spain

ISBN 978-981-16-4317-0 ISBN 978-981-16-4318-7 (eBook) ISBN 978-981-16-4319-4 (print and electronic bundle) https://doi.org/10.1007/978-981-16-4318-7 © Springer Nature Singapore Pte Ltd. 2022 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

A tribute to Dr. Tapaswini Tripathy, a young dedicated COVID warrior and a great academician who lost her battle of life to COVID in 2021, leaving behind her family and kids in ocean of sorrows.

Preface

The advent of DNA fingerprinting technology has revolutionized the process of criminal investigation. With the help of DNA report, it has become much easier for the investigating agencies, law practitioners, and honorable judges to conclude/opine in severely complicated cases. The beauty of this technology is that it does not only identify a culprit, but it has the potential to exonerate a wrongly convicted person. The scientific truth of DNA technology corroborating with the criminal justice system has been proven over the years throughout the globe. Generating DNA report of a few pages requires great expertise, skilled manpower, constant brainstorming in the SOPs, and use of advanced technologies. Due to varied nature of forensic samples, it becomes immensely difficult for DNA examiners to process these samples for generating a DNA profile. In this regard, the technology has seen much advancement over the years from RFLP to DNA phenotyping by using next-generation sequencing (NGS). The adoption of continuously evolving technology helps in processing the challenged samples, generating a DNA profile in much lesser time, as well as increasing the informativeness of a single analysis. Though law differs with countries, the technology used is the same. Thus, a comprehensive collection of advanced DNA technologies and case works has been included in this volume. This will be immensely useful for technology transfer and knowledge sharing among the examiners and other beneficiaries. Additionally, proper understanding of the technology will be ensured among the students, medical officers, investigating agencies, law practitioners, and honorable judges through real case studies. The edited volume includes the use of DNA fingerprinting technology in solving varieties of criminal as well as civil cases such as paternity dispute, identification of mutilated remains, culprit identification in sexual assault cases, and murder cases. Chapters on non-human studies are also included. The chapters are written by eminent DNA practitioners and academicians around the globe in simple English to help beginners grasp the topic with ease. This will be a huge asset not only for DNA examiners, investigating officers, law practitioners, and honorable judges but also for students of forensic science, genetics, law, and forensic medicine. New Delhi, India Sagar, India Granada, Spain April 2022

Hirak Ranjan Dash Pankaj Shrivastava J. A. Lorente vii

Contents

Volume 1 Part I

Principles of Forensic DNA Profiling . . . . . . . . . . . . . . . . . . . .

1

1

Forensic DNA Investigation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J. H. Smith

3

2

Introduction to Forensic DNA Typing and Current Trends . . . . . . Monika Chakravarty and Prateek Pandya

29

3

Biological Sources of DNA: The Target Materials for Forensic DNA Typing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pankaj Shrivastava, R. K. Kumawat, Pushpesh Kushwaha, and Manisha Rana

4

5

Collection, Preservation, and Transportation of Biological Evidences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hirak Ranjan Dash and Kamayani Vajpayee Forensic DNA: From New Approaches for the Bio-stain Identification to the Evaluation of the Genetics Evidence in Courtroom . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. D’Orio, P. Montagna, M. Mangione, and G. Francione

51

69

85

6

Tools and Techniques Used in Forensic DNA Typing . . . . . . . . . . . Akanksha Behl, Amarnath Mishra, and Indresh Kumar Mishra

119

7

Evaluation of the Autosomal STR Markers and Kits . . . . . . . . . . . Vikash Kumar

151

8

Forensic Human Y-Chromosome Markers: Principles and Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Arash Alipour Tabrizi

9

Haplodiploid Markers and Their Forensic Relevance . . . . . . . . . . Antonio Amorim and Nadia Pinto

185 219

ix

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Contents

10

Single-Nucleotide Polymorphism . . . . . . . . . . . . . . . . . . . . . . . . . . Anubha Gang and Vivek Kumar Shrivastav

11

Ethical Governance of Forensic DNA Databases in Southeast Asia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M. C. A. De Ungria and E. B. Jimenez

257

Using Laboratory Validation to Identify and Establish Limits to the Reliability of Probabilistic Genotyping Systems . . . . . . . . . . Dan E. Krane and M. Katherine Philpott

297

12

235

13

Potential of DNA Technique-Based Body Fluid Identification . . . . Aditi Mishra, Ulhas Gondhali, and Sumit Choudhary

321

14

Overview of Familial DNA and Forensic Phenotyping . . . . . . . . . . Samuel D. Hodge and John Meehan

339

15

A Glimpse of Famous Cases in History Solved by DNA Typing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hirak Ranjan Dash, Kamayani Vajpayee, and Radhika Agarwal

357

...

375

16

Role of the Molecular Anthropologist in the Forensic Context Elena Pilli

Part II Usefulness of Various Techniques of DNA Profiling in Solving Cases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Short Tandem Repeat Mutations in Paternity Analysis . . . . . . . . . Uthandaraman Mahalinga Raja, Usharani Munuswamy, Rajshree Raghunath, Thilaga Dhanapal, and Mahalakshmi Nithyanandam

18

Utility and Applications of Lineage Markers: Mitochondrial DNA and Y Chromosome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sara Palomo-Díez and Ana María López-Parra

395 397

423

19

Usefulness of the X-Chromosome on Forensic Science . . . . . . . . . . Cláudia Gomes and Eduardo Arroyo-Pardo

455

20

Using Mitochondrial DNA in Human Identification . . . . . . . . . . . Pankaj Shrivastava, Manisha Rana, Pushpesh Kushwaha, and D. S. Negi

479

21

Applications of NGS in DNA Analysis . . . . . . . . . . . . . . . . . . . . . . Kelly M. Elkins, Hannah E. Berry, and Kashiya R. Reese

501

22

Using Multiple Chromosomal Marker Analysis Tools, for DNA Profiling in Human Identification: New, Evolving and Productive Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rajendra V. E. Chilukuri

519

Contents

Part III 23

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Varied Nature of Cases Solved by DNA Profiling . . . . . . . .

The Effect of Consanguineous Marriages in Solving DNA Cases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Noora R. Al-Snan, Fatima J. AlBuarki, and Samreen S. Sayed

543 545

24

A Case Study on Murder Mystery Solved by DNA Typing . . . . . . Subhasish Sahoo and Rashmita Samal

559

25

Sexual Assault and Murder: When DNA Does Not Help Even Though It Is Present . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mark Benecke

567

26

DNA Profiling for Mass Disaster Victim Identification E. V. Soniya and U. Suresh Kumar

.........

575

27

Potential Use of DNA Profiling in Solving Terrorism Cases . . . . . . Noora R. Al-Snan

589

28

DNA Profiling in Forensic Odontology . . . . . . . . . . . . . . . . . . . . . . Pooja Puri, Mayank Kumar Dubey, and Naresh Kumar

605

Volume 2 Part IV

DNA Analysis in Disease Diagnosis . . . . . . . . . . . . . . . . . . .

29

Prenatal Diagnosis of Genetic Disorders by DNA Profiling . . . . . . Inusha Panigrahi and Priyanka Srivastava

30

CBU Posttransplant Chimerism Analysis Using ChimerMarker™ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Donato Madalese, Roberta Penta de Vera d’Aragona, Federica Schiano di Tunnariello, and Giovanna Maisto

31

Diagnosis of Genetic Disorders by DNA Analysis . . . . . . . . . . . . . Parag M. Tamhankar, Vasundhara P. Tamhankar, and Lakshmi Vasudevan

Part V Modifications in Routine Methodologies to Solve Challenging Cases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Challenges in DNA Extraction from Forensic Samples . . . . . . . . . Anna Barbaro

33

DNA Extraction in Human Bodies: From Fresh to Advanced Stages of Decomposition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Venusia Cortellini, Lorenzo Franceschetti, Heitor S. D. Correa, and Andrea Verzeletti

623 625

645

675

709 711

731

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Contents

34

DNA isolation from human remains . . . . . . . . . . . . . . . . . . . . . . . . María Saiz, Christian Haarkötter, X. Gálvez, L. J. Martinez-Gonzalez, M. I. Medina-Lozano, and Juan Carlos Alvarez

755

35

Usefulness of Quantitative PCR in Forensic Genetics . . . . . . . . . . Christian Haarkötter, M. J. Alvarez-Cubero, Juan Carlos Alvarez, and María Saiz

773

36

Logical Errors and Fallacies in DNA Evidence Interpretation Andrei Semikhodskii

...

799

Part VI

Non-human Case Studies . . . . . . . . . . . . . . . . . . . . . . . . . . .

821

37

Wildlife DNA Profiling and Its Forensic Relevance . . . . . . . . . . . . Ulhas Gondhali and Aditi Mishra

823

38

Barcoding of Plant DNA and Its Forensic Relevance . . . . . . . . . . . Gianmarco Ferri, Beatrice Corradini, Francesca Ferrari, and Enrico Silingardi

839

39

DNA-Based Analysis of Plant Material in Forensic Investigations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . James M. Robertson, Natalie Damaso, and Kelly A. Meiklejohn

857

Molecular Markers and Genomics for Food and Beverages Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rita Vignani, Monica Scali, and Pietro Liò

889

40

41

Use of DNA Barcoding for Plant Species Identification . . . . . . . . . Jaskirandeep Kaur Jossan and Rajinder Singh

911

42

Molecular Techniques in Microbial Forensics . . . . . . . . . . . . . . . . Neeti Kapoor, Pradnya Sulke, and Ashish Badiye

935

Part VII Quality Control and Challenges in Forensic DNA Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

957

43

Touch DNA Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sourabh Kumar Singh, Amarnath Mishra, and Akanksha Behl

959

44

The Use of Rapid DNA Technology in Forensic Science . . . . . . . . . Robert O’Brien

975

45

The Interpretation of Mixed DNA Samples . . . . . . . . . . . . . . . . . . Francesco Sessa, Monica Salerno, and Cristoforo Pomara

997

46

Current Status of DNA Databases in the Forensic Field . . . . . . . . 1019 Sachil Kumar, Saranya Ramesh Babu, and Shipra Rohatgi

47

InDel Loci in Forensic DNA Analysis . . . . . . . . . . . . . . . . . . . . . . . 1039 Tugba Unsal Sapan

Contents

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48

The Role of DNA Profiling in Landscape of Human Migration . . . 1051 J. A. Lorente, Christian Haarkötter, María Saiz, M. I. Medina-Lozano, X. Gálvez, M. J. Alvarez-Cubero, L. J. Martínez-González, B. Lorente-Remon, and Juan Carlos Alvarez

49

Challenges in the DNA Analysis of Compromised Samples . . . . . . 1067 Christian Haarkötter, María Saiz, M. J. Alvarez-Cubero, Juan Carlos Alvarez, and J. A. Lorente

50

Validating Forensic DNA Workflows Iman Muharam and Carla Paintner

51

Quality Control Measures in Short Tandem Repeat (STR) Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1107 Heather Miller Coyle

52

DNA Phenotyping: The Technique of the Future . . . . . . . . . . . . . . 1125 Kamayani Vajpayee and Ritesh Kumar Shukla

53

Swift DNA Technologies and Their Usefulness for Law Enforcement Agencies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1151 James Simpson

54

Tracing of Human Migration and Diversity by Forensic DNA Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1165 Nithyanandam Mahalaxmi, Avinash Chand Puri, Pawan Kumar Chouhan, and Alka Mishra

. . . . . . . . . . . . . . . . . . . . . . . 1087

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1185

About the Editors

Dr. Hirak Ranjan Dash is currently working as an Assistant Professor of Forensic Biology and Biotechnology at the National Forensic Sciences University, Delhi Campus, India. Besides teaching and research, he is actively involved in conducting training programs to various beneficiaries of forensic science. Before joining academics, he served as a forensic DNA expert at Madhya Pradesh Forensic Science Laboratory, India, for over 6 years. He has an experience of conducting DNA examination in more than 1500 complicated cases. He has completed his Ph.D. from the Department of Life Science, National Institute of Technology, Rourkela, India, He received his M.Sc. in Microbiology from Orissa University of Agriculture and Technology, Odisha, India. His research interests include forensic microbiology, thanatomicrobiome analysis, molecular microbiology, DNA fingerprinting, and genetic manipulation. He is one of the pioneers in India to work on NGS technology-based forensic DNA analysis. He has written 8 books and published 50 research papers, 14 book chapters, 12 conference proceedings, and 4 popular science articles. Dr. Pankaj Shrivastava received his Ph.D. in microbiology with a specialization in biotechnology from the Department of Biological Science, Rani Durgawati University, Jabalpur, Madhya Pradesh, India. He is in charge of the Forensic DNA Fingerprinting facility of the Govt. of Madhya Pradesh, India at Sagar. Dr. Shrivastava has more than 13 years of experience as a bench worker for examining and reporting a wide range of criminal cases using DNA technology and deposing the court evidence. His research interests include population DNA database, improvement of xv

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About the Editors

methods in forensic DNA typing, finding rapid protocols for the technology, and microbial forensics. He is a two-time recipient of the prestigious Pt. Govind Vallabh Pant Award from the Bureau of Police Research and Development, Ministry of Home Affairs, Govt. of India, and a recipient of the Anusrajan Award from AIISECT University, Bhopal, for his authored books. Dr. Shrivastava is also a recipient of the FICCI Smart Policing award for developing a fast DNA-typing protocol. He is a visiting faculty at many universities, police training institutes, and judicial officers training institutes. Dr. Shrivastava has written 13 books and published 65 research papers in journals of repute in forensic science, 21 book chapters, 84 conference proceedings, and 15 popular science articles. J. A. Lorente is Professor of Forensic Medicine at the University of Granada, Spain. After graduating from the Faculty of Medicine of Granada in 1985, he completed his Ph.D. (Honors) in medicine and surgery at the University of Granada, in 1989. Dr. Lorente has published over 170 peer-reviewed papers and several books and book chapters. He has a special focus on the use of DNA and its application to human rights (he launched the first-ever database to identify missing people, the Spanish Phoenix Program, back in 1999); he also created and launched the DNA-PROKIDS Program in 2004, and the DNA-Pro-ORGAN Program in 2016. His areas of interest in forensics also deal with population variability and analysis of old and ancient DNA samples and databases expansion and control. Dr. Lorente is also actively working in medical genomics, and he is the scientific director of the Center for Genomics and Oncological Research (GENYO), where his team focuses on liquid biopsy and cancer interception. Dr. Lorente is the founder and first president of the AICEF (Ibero-Latin American Network of Forensic Sciences) and an honorary member of the AFSN (Asian Forensic Sciences Network).

Contributors

Radhika Agarwal DNA Fingerprinting Unit, Forensic Science Laboratory, Bhopal, Madhya Pradesh, India Fatima J. AlBuarki Forensic Science Laboratory, Directorate of Forensic Science, Ministry of Interior, Manama, Kingdom of Bahrain Arash Alipour Tabrizi Legal Medicine Research Center, Legal Medicine Organization, Tehran, Iran Noora R. Al-Snan Forensic Science Laboratory, Directorate of Forensic Science, General Directorate of Criminal Investigation and Forensic Science, Ministry of Interior, Kingdom of Bahrain, Manama, Bahrain Juan Carlos Alvarez Laboratory of Genetic Identification, Department of Legal Medicine, Toxicology and Physical Anthropology, Faculty of Medicine, University of Granada, Granada, Spain M. J. Alvarez-Cubero Department of Biochemistry and Molecular Biology III, Faculty of Medicine, University of Granada, Granada, Spain Pfizer-University of Granada-Junta de Andalucía Centre for Genomics and Oncological Research (GENYO), Granada, Spain Antonio Amorim Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal Institute for Research and Innovation in Health (i3S), University of Porto, Porto, Portugal Faculty of Sciences of the University of Porto (FCUP), Porto, Portugal Eduardo Arroyo-Pardo Laboratory of Forensic and Population Genetics, Legal Medicine Psychiatry and Pathology Department, Medicine School of the Complutense University of Madrid, Madrid, Spain Forensic Sciences: Forensic Genetics and Toxicology Group, Instituto de Investigación Sanitaria del Hospital Clínico San Carlos IdISSC, Madrid, Spain

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Contributors

Saranya Ramesh Babu Department of Forensic Sciences, College of Criminal Justice, Naif Arab University for Security Sciences, Riyadh, Saudi Arabia Ashish Badiye Department of Forensic Science, Government Institute of Forensic Science, Nagpur, Maharashtra, India Anna Barbaro Department of Forensic Genetics, Studio Indagini Mediche E Forensi (SIMEF), President Worldwide Association of Women Forensic Experts (WAWFE), Reggio Calabria, Italy Akanksha Behl Amity Institute of Forensic Sciences, Amity University Uttar Pradesh, Noida, Uttar Pradesh, India Mark Benecke Forensic Biology Unit, International Forensic Research and Consulting, Cologne, Germany Hannah E. Berry TU Human Remains Identification Lab (THRIL), Department of Chemistry, Towson University, Towson, MD, USA Monika Chakravarty Forensic Science Laboratory, Home Department Government of NCT of Delhi, Delhi, India Rajendra V. E. Chilukuri SyberGENE Inc, San Jose, CA, USA Sumit Choudhary School of Forensic Science and Risk Management, National Security and Police University of India, Rashtriya Raksha University, Gandhinagar, Gujarat, India Pawan Kumar Chouhan Regional Forensic Science Laboratory, Indore, Madhya Pradesh, India Beatrice Corradini Department of Biomedical, Metabolic and Neural Sciences, Institute of Legal Medicine, University of Modena and Reggio Emilia, Modena, Italy Heitor S. D. Correa Department of Medical and Surgical Specialties, Radiological Sciences and Public Health, Forensic Medicine Unit, University of Brescia, Brescia, Italy Venusia Cortellini Department of Medical and Surgical Specialties, Radiological Sciences and Public Health, Forensic Medicine Unit, University of Brescia, Brescia, Italy E. D’Orio Bio Forensics Research Center, Angri, Italy Natalie Damaso Biological and Chemical Technologies, Massachusetts Institute of Technology Lincoln Laboratory, Lexington, MA, USA Hirak Ranjan Dash School of Forensic Science, National Forensic Sciences University, Delhi Campus, New Delhi, India M. C. A. De Ungria DNA Analysis Laboratory, Natural Sciences Research Institute, University of the Philippines Diliman, Quezon, Philippines

Contributors

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Program on Biodiversity, Ethnicity, and Forensics, Philippine Genome Center, University of the Philippines Diliman, Quezon, Philippines Thilaga Dhanapal Forensic Sciences Department, DNA Division, Chennai, India Mayank Kumar Dubey Senior Faculty-Forensic Science Department, Mody University, Laxmangarh, Rajasthan, India Kelly M. Elkins TU Human Remains Identification Lab (THRIL), Department of Chemistry, Towson University, Towson, MD, USA Francesca Ferrari Department of Biomedical, Metabolic and Neural Sciences, Institute of Legal Medicine, University of Modena and Reggio Emilia, Modena, Italy Gianmarco Ferri Department of Biomedical, Metabolic and Neural Sciences, Institute of Legal Medicine, University of Modena and Reggio Emilia, Modena, Italy Lorenzo Franceschetti Department of Medical and Surgical Specialties, Radiological Sciences and Public Health, Forensic Medicine Unit, University of Brescia, Brescia, Italy G. Francione Bio Forensics Research Center, Angri, Italy X. Gálvez Laboratory of Genetic Identification, Department of Legal Medicine, Toxicology and Physical Anthropology, Faculty of Medicine, University of Granada, Granada, Spain Anubha Gang Regional Forensic Science Laboratory, Indore, MP, India Cláudia Gomes Laboratory of Forensic and Population Genetics, Legal Medicine Psychiatry and Pathology Department, Medicine School of the Complutense University of Madrid, Madrid, Spain Forensic Sciences: Forensic Genetics and Toxicology Group, Instituto de Investigación Sanitaria del Hospital Clínico San Carlos IdISSC, Madrid, Spain Ulhas Gondhali Jindal Institute of Behavioural Sciences, O.P. Jindal Global University, Sonipat, India Christian Haarkötter Laboratory of Genetic Identification, Department of Legal Medicine, Toxicology and Physical Anthropology, Faculty of Medicine, University of Granada, Granada, Spain Samuel D. Hodge Temple University, Philadelphia, PA, USA E. B. Jimenez National Institutes of Health, University of the Philippines Manila, Manila, Philippines Jaskirandeep Kaur Jossan Department of Forensic Science, Punjabi University, Patiala, Punjab, India

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Contributors

Neeti Kapoor Department of Forensic Science, Government Institute of Forensic Science, Nagpur, Maharashtra, India Dan E. Krane Biological Sciences, Wright State University, Dayton, OH, USA Naresh Kumar Home Department, Government of NCT of Delhi, New Delhi, India Sachil Kumar Department of Forensic Sciences, College of Criminal Justice, Naif Arab University for Security Sciences, Riyadh, Saudi Arabia Vikash Kumar Centurion University of Technology and Management, Bhubaneswar, Odisha, India R. K. Kumawat DNA Division, State Forensic Science Laboratory, Jaipur, India Pushpesh Kushwaha Dr. APJ Abdul Kalam Institute of Forensic Science and Criminology, Bundelkhand University, Jhansi, India DNA Fingerprinting Unit, State Forensic Science Laboratory, Department of Home (Police), Government of Madhya Pradesh, Sagar, India Pietro Liò Department of Computer Science and Technology, University of Cambridge, Cambridge, UK Ana María López-Parra Laboratory of Forensic and Population Genetics, Legal Medicine, Psychiatry and Pathology Department, Medicine School, Complutense University of Madrid (UCM), Madrid, Spain Grupo de Ciencias Forenses: Genética y Toxicología forenses, Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC), Madrid, Spain J. A. Lorente Laboratory of Genetic Identification, Department of Legal Medicine, Toxicology and Physical Anthropology, Faculty of Medicine, University of Granada, Granada, Spain GENYO, Centre for Genomics and Oncological Research (Pfizer / University of Granada / Andalusian Regional Government), Granada, Spain B. Lorente-Remon Program International Liaison Officer (ILO), Department of Legal Medicine, Toxicology and Physical Anthropology, Faculty of Medicine, DNA-PROKIDS, Granada, Spain Donato Madalese Histocompatibility and Immunogenetics Laboratory, UOSD Cryopreservation and Ba.S.C.O, Oncohaematology Department, Pausilipon Hospital, A.O.R.N. Santobono-Pausilipon, Naples, Italy Nithyanandam Mahalaxmi Forensic Sciences Department, Chennai, Tamil Nadu, India Uthandaraman Mahalinga Raja Forensic Sciences Department, DNA Division, Chennai, India

Contributors

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Giovanna Maisto Flow Cytometry Laboratory, UOSD Cryopreservation and Ba.S. C.O, Oncohaematology Department, Pausilipon Hospital, A.O.R.N. SantobonoPausilipon, Naples, Italy M. Mangione Bio Forensics Research Center, Angri, Italy L. J. Martínez-González Department of Biochemistry and Molecular Biology III, Faculty of Medicine, University of Granada, Granada, Spain Pfizer-University of Granada-Junta de Andalucía Centre for Genomics and Oncological Research (GENYO), Granada, Spain M. I. Medina-Lozano Laboratory of Genetic Identification, Department of Legal Medicine, Toxicology and Physical Anthropology, Faculty of Medicine, University of Granada, Granada, Spain John Meehan Beasley School of Law at Temple University, Philadelphia, PA, USA Kelly A. Meiklejohn Department of Population Health and Pathobiology, North Carolina State University, Raleigh, NC, USA Heather Miller Coyle Forensic Science Department, Henry C. Lee College of Criminal Justice and Forensic Sciences, University of New Haven, West Haven, CT, USA Amarnath Mishra Amity Institute of Forensic Sciences, Amity University Uttar Pradesh, Noida, Uttar Pradesh, India Indresh Kumar Mishra Amity Institute of Forensic Sciences, Amity University Uttar Pradesh, Noida, Uttar Pradesh, India Forensic Science Laboratory, Rohini, Government of NCT, Delhi, India Aditi Mishra School of Forensic Science and Risk Management, National Security and Police University of India, Rashtriya Raksha University, Gandhinagar, Gujarat, India Alka Mishra Regional Forensic Science Laboratory, Gwalior, Madhya Pradesh, India P. Montagna Bio Forensics Research Center, Angri, Italy Iman Muharam HID Professional Services, Genetic Sciences Division, Thermo Fisher Scientific, Scoresby, VIC, Australia Usharani Munuswamy Forensic Sciences Department, DNA Division, Chennai, India D. S. Negi Centre for DNA Fingerprinting and Diagnostics (CDFD), Hyderabad, India Mahalakshmi Nithyanandam Forensic Sciences Department, DNA Division, Chennai, India

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Contributors

Robert O’Brien National Forensic Science Technology Center, Largo, FL, USA Carla Paintner HID Professional Services, Genetic Sciences Division, Thermo Fisher Scientific, Singapore, Singapore Sara Palomo-Díez Laboratory of Forensic and Population Genetics, Legal Medicine, Psychiatry and Pathology Department, Medicine School, Complutense University of Madrid (UCM), Madrid, Spain Grupo de Ciencias Forenses: Genética y Toxicología forenses, Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC), Madrid, Spain International University of La Rioja (UNIR), Logroño, Spain Prateek Pandya Amity Institute of Forensic Sciences, Amity University, Uttar Pradesh, India Inusha Panigrahi Genetic Metabolic Unit, Department of Pediatrics, APC, PGIMER, Chandigarh, India Roberta Penta de Vera d’Aragona Histocompatibility and Immunogenetics Laboratory, UOSD Cryopreservation and Ba.S.C.O, Oncohaematology Department, Pausilipon Hospital, A.O.R.N. Santobono-Pausilipon, Naples, Italy M. Katherine Philpott Dept. of Forensic Science, Virginia Commonwealth University, Richmond, VA, USA Elena Pilli Department of Biology, University of Florence, Laboratory of Anthropology - Forensic Molecular Anthropology Unit, Florence, Italy Nadia Pinto Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal Institute for Research and Innovation in Health (i3S), University of Porto, Porto, Portugal Centre of Mathematics of the University of Porto, Porto, Portugal Cristoforo Pomara Department of Medical, Surgical and Advanced Technologies “G.F. Ingrassia”, University of Catania, Catania, Italy Pooja Puri Amity Institute of Forensic Sciences, Amity University, Noida, India Avinash Chand Puri Regional Forensic Science Laboratory, Indore, Madhya Pradesh, India Rajshree Raghunath Forensic Sciences Department, DNA Division, Chennai, India Manisha Rana Department of Forensic Science, Guru Ghasidas University, Bilaspur, India DNA Fingerprinting Unit, State Forensic Science Laboratory, Department of Home (Police), Government of Madhya Pradesh, Sagar, India

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Kashiya R. Reese TU Human Remains Identification Lab (THRIL), Department of Chemistry, Towson University, Towson, MD, USA James M. Robertson Research Support Unit, Federal Bureau of Investigation Laboratory Division, Quantico, VA, USA Shipra Rohatgi Former Faculty/ Research Scholar, Amity Institute of Forensic Science, Amity University Gurugram/Noida, Noida, India Subhasish Sahoo HOD DNA Division, State Forensic Science Laboratory, Bhubaneswar, Odisha, India María Saiz Laboratory of Genetic Identification, Department of Legal Medicine, Toxicology and Physical Anthropology, Faculty of Medicine, University of Granada, Granada, Spain Monica Salerno Department of Medical, Surgical and Advanced Technologies “G.F. Ingrassia”, University of Catania, Catania, Italy Rashmita Samal DNA Division, State Forensic Science Lab, Rasulgarh, Bhubaneswar, India Samreen S. Sayed Forensic Science Laboratory, Directorate of Forensic Science, Ministry of Interior, Manama, Kingdom of Bahrain Monica Scali Department of Life Science, University of Siena, SIENA, Italy Federica Schiano di Tunnariello Manipulation and Cryopreservation Laboratory, UOSD Cryopreservation and Ba.S.C.O, Oncohaematology Department, Pausilipon Hospital, A.O.R.N. Santobono-Pausilipon, Naples, Italy Andrei Semikhodskii Medical Genomics Ltd., Tver, Russian Federation Francesco Sessa Department of Clinical and Experimental Medicine, University of Foggia, Foggia, Italy Vivek Kumar Shrivastav Regional Forensic Science Laboratory, Indore, MP, India Pankaj Shrivastava DNA Fingerprinting Unit, State Forensic Science Laboratory, Sagar, Madhya Pradesh, India Ritesh Kumar Shukla Biological and Life Sciences, School of Arts and Sciences, Ahmedabad University, Ahmedabad, Gujarat, India Enrico Silingardi Department of Biomedical, Metabolic and Neural Sciences, Institute of Legal Medicine, University of Modena and Reggio Emilia, Modena, Italy James Simpson Victoria University of Wellington, Tirohanga, New Zealand Rajinder Singh Department of Forensic Science, Punjabi University, Patiala, Punjab, India

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Sourabh Kumar Singh Amity Institute of Forensic Sciences, Amity University Uttar Pradesh, Noida, India J. H. Smith Forensic Services of the South African Police Service, Pretoria, South Africa E. V. Soniya Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India Priyanka Srivastava Genetic Metabolic Unit, Department of Pediatrics, APC, PGIMER, Chandigarh, India Pradnya Sulke Government Institute of Forensic Science, Nagpur, Maharashtra, India U. Suresh Kumar Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India Parag M. Tamhankar Centre for Medical Genetics, Mumbai, India Vasundhara P. Tamhankar Centre for Medical Genetics, Mumbai, India Tugba Unsal Sapan Institute of Addiction and Forensic Sciences, Uskudar University, Istanbul, Turkey Kamayani Vajpayee DNA Fingerprinting Unit, Forensic Science Laboratory, Bhopal, Madhya Pradesh, India School of Arts and Sciences, Ahmedabad University, Ahmedabad, India Biological and Life Sciences, School of Arts and Sciences, Ahmedabad University, Ahmedabad, Gujarat, India Lakshmi Vasudevan Centre for Medical Genetics, Mumbai, India Andrea Verzeletti Department of Medical and Surgical Specialties, Radiological Sciences and Public Health, Forensic Medicine Unit, University of Brescia, Brescia, Italy Rita Vignani Department of Life Science, University of Siena, SIENA, Italy Serge-genomics, SIENA, Italy

Part I Principles of Forensic DNA Profiling

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Contents Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Value of Forensic DNA Investigations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evidential Value . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Forensic DNA Evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Forensic DNA Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Forensic DNA Databases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Use of Forensic Lead Investigative Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Familial DNA Searches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Modus Operandi and Signature of Serial Offenders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Offender Profiling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Best Practices Using Forensic Investigative Leads as Effective Investigative Tool . . . . . . . . . . . . Step 1: Notification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Step 2: The Investigation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Step 3: After the Arrest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reasons for Unsuccessful Investigations of Serial Casework . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pretrial Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Erroneous Forensic DNA Findings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Forensic DNA evidence is assisting in resolving crime and is now commonly used in most countries. It assists in exonerating the innocent, identification of perpetrators of crime, establishes paternity, and identification of human remains. When processing and testing exhibit material, the crime scene and laboratory examiners must be mindful of the evidential value of DNA physical evidence. There has been no other technology that has been so voraciously scrutinized for J. H. Smith (*) Forensic Services of the South African Police Service, Pretoria, South Africa e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 H. R. Dash et al. (eds.), Handbook of DNA Profiling, https://doi.org/10.1007/978-981-16-4318-7_57

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acceptance in the courts as forensic DNA evidence. Since the introduction of forensic DNA evidence to the courts, there have been many reported incidences where errors have occurred during the collecting and testing phase of DNA exhibit material and the interpreting of the results. The use of forensic DNA evidence is based on reliable technology: provided that the necessary quality assurance and quality control measures are consistently applied and the value of forensic DNA findings is not overstated. Forensic databases are assisting investigators in the investigation of cases where serial and multiple offenders are involved. Verified forensic DNA investigative lead reports that are generated from the comparison searches conducted on the forensic DNA databases can either link a known person on the database to crime scenes or link various crime scenes where the person’s forensic DNA profiles are not yet uploaded to the database. Cross-forensic investigative lead products can also be used to check for certain common attributes of value that is shared between different types of forensic investigative leads to aid with the resolution of linked cases.

Keywords

Forensic DNA · Crime scene · Exhibit material · DNA databases · Short term repeat · DNA findings · Erroneous DNA findings · Evidential value · Probative value · Value of DNA Evidence · Physical evidence · Forensic investigative leads · Forensic DNA investigative leads · Cross-forensic investigative leads · Erroneous forensic DNA findings · Best practices using forensic investigative leads as effective · Serial murder cases · Serial rape cases · Modus operandi Abbreviations

DNA IBIS IMEI ISO SMS STR

Deoxyribonucleic acids Integrated ballistic identification system The unique International Mobile Equipment Identity number on cell phones International Organization for Standardization Small message service available on cell phones Short tandem repeat

Definitions • Competence: the demonstrated ability to apply knowledge and skills. • Chain of Custody: a physical log(s) for a single piece of evidence material that documents who had possession and when the evidential material was in his or her possession during the different stages of processing the evidential material (e.g., from the collection, storage, transport, receipt, and analysis phases and the disposition thereof and delivered to court).

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• Defendant’s fallacy: the fallacy by arguing that because many people in the population may share the forensic DNA profile, thus the DNA evidence is worthless; or arguing that if a profile frequency is reported as 1 in 1 million, then it is expected that the number of people in the population with a matching profile is 100 and the probability that the defendant is the culprit is only 1 in 100 (1%). • Low copy number (LCN): particularly for current short tandem repeat (STR) typing, refers to the analysis of any sample that contains less than 200 pg. of template DNA. • LCN typing: simply can be defined as the analysis of any DNA sample where the results are below the stochastic threshold for reliable interpretation. • Modus Operandi: the style or method that a perpetrator uses in the committing of his or her crime. • Prosecutor’s fallacy: the statistical reasoning typically used by a prosecutor overstates DNA evidence, for example, match probability and the probability that the defendant is innocent is confused. The prosecutor uses the match probability and states that there is 1 in x probability/chance or occurrence that the genetic material (DNA) is from the defendant. The “prosecutor’s fallacy” has compromised the use of DNA evidence for a fair trial. This fallacy suggests that the rarity of a profile is interchangeable with the probability that the defendant is innocent (e.g., the rarity of a one in a million match produces the false conclusion that the chance of the defendant being innocent is one in a million). • Stochastic effects: the observation of intra-locus peak imbalance and/or allele dropout resulting from random, disproportionate amplification of alleles in low-quantity template analytical thresholds should establish a balance between allele preservation and artifact editing, resulting in confident, accurate allele calls. • Restriction enzyme: an enzyme with endonuclease activity that cuts DNA at specific sequences. • Restriction fragment length polymorphism (RFLP): the detection of length or sequence variation after cleaving DNA with a restriction enzyme and detection using a small section of DNA within the sequence between two restriction sites. RFLP testing was in the detection of minisatellites. • Short tandem repeat (STR): polymorphic region of DNA where alleles differ in the number of tandemly arranged core repeats. STR allele typically ranges in size between 100 bp and 400 bp. Also known as microsatellites. • Southern blot: a technique that is used to transfer DNA from a gel onto a nylon membrane. • Variable number tandem repeat (VNTR): polymorphic region of DNA where alleles differ in the number around 500 bo to over 20 kb. Also known as minisatellites. • Forensic investigative leads: Verified outcome linking different crime scenes or linking a person to a crime scene after performing a comparison search on a forensic database such as DNA database or a fingerprint database or a ballistic biometric database.

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Introduction Edmond Locard (1877–1966) is considered to be one of the main persons that laid the foundations for the development of modern forensic science. He is well-known for his “Locard Exchange Principle” that is based on the fact that there is an interaction between different physical entities when they come into contact. He made the inference “that every contact leaves a trace value” (Saferstein 2011). There is a good likelihood that the genetic material (DNA) originating from a perpetrator will be left behind on the crime scene due to him or her coming into direct contact touch with an object at the crime scene Physical evidence, such as genetic material (DNA), found at crime scenes has proven to be a powerful tool in the fight against crime (Goodwin et al. 2011). The genetic material (DNA) can identify perpetrators, convict the guilty, and exonerate the innocent (Meintjes-Van der Walt 2010). Forensic DNA profiling has undergone significant development since the first case where it was applied as an investigative tool to assist with the identification and conviction of a murderer and rapist (Butler 2010). Initially, it was only possible to obtain forensic DNA from large visible stains such as blood or semen. Today, forensic DNA profiles are derived from trace invisible amounts of DNA, such as skin cells left from touch transfer (Van Oorschot and Jones 1997). The early DNA profiling techniques were labor intensive and time-consuming compared to the available techniques deployed in the modern forensic laboratory. The forensic multiplex chemistries used in the last decade have seen a significant increase in the number of markers deployed to provide more discrimination power in forensic DNA profile identification, to facilitate international sharing of forensic DNA profiles, and to minimize incidental outcomes from comparison searches on forensic DNA databases (Li 2015). The commercial kits available today utilize multiplex analysis of multi-allelic short tandem (STR) markers located on different chromosomes for high power of discrimination, and many countries are routinely using STRs with at least 20 loci (Goodwin et al. 2011) Additional DNA techniques that can be useful under the appropriate circumstances have been developed, such as analysis of mitochondrial DNA and the Y-chromosome (Butler et al. 2002), as well as low copy number (LCN) DNA testing (Goodwin et al. 2011). The main questions that a forensic DNA examiner is asked to address are (Saferstein 2011; Manamela et al. 2015): • • • •

Who left the genetic material (DNA) at the crime scene? From what body uid did the genetic material originate from? How was the genetic material deposited? Has the necessary quality assurance and quality control been applied during the processing and analyses of the exhibit material containing the genetic material? • Have the results been objectively and fairly interpreted and reported? The interpretation of a single-source forensic DNA profile obtained from exhibit material is simple and can provide powerful scientific evidence either to exclude or

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to include any person as a possible source of that genetic material (DNA) (Balding 2005; Goodwin et al. 2011). When exhibit material contains genetic material (DNA) from more than one contributing person, the interpretation of who is the source of this DNA can become difficult or even impossible to attribute to with confidence (Balding 2005; Goodwin et al. 2011). Technological improvements in forensic DNA analysis have resulted in the ability to analyze even smaller quantities of DNA with increased discrimination power. The advent in the establishment of forensic DNA databases for comparison searching has assisted forensic examiners in collaboration with law enforcement to resolve many casework linking serial (see Fig. 1: Example of DNA conviction and mass screening case) and multiple offenders (Meintjes-Van der Walt 2010; Smith 2020).

Value of Forensic DNA Investigations Today, some wrongly convicted people have been exonerated because of forensic DNA evidence (Gould et al. 2013). Furthermore, in casework, persons are excluded routinely. Forensic DNA has proved to be a powerful investigative tool in: • Solving crime where evidence is matched with a perpetrator • In deciding who the father in paternity testing is • Mass disaster identification of victims in natural disasters (such as the 2004 Indian Ocean Tsunami that killed about 225,000 people) • Terrorism acts (e.g., almost 3000 people were killed in the attack on the World Trade Center on September 11, 2001) • Identification of unidentified remains in structural building collapses (e.g., on September 12, 2014, a guesthouse located within a church premise in Nigeria completely collapsed to the ground and killing 115 persons) • Identification of the “unknown” soldier • Helping in working out inheritance claims The physical evidence will not be accepted in court if the exhibit integrity is compromised due to a shortcoming in one of the aforementioned aspects. Biological contamination is the compromise of the integrity of the exhibit where the unwanted transfer of genetic material (DNA) from an unknown source to a piece of physical evidence. In the process, the original condition of the exhibit material is changed and therefore also the future analysis result to be obtained. The adding or removal of extraneous material from the exhibit material (i.e., contamination) is usually unknown to the investigator or forensic examiner. Therefore, the in uence on the analysis result will be unknown as well. Measures to prevent contamination must be in place when processing a crime scene and when handling exhibits. Contamination can be prevented by following basic contamination prevention steps. For example, the wearing of personal protective

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clothing and the correct use of packaging material for exhibit material collected are critical contamination preventive measures. The consequences, if contamination does occur with exhibit material, will negatively impact on the resolution of the case in question. In 1987 Colin Pitchfork was the first person to be convicted using forensic DNA profiling. It was also the first case where mass screening identification was applied to a population to assist identification in a criminal investigation. On 21st November 1983, a 15-year-old Lynda Mann’s body was found on a remote footpath after she did not return from visiting her friend. The post-mortem revealed that she had been sexually assaulted and strangled. A vaginal sample was retrieved from her body. Three years later, another 15-year-old girl, Dawn Ashworth’s body was found in a wooded area. She was also been raped and strangled. Richard Buckland, a seventeen-year-old who had a learning disability was arrested for the murder of Ashworth. He had some knowledge of the crime that was not in the public domain. Whilst being questioned, Buckland would confess to the crime, but he later withdrew his confession. He, however, would consistently deny that he committed the first murder. Dr. Peter Gill from the Forensic Science Service in the United Kingdom developed a preferential extraction method to separate spermatozoa from vaginal cells. This method was used to extract the spermatozoa from the vaginal samples that were collected from the bodies of the two girls. Subsequently, Professor Alex Jeffreys from the University of Leicester applied his method of using distinguishable patterns in a person’s DNA to help solve the cases. Professor Jeffreys discovered that certain regions of DNA contained DNA sequences that were repeated many times next to each other. He observed that the number of repeated sections could differ from one person to the next. These DNA repeat regions became known as a variable number of tandem repeats. His technique visualised these repeated sections as distinguishable patterns (resembling that of a barcode). Using the restriction digestion length polymorphism (RFLP) the first DNA profile was created by Jeffreys. Professor Jeffreys first performed restriction digestion on the DNA extract using REase and separated the various DNA fragments by making use of agarose gel electrophoresis. To perform southern hybridization, the separated DNA fragments were transferred to a nylon sheet. The detection of the various fragments was made possible by hybridizing the radio-labelled probes to the various fragments. This original RFLP method resulted in many bands per sample on an autoradiogram, so giving the resemblance of a barcode. Unfortunately, his method also required a relatively large quantity of source DNA to generate a forensic DNA profile (Goodwin et al. 2011). It was clear that the same person had raped and murdered both the girls. By doing so, it was evident that Buckland’s DNA was different and he was exonerated by using this novel forensic DNA profiling technique of Jeffreys. The Leicestershire police and the Forensic Science Service then embarked in performing mass screening by collecting DNA samples from 5000 volunteering local men. Unfortunately, after almost six months of DNA examinations, no match could be were found. A DNA sample was only later collected from Pitchforth after a man named Ian Kelly was heard bragging in a Bar that he had given a sample while masquerading as the friend of Pitchfork. Later, Pitchforth’s DNA was found to match the crime samples. Pitchforth confessed to committing both crimes. Fig. 1 Example of DNA conviction and mass screening case

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Evidential Value Evidential value, or sometimes referred to as probative evidence, is the key to examining crime scenes and exhibit material effectively. The evidential value determines which exhibits to select for collection and examination. Evidence must be relevant if it is to be admitted into in court. This means it must be material (i.e., related to the specific crime under investigation) and probative (actually prove something). The exhibit material selected should contribute toward putting the pieces of the puzzle together to assist with the forensic investigation. Three key factors play a role in this selection (Manamela et al. 2015): • What type of exhibit is it? • Where was the exhibit found (location or the place where and/or position of the exhibit material and/or stain of the exhibit material)? • To whom does the exhibit belong (ownership)? A crime scene examiner, for example, may find it difficult as to what possible exhibit material to collect at a crime scene where the property was stolen. In illustrating the point, let’s use the example where a factory was burgled and the perpetrator was not injured and left any bloodstains. The crime scene examiner will carefully examine the crime scene and identify entry and exit points, possible areas where the perpetrator had been or came into contact with the examiner. The crime scene examiner may have found that the perpetrator gained entrance to the factory by opening the main entrance door. To collect “skin cells” (epithelial cells/ touch DNA samples) from the handle of the main entrance door would not be of much value, since several people regularly during the day touch it to open the door. The door handle will contain a mixture of genetic material left by several contributing people, and it will be extremely difficult to interpret the forensic DNA profile. The forensic examiner will have to perform a thorough investigation and, apart from searching for other types of physical evidence, will need to identify other possible areas, which are most likely to be limited to the touch of the perpetrator. The crime scene examiner must also consider other different types of physical evidence and the different types of forensic examination types (such as trace analysis such as paint and fiber, ballistic examinations, disputed document examinations, chemical analysis) that may be performed on the physical evidence. Evidential value of evidence means evidence which is sufficiently useful to prove something important in the crime investigation by either linking a perpetrator or providing evidence to eliminate a person from the criminal investigation of an offence or either to prove innocence or guilt of a person. To establish how the crime was committed, the crime scene examiner must collect and observe the clues and information (including information from eyewitness and tenants) at the crime scene, which will contextualize and reconstructs the events leading to the offence. Physical evidence is considered to be of little use without an interpretation of the

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Scenario: A woman is raped on the bed of her own apartment by an unknown male perpetrator. There are no signs of forced entry. The perpetrator is injured and scratched during the incident by the victim. The perpetrator left semen, hair (with roots) and his own blood (stains) on the linen of the victims’ bed. She does not sustain any major injuries apart from bruise marks on the body and genital injuries. Analysis: The crime occurred in the daily environment of the victim, where she continually deposits her own genetic material. The victim was not injured with a weapon and thus no bloodstain pattern marks will be observed. Consequently, the crime scene examiner will not collect genetic material deposited by the victim at the crime scene. Expect that the perpetrator will leave genetic material behind at the crime scene. The genetic material left will place the perpetrator on the scene where he had no earlier legal access to. Evaluate the evidential value of any object containing the perpetrator’s genetic material (DNA) such as semen, saliva, hair root or blood (if he was injured), or if it links the perpetrator to the scene. Conclusion: The post-coital (rape sample) and nail scrapings from the victim, and the bloodstain, hair and semen stains left by the perpetrator on the linen will have evidential value and link the perpetrator to the crime scene. The exhibit material will give an excellent opportunity to get forensic DNA profiles for comparison matching. Fig. 2 Scenario of a rape case to illustrate evidential value

significance of its presence and/or the information that can be obtained for criminal investigation. The value of physical evidence can be measured either in generic terms, which may ultimately be subjective or in contextual terms (see Fig. 2: Scenario of a rape case to illustrate evidential value). Thus, a crime scene examiner and the forensic examiner are guided by the relevancy test about what to collect or what to the examiner. Consider the opportunity to get a forensic DNA profile needs after determining the evidential value of which exhibit material needs to be collected for DNA analysis. Hence, the usefulness and evidential value of forensic analyses on any exhibit material are determined by the type of exhibit, ownership, and the location (the place where and/or position) of the exhibit material and/or stain. It is important to show “ownership” when approaching forensic investigations. “Ownership” is determined by who was responsible for leaving the genetic material (DNA) at the scene or on the exhibit material. In forensic DNA analysis in the case of forensic biological evidence, it refers to the donor of the biological material such as blood, semen, “skin cells” (epithelial cells), and hair on a crime scene or object found on the crime scene. Compare the forensic DNA profile of the crime samples with the forensic DNA reference samples of the suspected perpetrator in the alleged crime to prove “ownership.” Concerning evidential value, it is critical to show whether a specific person had earlier access to a crime scene environment. Thus, establish the “location,” the position where the exhibit (sample) is found in with the parties involved. The crime scene environment must be evaluated to determine whether it is a victim, perpetrator, or neutral environment. This is a crucial step to identify which donor’s genetic material needs to be identified to prove the donor’s presence on the crime scene during the commissioning of the alleged offence. The crime scene examiner

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Example of physical evidence in a sexual assault or rape incident that may aid the investigation: Perpetrator’s semen in the victim’s orifices or deposited on an item at the crime scene Hair (roots) of perpetrator left at the crime scene A weapon with transfer evidence of some kind (for example “skin cells” from the perpetrator) Wound patterns on the victim Torn pieces of clothing The condom of the perpetrator left at the crime scene Victim’s blood at the crime scene Fibres from ligatures used by the perpetrator to bind the victim Hair/Fibres of the victim in the perpetrator’s vehicle Victims blood on the perpetrator’s weapon Example of physical evidence in property crime: Tool marks, fingerprints, blood (if injured), “skin cells” left at the point of entry or at strategic points where the item was handled by the perpetrator Broken doors/ windows (direction of broken glass) Missing valuables Footwear impressions Fig. 3 Examples of physical evidence

must also examine the crime scene for physical evidence that may be submitted to other forensic science examination types. Thoroughly search the crime scene for physical evidence to determine that a crime was committed. Examples of physical evidence that may have evidential value are illustrated in Fig. 3: Examples of physical evidence. It may not be possible to scientifically find the age (time since deposition) of a body uid stain found at the crime scene. It is, however, possible after prolong periods after deposit to detect body uid stains. The mere fact that a person’s genetic material (DNA) is found at a crime scene does not conclude that the person is guilty or innocent of committing a crime (Hodge 2018). Forensic examiners must limit their court testimony to the value of DNA and not venture into opinions on how the genetic material (DNA) was left at the crime scenes or on the propositions or activities or on the way the DNA was transferred (Gill et al. 2020).

Forensic DNA Evidence Deoxyribonucleic acid (DNA) is the blueprint of life. Genetic material (DNA) is the fundamental building block for a person’s entire genetic makeup that contains the informational code for replicating the cell and constructing the needed proteins. It is part of virtually every cell in the human body, and a person’s genetic material (DNA) is the same in every cell. Hence, the genetic material (DNA) in a person’s blood is the same as the genetic material (DNA) in his or her skin cells, saliva, and other

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biological material. Genetic material (DNA) is found in most cells of the body, including white blood cells, semen, and body tissue and hair roots. Traces of DNA can also be detected in body uids, such as saliva and perspiration, due to the presence of epithelial cells therein. The entire human genome has been sequenced, and it found that most of the human genome is the same for all people; it was demonstrated that only a small percentage of the human genome displays variation between persons. Thus, forensic examiners only need to use chemistries that focus on these regions of variation. For this reason, forensic DNA analysis is a powerful tool to link persons to crime scenes (Jakovski et al. 2017). By performing forensic DNA analysis, the identity of the donor of genetic material (DNA) is determined. A person can be linked to a crime scene by matching the forensic DNA profile derived from the exhibit material collected from the crime scene to the forensic DNA profile derived from a reference sample (buccal sample) collected from an identification subject (such as a suspected perpetrator). A forensic DNA profile is a string of alphanumeric characters, which donate identity. This sequence of alphanumeric characters is nothing more than a biometric containing information of a purely objective and irrefutable character. In a forensic DNA database, the forensic DNA profile derived from an identification subject only has value when comparing it with another profile derived from the database: if the subjects’ forensic DNA profile matches that of a forensic DNA profile derived from the exhibit material collected from the crime scene. The human genome has variable regions that make it possible to use the genetic material (DNA) information in forensic identity applications. DNA variation occurs in the form of different alleles or various possibilities at a particular locus. The two primary forms of a variation are possible at the DNA level: sequence polymorphisms and length polymorphisms (Butler 2012). Forensic DNA typing can be historically classified into fragment length polymorphism methods and polymerase chain reaction-based methods. Many countries are now using forensic DNA analysis based on STRs (Balding 2005). More than 97% of the human genome is noncoding and repetitive. Forensic DNA analysis uses the repetitive nature and polymorphic properties of junk DNA. The number of repeats and the sequence structure of those regions vary between persons and organisms based on the entire genetic material (DNA). Half of a person’s genetic code comes from his or her mother, and half comes from his or her father. The genetic material (DNA) of close biological relatives can thus be used in paternity and kinship analysis (Saferstein 2011). Many forensic laboratories deploy standard chemistries to generate forensic DNA profiles that contain no or very limited medical information of a person’s predisposition or any physical characteristics (such as the color of the eyes or hair or how tall or short you are). There are trending chemistries becoming available in the forensic field to complement the arsenal testing chemistries available to a forensic laboratory. These chemistries can determine the additional attributes of the donor of the genetic material (DNA) on the exhibit material (Kayser 2015; Wienroth 2018). Recent innovations and developments in forensic DNA testing in the criminal field are related to the techniques of forensic DNA phenotyping, the use of ancestry-

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informative markers, and familial searching. Forensic DNA phenotyping can be described as a set of techniques that aims to infer human externally visible physical features, such as the eye, hair, and skin color, and continental-based biogeographical ancestry of criminal suspects based on analysis of biological materials collected at the crime scenes (Machado and Silva 2019). The use of these additional chemistries may be limited and restricted in a laboratory by the regulatory framework of a particular country or region due to possible contravention of a human or constitutional right. Adhere to the set of legal requirements and proper quality assurance standards when handling physical and presenting evidence in court. The regulatory framework and the prescripts of a particular jurisdiction give the guidance of how to process crime scene investigation and the seizure of exhibit material. Additionally, the international ISO standards exist and need compliance for implementing the quality management system for the processing and analyzing of crime scenes and exhibit material in laboratories. Aspects, like search warrants, the chain of custody, contamination issues, quality controls, and quality assurance, must have adhered to when physical evidence is collected, handled, and analyzed (Meintjes-Van der Walt 2010). No other forensic field has received so much scrutiny in court and through peer review as to what forensic DNA testing has been exposed to. It is for this reason that the quality assurance standards based on the ISO17025 standard for forensic DNA testing are very stringent and must meet the many guidelines required by the forensic community and by the court system. During the forensic examination process at the crime scene or in the laboratory, the sources of genetic material (DNA) with evidential value need to be identified, documented, collected, and preserved to support the forensic DNA analysis process.

Forensic DNA Analysis The forensic examiner must find the nature and donor of the DNA exhibit material (such as blood, semen, saliva, tissue, bones, and hair). The forensic examiner will analyze biological exhibit material with the aid of scientifically validated methods to: • • • • • • •

Determine the evidential value of exhibit material. Screening testing of exhibits using presumptive tests. Forensic DNA analysis. Bloodstain pattern analysis. Facial reconstruction. Anthropological examinations. The attending of crime scenes to aid the investigating officer with a reconstruction based on bloodstain pattern analysis.

Evidence recovery and presumptive testing are normally the first step in the laboratory process to analyze the exhibit material after being received. The analysis aims to:

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• Document the chain of evidence of the exhibit material by documenting the condition of the packaging material, including the unique seal number and inscriptions on the packaging material. • Document (photographing and/or sketches) the appearance and description of the exhibit material. • Screening analysis, with the aid of presumptive test methods, is performed on the exhibits to find, whether any possible blood or semen is present on the exhibits (Exhibits which have been tested by the crime scene examiner may not be required to be retested.). • Determine the evidential value of biological evidence identified on exhibit material. • Removal of biological evidence from exhibit material for forensic DNA analysis. After evidence recovery, including the type identification of the body uid through presumptive and/or confirmation tests, is performed on exhibit material, a small stain of the exhibit material is subjected to forensic DNA analysis to give the forensic DNA profile (see Fig. 4: Steps of forensic analysis). After obtaining forensic

DNA Isolation (Extraction) process: Possible biological material identified during the preliminary testing phase, such as possible blood and possible semen, are then isolated by using the proper extraction method. The DNA isolation (extraction) process entails the release of DNA for the cells within the sample.

Quantification process: The extracted samples then undergo a process called quantification PCR (QPCR), this is done to determine the quantity of DNA present as well as if male DNA is present.

Amplification: If a sufficient quantity of DNA is present, the samples are then amplified by a process called PCR (Polymerase Chain Reaction). Amplification is done to multiply the targeted area of DNA to enhance the visualization.

Electrophoresis: • The DNA segments (alleles) are then separated according to size by a process called electrophoresis.

Expert software systems and analysis: The separated DNA segments (alleles) are interpreted by examiners using expert software systems to provide a forensic DNA profile.

Fig. 4 Steps of forensic DNA analyses

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DNA profiles from the exhibit material and reference sample from the subject or perpetrator, the forensic examiner must interpret the results and compile a forensic match report. The match (either “inclusion” or “exclusion” report) may exclude a perpetrator or provide a match between the perpetrator and the crime scene exhibit material. The inclusion match is supported with some statistical analyses to contextualize the results (statistical probabilities of a random match or likelihood ratios) that are used (Butler 2015). If required, the forensic examiner may be required to present the evidence in person to the court of law.

Forensic DNA Databases Although forensic DNA technology has been used worldwide since 1985 to resolve crime, forensic DNA analysis was predominantly used as a prosecutorial tool for criminal and paternity casework on a case-by-case basis. The United Kingdom established the first forensic DNA database in 1995. A forensic DNA database is a collection of forensic DNA profiles obtained from crime samples and donors, such as persons arrested and convicted offenders and used to perform comparative searches to establish an investigative lead. The value of a forensic DNA database as an investigative tool grows as the size of the database increases. The effectiveness of the forensic DNA database lies in the fact that many of crimes, such as sexual assault and property crimes, are committed by repeat offenders and that there is a likelihood that the person who committed the crime being investigated was convicted of a similar crime and their forensic DNA profile may already be loaded onto the database (see Fig. 5: Case study illustrating the effectiveness of a forensic DNA database). Access to forensic databases is mostly restricted. By 2019, more than 69 countries have followed suit and established national forensic DNA databases to assist in resolving crime. The establishment of forensic DNA databases has raised ethical and human rights issues on the criteria of inclusion and retention framework of both samples taken from persons and forensic DNA profiles derived from these samples (Kumar et al. 2015). There are many reported instances where genetic information has been abused by governments to the detriment of its citizens. This has often included stigmatization and discrimination in areas such as insurance qualification, employment, health care, and education (National Research Council 1992; Coodly 2019). It is evident that the ongoing public and policy debate is impacting the use and expansion of forensic DNA databases. Some safeguards are implemented at the national or regional level, but there is a lack of global standards and a need for more societal engagement and debate (Wallace et al. 2014). It is thus important that countries should have detailed legislative framework that articulates the establishment and purpose for which a forensic national database may be used for, including aspects such as the retention periods for sample storage and profiles, access and security issues, the manner and from whom in which samples are collected, and how comparative searches is performed and reported.

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CASE STUDY: EFFECTIVENESS OF A FORENSIC DNA DATABASE The National Forensic DNA Database of South Africa (NFDD) was established in 2015. Forensic DNA profiles derived from crime scenes and certain categories of persons, such as persons arrested and charged; convicted offenders; persons under investigation and not arrested; missing persons and unidentified bodies are stored in different indices for comparative searches. With the creation of the NFDD, comparative searches on the NFDD very quickly resulted in numerous person-to-crime forensic DNA investigative leads and several hundred crime-to-crime forensic DNA investigative leads. For example in early 2015, the forensic DNA profile of an unknown male was linked on the NFDD to 30 separate rape cases. Sikhangele Miki, a 34-year-old male was charged with several offences committed between January 2011 and November 2015 in the areas of Delft and Khayelitsha, Cape Town, South Africa. His Modus Operandi, described by the court which ultimately convicted him, was “the signature of a person who is cruel, vicious and lacking any sense of empathy”. Miki would during the evenings, or early mornings would follow his victims, grab hold of them, threaten them at knifepoint by holding a knife to their necks and would demand money and cellular phones. He would then take them to an isolated place and rape them. Often he would either punch or hit his victims with the back of the knife handle or in some cases he stabbed them to stop them from struggling or crying for help. Nine of Miki’s thirty victims were under the age of 16. One victim was even dragged to a space behind a police station where she was raped. In 2014, Miki was arrested and convicted for an unrelated charge of assault with intent to cause grievous bodily harm for which he served 11 months in prison. During 2015, whilst serving time in prison a buccal sample was collected from him and submitted for forensic DNA analysis for uploading to the NFDD for comparative searching. The outcome of the comparative search resulted in linking Miki to the 30 unsolved rapes. The case was brought before the High Court of the Western Cape where Deputy Judge President Patricia Goliath compared the modus operandi of Miki to a “monster lurking in the shadows, attacking, robbing and raping girls, exploiting the vulnerability of his victims.” She went on to say that Miki derived pleasure in the degradation and pain inflicted his victims. She further stated, “The accused is a serial sexual predator. Even in the event of the remote possibility of rehabilitation, it is clear that he is a danger to society especially women and young girls.” Due to the overwhelming DNA evidence against the accused, Miki pleaded guilty to 84 charges which included 30 counts of rape, 27 of kidnapping, and 12 of robbery with aggravating circumstances. Miki was ultimately sentenced Miki to 15 life terms and an additional 120 years, to run concurrently: Without the power of the NFDD and the DNA legislation which allowed DNA samples to be collected from the accused whilst he was serving time in prison as a convicted offender, it is unquestionable that Miki would have continued his reign of terror against vulnerable women in the future had he not been linked to his previous crimes by the NFDD. Fig. 5 Case study illustrating the effectiveness of a forensic DNA database

Forensic DNA databases have now proven to be an effective investigative tool to resolve crime, which involves recidivism, and may eventually contribute to crime prevention and deterrence (Van der Beek 2015; Jakovski et al. 2017). Studies in the United Kingdom (which launched its database in 1995) have shown that more than 60% of persons sent to prison for violent offenses and then released were rearrested for a similar offense in less than 3 years. Also, many “cold cases” before the existence of any forensic DNA databases can now potentially be linked to

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an offender/perpetrator of another crime. Furthermore, when crime scene examiners focus and there is a significant increase in the collecting of genetic material (DNA) with evidential value at crime scenes such as property crime, the database becomes an effective investigative tool. This has then a profound positive impact on improving the conviction rates of the country (Asplen 2004). In other words, not only will a forensic DNA database increase the likelihood of identifying known perpetrators, but it will also increase the possibility of linking perpetrators to multiple crime scenes.

Use of Forensic Lead Investigative Information Several forensic databases, such as DNA, ballistic, and fingerprints, have been established to assist law enforcement to investigate and resolve the crime. After performing comparison searches between the biometric information loaded onto these databases, either a person is linked to a crime scene or different crime scenes are linked. Verified forensic investigative leads are subsequently issued to investigators to follow-up and investigate. The mere fact that a person is linked through a database to a crime scene does not necessarily imply that the person is the perpetrator of the crime. There may exist legitimate reasons as to why the person’s biometric information was found at the crime scene. The person may have had legal and regular access to premises where the crime was committed. For example, the person may be the owner or tenant or a regular visitor to the premises where the crime was committed. The fact that a person’s genetic material (DNA) is found on a postcoital rape sample does not imply that the person is a rapist – the sexual intercourse may have been from consent. Other data left on surveillance cameras or cell phone data or cell phone tracking data or geographical location data linked to images could be systematically exploited in the investigation. The use of forensic case data combined with temporal and graphical dimension may increase the likelihood of apprehending the perpetrator. The recent technological innovation permits the data extraction of frames delineating more than one person. Software is now available that can assist to identify different perpetrators in complex DNA mixture results. A putative forensic DNA profile derived from a family member on the database can be used to link a perpetrator to the crime scene exhibit material. In South Africa, forensic examiners routinely check for cross-leads between different types of forensic investigative leads (verified outcomes of comparison searches conducted on a forensic database such as DNA, Ballistic, and fingerprint databases (Smith 2020). A cross-lead is considered where there is common information, such as the case reference details (station and case number) and/ or the name of the person linked in the forensic investigative leads. By combining this biometric information and sharing it with the investigating officer, the number of cases linked is increased. Thus, there is a better opportunity for success in resolving the different cases by arresting a perpetrator. By increasing and simultaneous prosecuting the

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number of cases, the perpetrator is more likely to be found guilty and convicted with a heavier sentence than when the cases are prosecuted in a piecemeal fashion.

Familial DNA Searches The use of familial DNA searching can prove to be useful in a criminal investigation. Familial DNA searching assists investigators to point them toward a possible perpetrator. Persons who are close relatives tend to share more genetic (DNA) than unrelated persons. This can be used in criminal investigations by performing a familial search of exhibit material against a person’s genetic information (DNA profile) on DNA databases or genetic genealogy database and to identify a possible relative of the genetic material (DNA) left by the perpetrator at the crime scene (Ram et al. 2018). Familial DNA matches are partial, or near, matches that require further investigation (Butler 2015). Many support the use of familial searching as an investigative tool and argue that it has the potential to facilitate the identification and conviction of perpetrators, prevent crime, resolve cold cases, exonerate wrongfully convicted persons, and improve public safety; however, its use also raises important constitutional, ethical, and practical considerations that need to be taken into account by law enforcement (Niedzwiecki et al. 2017). The arguments for and justifying why DNA samples must be taken from certain categories of persons, such as convicted offenders and arrestees, are that they have a diminished expectation of privacy as a result of their conviction or arrest. This argument cannot be applied to why offenders’ relatives need to be linked through a database search. Familial searches may uncover differences in genetic and social family relationships. Genetic data from distant relatives in public genetic genealogy databases have aided many cold case investigations. For example, the use of the private heritage of genetic genealogy databases was applied to identify and led to the arrest of alleged “Golden State Killer” Joseph De Angelo (Fuller 2018].

Modus Operandi and Signature of Serial Offenders Although a comparison search on the forensic DNA database may not provide the specific identity of a known person whose forensic DNA profile is already uploaded, different crime scenes may be linked due to the forensic DNA profile matches. Offender profiling, modus operandi, and signatures left by serial perpetrators become useful during the investigation of the linked crimes (Baker et al. 2014). The modus operandi of repeat or serial offenders is an important consideration in the investigation of crime. It is simply the way a particular perpetrator operates. Modus operandi accounts for the type of crime and property or persons attacked, the

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tools or weapons used in committing the crime, and the way the perpetrator gained entrance and the victim was approached (including disguises or props used), how the perpetrator enters the crime scene, and the time and place the crime was used. The modus operandi of these perpetrators often remains stable or changes very little over the seriousness of crimes committed. In addition to the modus operandi, the signature of the serial perpetrator provides an additional information in the criminal investigation. The signature of the serial perpetrator is the imprint or expression that is used to re ect their unique identity that feeds toward the feeding of their fantasies and satisfaction. The serial murder may, for example, leave specific markings on the body or display in a bizarre position or have a particular ligature to type his victims (Baker et al. 2014).

Offender Profiling The purpose of offender profiling is to assist investigators to narrow their search for the field of the perpetrator. This is based on the characteristics of the crime scene and initial investigative information. Information descriptors include the following: sex, race, approximate age, criminal history, residency concerning the crime scene, employment history, social adjustment, sexual abnormities or adjustment, use of narcotics or stimulants, educational level, and interpersonal skills. Although profiling may assist in sentencing mitigation, it is rather used as an aid than as evidence in court. It has proven to be vital during the investigative phase, especially in the investigation of serial murder casework (Baker et al. 2014).

Best Practices Using Forensic Investigative Leads as Effective Investigative Tool It is important for every investigating officer, who receives a forensic investigative lead, to immediately make contact with the other investigating officers, working on the other linked cases. A forensic investigative lead will provide valuable information for a case under current investigation that has been linked to another case(s). This means that, for example, the evidence in case A matches the DNA case B and other cases (where a perpetrator may or may not have been arrested). The information indicates this is now a serial rape or serial murder investigation. A serial rapist is a perpetrator who rapes two or more victims. A serial murderer is a person who murders two or more victims because of an inner desire/urge to kill them. There may be information that indicates a serial rapist or serial murderer is active in the area because of a similar modus operandi. Similar information or investigative leads may have been provided on fingerprint evidence or IBIS (ballistic database) links that can be of assistance for solving the relevant cases as identified. Certain best practices need to be followed to ensure a successful arrest and prosecution.

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Step 1: Notification Notify the relevant persons and stakeholders within your district or region and nationally of the fact that a serial offender is active. This will include making contact with police commanders and responsible person at behavioral investigative units, which specializes in assisting investigators in investigating serial offender linked cases. Warn the public that a serial rapist or serial murderer is active in the area. This should be done only after approval has been obtained to involve the media. Convey this information to local newspapers, radio stations, and television.

Step 2: The Investigation Certain investigative steps need to be taken to effectively investigate a series. The most effective way to investigate serial related cases is to form a task team. All linked cases, whether through DNA evidence, fingerprints, cellular phone data, or identikits, should be consolidated under the task team, one dedicated investigator detective. This step is recommended, even if some cases are outside the investigator’s area of responsibility and investigation, for example, if a case is from another region.

Appeal to Victims to Come Forward Since only a few cases of rape are reported to law enforcement, it means that there were other victims of the serial rapist that were not reported. These victims might have valuable information that could help solve the series. An appeal in the media could lead to these victims coming forward and opening cases. The modus operandi and identikit can be put in the local newspapers and appear on television news with contact details, asking for victims to come forward. Even if the victim does not want to open a case, valuable information can be obtained in this way. Search for Similar Cases While DNA evidence (genetic material) can be a way to identify other cases, the investigator must not only rely on DNA evidence (genetic material) only to help identify other cases that are the work of the same perpetrator. The investigator should enquire from colleagues whether they have cases with similar modus operandi or to check with crime intelligence officials to check whether other cases of rape or murder have been reported in the same area. The investigator must examine case files relating to assault, attempted rape, robbery, and even murder that occurred in the same geographical area of the linked cases. These cases might also be linked to the same perpetrator but maybe were not registered as rape cases. Serial rapists and serial murderers like to use a small geographic area in which to commit their crimes. Check for cross-forensic investigative leads.

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It should be mindful that certain information collected may be sensitive or classified material requiring protection and of which unauthorized disclosure or loss could reasonably be expected to be prejudicial to lawful methods for the protection of public safety.

Revisit the Crime Scenes It is essential to revisit the crime scenes as soon as possible. In rape cases, investigating officers often do not revisit the crime scene with the victim to locate exhibits and determine exactly where the crime had occurred. The investigator should look for evidence such as condoms, semen, and tissues the perpetrator had used and for belongings of the victim that might have the perpetrator’s fingerprints on them. Consideration may also be given to using dogs that have been trained to identify biological to assist in locating exhibits that may have the perpetrator’s semen on it at crime scenes. Revisit the crime scene as soon as possible, should a perpetrator agree to do pointing out later, after the arrest. A perpetrator might only be arrested a year after the rape and the area might look very different then. Ideally, investigators should record the crime scene location by using GPS coordinates and mapped. Since serial criminals often continue to use the same geographical area, it will be a good idea to keep the area under observation, should the perpetrator return. Exhibits Material All sexual assault evidence collection kits and other exhibit material must be sent to the forensic science testing laboratory for immediate processing, and not only once a perpetrator has been identified. The investigator must maintain adequate documentation and chain of custody documentation. Ensure that the chain of evidence statements is obtained immediately, and not later. Reinterview Victims Investigators should remain in contact with victims and victims in other linked cases, to ensure that the victim is available for the court process. All victims need to be reinterviewed and informed that the cases are still under investigation. Often victims see the perpetrator again because serial offenders use the same areas to commit future crimes. If the victim has the investigating officer’s contact number, it can assist in apprehending the perpetrator. This has happened in many serial cases because the perpetrator likes to use the same geographical area again. Sending an SMS to the victim once a week, indicating that the case is still under investigation or keep the victim informed of the progress made, will encourage the victim to remain in contact and to proceed to trial when the perpetrator is arrested. Cellular Phones Most serial rapists or serial murderers take the victim’s phone. It is essential that detailed billing for the victim’s number should be obtained from the day before the perpetrator made contact with the victim up until the date of the court order

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application to obtain direct access to telephone records. Sometimes the perpetrator uses the victim’s phone and SIM card to make a call, before throwing away the victim’s SIM card. The number that the perpetrator called can then also be identified through the court order to see who was called. More likely, the perpetrator will replace the victim’s SIM card with his own SIM card and that number should also be identified through the court order. Besides the detailed billing of the SIM card(s), also obtain a court order for the handset profile for the stolen cellular phone for the same period as the detailed billing. It will provide information on the SIM cards that have been used in the stolen phone. The cellular phone information that is obtained through the court order should be analyzed to see how it can assist with the investigation and should not only be filed. It is important to note that the victim does not need to know their phone’s IMEI number. The IMEI number can be obtained from the detailed billing of the SIM card number. Also, note that detailed billing and a handset profile can be obtained for a pay-as-you-go number.

Step 3: After the Arrest Once a perpetrator has been identified and arrested, the perpetrator’s DNA reference (buccal) sample should be immediately submitted for forensic DNA analysis.

Preparation for the Trial All cases linked to one perpetrator must be tried together in one trial as this improves the chances of obtaining a successful conviction. If cases are from different court jurisdictions, areas, make an application to centralize the cases for trial in the same court. This can even be done for cases outside the province. It is important not to only prosecute in cases where there is DNA evidence, if the modus operandi is the same, but also consider using the modus operandi (similar fact evidence) to obtain convictions in those cases that are similar but without the physical evidence as proof. The investigators from behavior science units can be approached to also give evidence in supporting the case investigation. Sentencing For sentencing, the testimony must be given about the fact that the accused is a serial rapist and/or serial murderer and he or she is a danger to society. The investigators from behavior science units can give evidence in this regard. The comments of the presiding officer, along with any sentencing reports, should accompany the convicted offender to the Department of Correctional Services and should be put on the perpetrator’s file at the Department of Correctional Services. This is important because one day the offender will appear before a parole board, and unless this information is in the offender’s file, the Department of Correctional Services or Parole Board cannot make a properly informed decision on the parole matter.

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Reasons for Unsuccessful Investigations of Serial Casework Investigators often become very despondent when there is a negative court outcome in serial offender casework. The following are some of the reasons why serial investigations may not have the expected outcome:

Serial Related Cases Not Consolidated Sometimes forensic DNA-linked cases may be investigated by different units or investigators. For example, rape that occurred outside in an open field will usually be investigated by one unit; however, a rape inside a victim’s home might be investigated as a house robbery and dealt with by the organized crime investigators. Rape and murder might be investigated by different independent investigators, even though all of these cases are linked to the same perpetrator based on the genetic material (DNA) evidence. The best practice would be to form a task team to deal with these cases and work together, using all the information that is available in different cases. Serial Casework Investigated by Inexperienced Detectives Inactivity in follow-up with investigative leads will lead to the perpetrator committing more crimes. The most experienced investigator should be in charge of the investigation. Junior investigators can still be involved to learn from the experienced investigator(s). Cases Closed as Undetected Are Not Reopened Even if a case had already been closed as undetected by the time the forensic DNA report stating that these cases are linked, the cases should be reopened and included in the serial investigation. Often these cases have valuable leads that were not properly followed up at the time. Informal Identity Parades If an investigating officer identifies a possible perpetrator and takes the perpetrator to the victim and asks the victim whether he/she indeed was the correct perpetrator, the case investigation may become compromised. Besides the fact that this is not the correct procedure, it has no evidential value. Investigators must hold formal identification parades according to the prescripts so that this identification evidence can be used in court.

Pretrial Preparation Forensic examiners must always be objective and impartial when dealing with forensic evidence. Examination test methods used and interpretation of results must be processed following international acceptance criteria. In the interest of good justice, it is important that both defense lawyers and prosecutors adequately

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prepare for a trial. Smith (2019) from the Section Forensic Database Management of the South African Police, who is responsible for managing the DNA database, and Meintjes-Van der Walt (2010), an experienced prosecutor in criminal casework, propose the following set of questions in preparing for court cases where forensic DNA evidence is relevant: • Has the chain of custody of key evidential material been proved and supported with documentation? • Has the possibility of contamination been considered and eliminated? • Are all the forensic DNA test examinations supported with the required documentation? • Was there adequate discovery? • Was the accused able to exercise his or her right to a fair trial? • What are the standard operating procedures of the testing laboratory? Are they available for the defense? Have they complied within the specific case? • Who are the crime scene and forensic examiners that performed the tests on the exhibit material? • What is the competency (including qualifications and training) of the crime scene and forensic examiners? • What are the proficiency test results of the forensic examiners that were involved with the examination of exhibit material in the specific case? • Has the laboratory got a quality management system based on the ISO17025 international standard? Is the laboratory accredited and been subjected to internal and external audits? • Could the DNA exhibit material (including work and storage areas) and laboratory equipment been contaminated during the handling and testing thereof? • What are the controlling measures, including quality controls, during the methods tests to monitor contamination? Was contamination excluded in the specific case? Was a possibility of the exhibits been contaminated by the crime scene and forensic examiners excluded? • Are all the forensic DNA test methods validated? Are the validation studies available? • What equipment (with serial numbers) were used in the forensic DNA examination of the exhibit material? Are the forensic DNA equipment calibrated? Are the calibration certificates available on request? • Has potential examiner bias been addressed and excluded in the interpretation of the results in the specific case? Was the forensic DNA results and report subjected to administrative and technical review before releasing the report? • Does the defense have access to STR electropherograms? Have the allele designations been processed correctly? Is there any ambiguity with the electropherograms? • Were the isolated DNAs from the exhibit material quantified? What was the quantity of the DNA used for amplification?

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• Is the profile of the exhibit material a single source or a mixture? What are the criteria for making a match? Consider requesting a qualitative analysis approach with the genotypes contributing to the mixture. • What is the possibility that a close relative was involved in the crime? • Did the accused recently have a bone marrow transplant? • What statistical method was used to support the DNA results? Was the statistical value of the exhibit material calculated correctly and according to international acceptable practice? • What is the possibility of an additional contributor with mixture results? • What other physical and other evidence is there to support the DNA evidence?

Erroneous Forensic DNA Findings Forensic DNA evidence is based on accepted scientific principles and courts have “judicially” noticed DNA as a reliable scientific test. There are, however, many instances where reported forensic DNA findings have proved to be erroneous. Instances of incompetence or deliberate fraudulent action have occurred, knowingly excluding information or removing information from an exculpatory report of the accused, knowingly giving incorrect oral, purposefully concealing that an error occurred in practice, fabricating competency and qualifications, providing evidence to tests that were never performed, concealing sample switches or contamination, falsifying laboratory reports, or providing results based on non-validated test methods, wrongfully “reading” and including an accused into a mixture forensic DNA profile, and overstating the significance of DNA results (Cooley 2010). DNA evidence may be presented in a misleading way, where it is either exaggerated, such in the case of the prosecutor fallacy, or underestimated, such as in the defendant fallacy: It is important for all parties (expert witnesses, lawyers, and presiding officers) concerned to understand the mathematical principals supporting forensic DNA evidence. Forensic evidence is often given in terms of a mathematical probability. The forensic examiner must be cautious that they do not make the error of “the prosecutor’s fallacy,” in the interpretation of such probability (Thompson 2009): DNA analysis is subject to human error based on the interpretation of the DNA results. These errors are often made when dealing with mixture samples, low copy number DNA (Gabel 2014), and degraded evidence (Hodge 2018). Low Copy Number DNA refers to trace DNA from which it is difficult to obtain a full profile such as availability of limited, damaged, or degraded DNA, oligospermic or aspermic perpetrators or from extended interval post-coital samples. Forensic science testing laboratories must implement a quality management system that is open to peer review. The quality management system will include various quality control measures to cover the different activities and processes of the forensic DNA laboratory (Wienroth 2018).

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A forensic examiner must be committed to professional ethics and seek the truth (Li 2015). His or her conduct must be impartial and without bias. The forensic examiner must embrace science and always ask if it is good science that is being practiced. Practicing good science requires an understanding of the principles of the scientific method and the limitation of the forensic method in supporting the forensic result and interpretation.

Conclusion Although forensic DNA testing is a powerful investigative tool, it is not always the silver bullet in case examination and investigators must not neglect other forensic examination methods and database capabilities to resolve their casework. Forensic DNA testing has proved to be very useful in resolving criminal casework to assist with the: • • • • • • •

Conviction of a perpetrator. Exoneration of an innocent person. Identification of a poacher or the determination that poaching has occurred. Proving of championship pedigree. Identification of the bodies and remains in mass disaster incidents. Determination of paternity. Determining who was driving a vehicle during an accident if the blood of the driver is found on the windscreen. • Linking a perpetrator to several crime scenes by performing comparative searches on a DNA database. The mere fact that a person’s DNA is found on a crime scene or an exhibit item or rape sample does not in itself imply that he or she is the perpetrator of a crime. There may be an innocent explanation as to why the DNA person is deposited at a crime scene. The person may have had a legal reason or is the resident at the crime scene. Furthermore, in rape cases, sexual intercourse may have been a consensual sexual intercourse. With the advent of uploading forensic DNA profiles to DNA databases, it became possible to perform comparison searches and identify serial offenders, or multiple offenders, and to distinguish copycat offenders. In most countries, on the one hand, when an offender commits two or more of the same offenses at different time intervals, the offender is known as a serial offender. This is particularly important, given the recidivist nature of sexual predators. On the other hand, an opportunist offender that commits different offenses is known as a multiple offender. Forensic DNA investigative leads and cross-leads have become important evidence in the arsenal to fight crime. There is a growing trend for forensic DNA products to be delivered as intelligence products over and above the direct evidence presented in the courts. These

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trends are opening a very exciting future of using technology to support criminal investigations and the resolution thereof. Forensic DNA analysis must be performed efficiently and reproducibly while complying with the regulatory framework, and the forensic examiner must maintain scientific objectivity so that the evidence holds up in the courts.

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Introduction to Forensic DNA Typing and Current Trends Monika Chakravarty and Prateek Pandya

Contents From Sampling to Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Improved Software Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Massively Parallel Sequencing/Next-Generation Sequencing: Advanced Human Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The NGS Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Comparing CE and NGS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Forensic Applications of NGS Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . STR and SNP Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondrial DNA Sequencing in Forensics Using NGS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA Intelligence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Forensic DNA Phenotyping (FDP): Visible Phenotype Estimation . . . . . . . . . . . . . . . . . . . . . . . . . Ancestry Informative Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Degraded Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Identifying Monozygotic Twins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Emerging Applications of NGS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Utility of NGS/MPS over CE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Forensic Genetic Genealogy: The New Means of Genetic Identification . . . . . . . . . . . . . . . . . . . . . . . The Ekeby Man Case . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Golden State Killer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Rapid DNA Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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M. Chakravarty Forensic Science Laboratory, Home Department Government of NCT of Delhi, Delhi, India P. Pandya (*) Amity Institute of Forensic Sciences, Amity University, Uttar Pradesh, India e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 H. R. Dash et al. (eds.), Handbook of DNA Profiling, https://doi.org/10.1007/978-981-16-4318-7_1

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Abstract

DNA profiling in forensic context has seen tremendous growth over the last almost four decades. Forensic DNA fingerprinting started with Alec Jefferey’s single and multilocus probes via autoradiography followed by uorescencebased PCR-STRs. Lately, there has been a significant change in technology for human identification. Chip-based massively parallel sequencing is the most recent advancement. Drawing investigative leads, disaster victim identification, and unsolved cold cases created a requirement for more efficient ways of extraction, rapid DNA testing, and genetic genealogy. Pooling forensic genetic genealogy, mitochondrial DNA for challenging sample analysis and STR-SNP sequencing has opened more avenues to generate the data leading to identification. This chapter highlights some of the advanced methods adopted in forensic DNA profiling and a few appropriate cases incorporating such methods. Keywords

InDels · Massively parallel sequencing · Forensic DNA phenotyping · Biogeographic ancestry · Genetic genealogy · GEDmatch · Rapid DNA

From Sampling to Identification Human identification is an essential component of forensic science. In the earlier times, the emphasis was on the phenotypic aspects of human body such as anthropometric measurements, color, and related features. However, since the advent of DNA technology, the field of human identification has grown significantly in terms of establishing individuality, relationships, and cultural and ethnic correlations. In addition, DNA technology has also provided various interesting and useful avenues such as establishing the genetic basis of various diseases through marker identification, evolutionary biology, gene therapy, mapping, subspecies level identification, habitat correlation, and related areas. The journey of DNA as an identification tool in forensics for the past three decades has been comparatively simple. Generally, a crime scene sample is profiled using existing methods and compared to a suspect or uploaded to a forensic DNA database of convicted offenders. Crime investigating laboratories mostly apply polymerase chain reaction (PCR) and uorescence-based capillary electrophoresis (CE) to detect length variations in short tandem repeats (STRs). Although current capillary electrophoresis is the gold standard for analysis of forensic samples and is the method of choice for separating STRs for most forensic laboratories, the advent of massively parallel sequencing (MPS) or nextgeneration sequencing (NGS) has shown new avenues for detailed genetic analysis. NGS technology is evolving rapidly over the last decade and has proved

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advantageous in challenging forensic samples, including mixtures, low copy number DNA, and degraded samples. NGS technology offered comprehensive results in case of mismatches observed in disputed paternity cases (Ma et al., 2016).

Improved Software Tools Software tools for data collection emanating from capillary electrophoresis are an essential component of DNA profiling technology work ow. Advanced data collection and analysis software could affect data analysis in capillary electrophoresis. This could facilitate data processing by reducing off-scale data, thereby increasing laboratory’s output. Stochastic effects like allele dropouts, allele drop in, sister allele imbalance, and stutters occur more often in low DNA samples and may lead to stutter amplification. Upgraded 3500 Data Collection Software v4.0.1 (User Bulletin Publication Number 100075298, Thermo Fisher Scientific) provides new features to reduce spectral pull ups. Gene Mapper™ v1.6, automated genotyping software (User Bulletin Publication Number 100073905, Thermo Fisher Scientific), cuts off threshold limit from 33,000 RFU (Relative Fluorescence Unit) to 66,000 RFU and enables accurate evaluation of peak heights to determine minor or low-level contributor. It reduces off-scale data and is useful both for routine casework and database samples. Improved software provides profile comparison feature, improved marker labeling along with stutter filters so that the stutter peaks are not exported. Recently, there have been integrated CE and NGS case management work ows having data concordance (Converge software, Thermo Fisher Scientific) in which the samples can be analyzed both for CE and NGS at the same platform. Improved software tools have population statistics included in the software itself; however, the databases can be customized according to the region. Variants can be accurately detected, and samples that have been run on the same sequencer can be compared. Fast, automated analysis of complex data and multiple data export options have streamlined the analysis. Some laboratories are also in the process of developing probabilistic genotype software to estimate the number of male and female contributors (Coble and Bright 2019). CE Data Interpretation Improvement • Pull ups reduced. • Signal optimization across the capillaries. • Reduced off-scale data. • Incorporation of 6-dye chemistry. • Automatic spatial calibration. • RFID tracking. • One reagent cartridge. • Common array length and single polymer for all applications. • Small bench top instruments, touch display.

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Massively Parallel Sequencing/Next-Generation Sequencing: Advanced Human Identification DNA sequencing consists of identifying base sequence of certain sections or entire length of DNA molecule. Sequencing technology has evolved tremendously since first-generation technology started in the 1960s (Heather and Chain 2016). Since then, the technology has improved significantly while jumping from first-generation (Maxam and Gilbert 1977; Sanger, Nicklen, and Coulson 1977) to second- (Hyman 1988; Shendure and Ji 2008) and third-generation tools (Niedringhaus et al. 2011). Conventional capillary electrophoresis has been the chosen method for forensic laboratories to identify the perpetrators of crime and to exonerate individuals (Chakravarty et al., 2019; Shrivastava et al., 2012). Molecular biology in association with population genetics principles paved the way for excellent human identification and construction of large number of DNA databases (Dixit et al. 2019; Srivastava et al. 2020). Short tandem repeats (Autosomal STRs, Y-STRs, X-STRs, and miniSTRs,) mitochondrial DNA, and single-nucleotide polymorphisms (SNPs) are the tools used for CE fragment analysis (Fig. 1). With recent developments in next-generation sequencing (NGS), novel methods have been devised within the forensic community to make way for the investigative proceedings when CE-STR analysis fails to produce results. Several forensic laboratories are in the process of applying the MPS technology for the analysis of conventional STR markers and mitochondrial control DNA region and the possible uses of other DNA markers not frequent in casework such as next-generation STR kits, single-nucleotide polymorphisms (SNPs), insertion/deletion (InDel) markers, and mitochondrial DNA sequence (Phillips et al., 2007). With NGS we can generate a lot of data from a single sample, viz., autosomal, Y-STRs, X-STRs, identity SNPs, as well as phenotype and biogeography ancestry (Phillips, 2015) (Fig. 2).

The NGS Technology With the initiation of Sanger sequencing method in the 1970s (Sanger et al. 1977), DNA sequencing technology has come a long way. Several genome projects have been completed using Sanger technology. However low throughput and high cost pose a limitation to its use in more complex genome analyses (Fullwood et al., 2009). The recently introduced NGS technology has superseded these problems and

Fig. 1 Current tools for routine, degraded, kinship, and sexual assault samples

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Fig. 2 NGS: Multiple markers, one amplification

Fig. 3 Sanger sequencing versus massively parallel sequencing

is being used in forensics (Weber-Lehmann et al. 2014), diagnosing diseases (McCarthy et al., 2013), and ancient DNA analysis (Poinar et al. 2006). With Sanger sequencing, both the strands of a PCR product are sequenced, i.e., we read the sequence in one direction followed by reading in other direction, eventually sequencing each base twice. In MPS/NGS each base is sequenced many times yielding much more information about a sequence (Yang 2014). Billions of molecules can be sequenced in parallel, hence the name massively parallel sequencing (Fig. 3). Sequencing multiple reads at the same time leads to reduction in time as well as cost. Roche introduced the world’s first high-throughput sequencing system utilizing pyrosequencing based on sequencing by synthesis in 2005 (Margulies et al. 2005) followed by the technologies offered by Thermo Fisher Scientific, Illumina, Ion Torrent Inc., and Pacific Biosciences (PacBio) to name a few. Since then, the NGS technology has been offering new horizons for forensic genetics.

Comparing CE and NGS Extracted DNA in CE technology is PCR amplified using commercially available kits with uorescent dye labeled primers to multiplex STRs with overlapping size ranges. PCR products are then separated through CE according to their molecular weight. The result is in the form of electropherogram comprising peaks with their sizes expressed as base pairs and height expressed in relative uorescent units (Fig. 4) (Riman et al., 2020). NGS work ows (Fig. 5) also have PCR amplification to enrich STR markers. The target-specific primers contrarily are not uorescently labeled but are taken for library construction. PCR products have adapters attached

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Fig. 4 Work ow of a traditional DNA Analysis

at both the ends producing DNA libraries which can be sequenced (Müller et al. 2018). Raw digital data of sequencing reads is obtained as a result of sequenced DNA libraries (Mardis 2017). Multiple libraries can be pooled into one reaction as

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Fig. 5 NGS Work ow

Table 1 CE versus NGS Parameters Variation Primers Random matching probability Population studies

Capillary electrophoresis Length based Fluorescent Length-based discrimination Uses allele frequencies

NGS/MPS Sequence based Non- uorescent Sequence -based discrimination Uses frequencies for sequence-based alleles

Fig. 6 Similarities between CE and NGS/MPS methodology

the samples are barcoded. Available algorithms or software can determine the sequence and length-based polymorphisms (Woerner et al., 2017). Table 1 shows differences between CE and NGS technologies, whereas Figs. 6 and 7 depict the similarities between methodologies adopted in the processes.

Forensic Applications of NGS Technology The application of massively parallel sequencing is increasing in forensic DNA analysis as the crime investigation laboratories are looking for methodologies to obtain maximum information from a trace or degraded forensic sample (Fig. 8) (Montano et al. 2018). Presently it is possible to map whole genomes with constantly increasing speed and decreasing costs (Børsting and Morling 2015). The true diversity in core forensic loci has been explored, thereby adding statistical weightage to the evidence.

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Capillary Electrophoresis

MPS/NGS

Check size standard and allelic ladder

Check run quality

Check positive and negative controls

Check positive and negative controls

Check for the stutter and artefact peaks

Review quality control flags

Make edits, rename the peaks

Make edits, rename alleles

Generate and download reports

Generate and download reports

Fig. 7 Data analysis similarities between CE and NGS

STR and SNP Sequencing CE explores length based, whereas NGS is sequence-based variation (Gettings et al. 2015). Multi-application sequencing has helped in achieving new heights. With NGS, it is possible to derive full sequence information (SNPs and InDels) within the STR loci to derive investigative leads and to estimate mutational events in kinship testing (Dalsgaard et al., 2013; Gettings et al., 2015). Insertion/deletion polymorphisms (InDels) which comprise the characteristics of both STRs and SNPs (Kidd et al. 2012) have now been used for forensic case work examination, databasing, and anthropological studies (Liu et al., 2020). SNPs and mitochondrial DNA (mtDNA) provide an effective accompaniment to traditional CE-STR analysis by increasing the amount and kind of genetic information that a single sample may yield. By putting together this information, a powerful investigative profile can be generated for use with missing persons and mass disaster victim identification (DVI) and to determine the number of contributors in a mixture (Petrovick et al. 2020) and cold cases. It is helpful particularly in those cases where traditional CE-based methods do not provide a full profile. Isometric alleles having identical size, but different sequence, could be identified. MPS offers high resolution as well as high power of discrimination, and degraded sample can still be used. Lots of sequence variation can be analyzed within an

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Fig. 8 NGS facilitated forensic tools

allele. NGS technology can target just a few genes to all the nucleotides in a whole genome (Guo et al., 2017). A large number of panels have been developed for major as well as admixed populations. Successfully reliable and responsible sequencing of autosomal as well as Y-STR loci leads to conviction of a rape suspect by a court in Amsterdam, Netherlands. This case worldwide was the first case where conviction was based on MPS.

Mitochondrial DNA Sequencing in Forensics Using NGS Mitochondrial genome has higher copy numbers as compared to nuclear DNA and is less prone to degradation. Degraded samples could yield good results using mt DNA. Challenging samples are difficult to analyze with nuclear DNA; forensically fruitful information can be obtained by means of mitochondrial (mt) DNA (Holland & Parsons, 1999). It is the only source of information from samples like hair shafts

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where nuclear DNA is generally depleted (Higuchi et al., 1988). Mt. DNA analysis is an indispensable tool in forensic DNA examination (Holland et al., 2019); Mt. DNA is important for lineage, ancient DNA applications, and maternal inheritance for bio-ancestry. Capillary electrophoresis-based Sanger sequencing has been the gold standard for the last few decades (Berglund et al., 2011). But the technique is time-consuming and laborious and can focus only a portion of mitochondrial genome, i.e., HVI and HVII. Detection of heteroplasmy and occurrence of two or more mitochondrial genotypes in a cell can increase the discrimination power of analysis to a great extent. Analyzing heteroplasmy with Sanger sequencing is exceedingly difficult. Introduction of NGS technologies has revolutionized the arena of genomics (Kircher et al., 2012;). Last few decades have witnessed the utility of varied NGS technologies in assessing mtDNA, viz., Roche’s 454 (Payne et al. 2013; Illumina’s GAII (Li et al. 2010), Illumina’s HiSeq 2000 (Tang et al. 2013), and Ion Torrent’s Personal Genome Machine (PGM) (Parson et al. 2013). MPS addresses the limitations of traditional sequencing and sequences the entire mtDNA in one reaction without consuming much sample. MPS has large multiplex panels which makes it feasible to sequence the entire mitochondrial genome. With MPS complete, mtDNA sequence can be deduced from hair samples that come across in forensic case work (Parson et al. 2015). For aged and ancient genetic material, quantification and estimation of the level of degradation is the correct approach. In the highly degraded sample where nuclear DNA profile is not possible, NGS/SNP and mitochondrial DNA is the best alternate method. Tsunami victims case where high temperature and moisture resulted in degradation, the Russian Czar family missing children, and missing Mexican students of 2014 where apparently the individuals were burnt and killed and the remains were not amenable to DNA typing were some of the cases solved using mitochondrial analysis.

DNA Intelligence DNA intelligence or forensic DNA phenotyping (FDP) facilitates the prediction of bio-geographic ancestry and external visible characteristics of the donors of forensic samples (Fig. 9). This sort of intelligence administers priceless investigative leads in cases where database matches do not yield results from STR analysis (Phillips et al., 2019).

Forensic DNA Phenotyping (FDP): Visible Phenotype Estimation STR analysis fails to identify a person whose profile is unknown to the analysts. The drawbacks of comparative STR analysis lead to the birth of a new field in forensic genetics, i.e., forensic DNA phenotyping (FDP) (Kayser and De Knijff; 2011). Forensic DNA Phenotyping implies to the prediction of human appearances from traits such as hair color, eye color, and skin color from unknown crime scene samples

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Fig. 9 DNA intelligence

(Kayser 2015). Investigative leads can be obtained from unknown sample donors, unidentified with the current CE technology. The technology can be of great help in disaster victim identification and missing person identification cases.

Ancestry Informative Markers Bio-geographic ancestry (BGA) can be predicted using ancestry informative markers (AIM) (Xavier et al., 2020). During the course of the past few years, a large number of panels of the ancestry informative markers have been recommended for analyzing population genetic structure (Jiang et al. 2018). Also, ancestry can be established from the components of mixtures.

Degraded Samples Conventional forensic analysis has a limitation of allele size with respect to degraded samples. To overcome this, shorter markers (MiniSTRs) were adopted (Martín et al., 2006). Currently SNPs have been incorporated in sequencing panels (Gettings et al., 2015). In CE, as a ski slope is obtained for a degraded sample, but NGS is not size based, so interpretation of results is easier, and also it also works with less amount of sample.

Identifying Monozygotic Twins Monozygotic twins, having the same genetic structure, cannot be differentiated by conventional techniques like STR, SNP, and mitochondrial DNA analysis.

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Identification of extremely rare mutations by NGS can differentiate between monozygotic twins (Weber-Lehmann et al., 2014).

Emerging Applications of NGS Species origin of a sample, age range of contributors, metagenomics or human microbiome analysis, and methylation analysis are some of the emerging applications of NGS. Microbiome gives unique identity to an individual and can be used to determine the site of origin of the sample (Tozzo et al., 2020). NGS has a wide variety of application in body uid and tissue identification via mRNA and methylation studies (Ingold et al. 2018). Studies have reported the use of miRNAs (microRNAs), a group of small noncoding RNAs having applicability in age prediction for body uids or crime scene stains using MPS (Fang et al. 2020). NGS is also useful in wildlife forensics.

Utility of NGS/MPS over CE The analysis of conventional STR markers using MPS provide a number of benefits over standard CE analysis, namely, particularly increased number of loci, higher discrimination power, and shorter amplicon length for a conclusive analysis of degraded and trace DNA evidence: • • • • • • • • • • •

Low DNA samples. Male minor contributor. Degraded samples. Unknown tissue origin. Cold cases. Suspect untraceable cases. Failed CE cases. No STR profile match in DNA database. Cold homicide cases. Mixture interpretation. Complex kinship cases or familial search.

The technique has certain drawbacks also, such as being expensive, lacking standardization, requiring huge resources for bioinformatics, and data storage besides accumulating sensitive personal data which is difficult to protect as entire genome including the coding regions can be sequenced. Although CE has a limitation of panels being limited by size ranges and uorescent labels, still CE-based STR typing will remain the standard casework and database application as it is cheap, fast, and reliable and can be undertaken on regular basis, whereas MPS-based STR typing represents a specialized tool in forensics and can be used for specific cases. It involves time taking, tedious steps in library and DNA template preparation,

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effective bioinformatics tools for sequence alignment, efficient data storage servers for the storage of sequence data files, and lack of standardization. The said shortcomings are being worked upon and do not seem to be a major long-term issue (Alonso et al. 2018). The technology guarantees endless advancement and may soon become the standard benchmark of quality. Today NGS platforms are being used not only in forensics (Fordyce et al. 2011) but also in microbial (Caporaso et al. 2012) and cancer research (Kirsch and Klein 2012).

Forensic Genetic Genealogy: The New Means of Genetic Identification DNA analysis is being widely used in human identification, missing person/disaster victim identification and kinship testing (Fig. 10). A CE-STR based analysis involves uploading a crime scene profile to a database to obtain a hit or match (Fig. 11), whereas forensic genealogy is based on SNP testing and uploading the results to genealogy database to measure the genetic relatedness (Fig. 12). Genetic genealogy has helped solve dozens of cold cases that would not have been cracked otherwise (Greytak et al., 2019). Matching is done by searching for DNA segments which are shared among the relatives. Genome-wide SNP data is used to measure the genetic relatedness (Fig. 13) (Kling and Tillmar 2019). Genetic genealogy though is not linked to CODIS but cases end the same way. In its evolutionary stage, genetic genealogy started with Y STR analysis. For the last 2 years, over 100 cases have been solved that have not been solved over decades. It has proved to be extremely accurate. Family trees have been constructed to find birth

Fig. 10 Applications of DNA in legal field

Fig. 11 Flow chart for CESTR-based analysis

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Fig. 12 Flow chart for forensic genetic genealogy

Fig. 13 From remains to database search: protocol for FGG

families by genealogists. Reference samples are compared with evidence material to confirm identity. Cold cases, missing person identification, unsolved heinous crimes, and exoneration of the innocent can be solved by forensic genetic genealogy. Voluntarily submitted DNA data for forensic purposes is being used by “Genealogy Data Matching” (GEDmatch, Verogen, Inc.) software to generate investigative leads. GEDmatch allows the user to upload and compare SNP data in database. Autosomal profiles are uploaded to GEDmatch, to find out a potential match, and most recent common ancestors of a suspect were found on family tree, although there still exist issues in genetic genealogy relating to privacy notwithstanding the advancements (Court S. Denise, 2018; Wickenheiser, 2019). Moreover, the technique is expensive, time intensive, and resource intensive as is outsourced by forensic agencies and requires specialized knowledge. Low-quality uploads result in false genealogy connections. A cold case, a 16-year-old double homicide case, was solved by FGG in Sweden. SNP profiles were generated from the crime scene samples using whole genome sequencing and advanced bioinformatics tools. Relatives were searched in GEDmatch, and family tree DNA databases were constructed ultimately giving leads. With the arrival of IGG, alternate route for DNA analysis based on SNPs has been described. The following are the technical requirements for SNP testing: • At least 1 ng of DNA. • Single source DNA samples preferred. • Degradation of samples could be a problem.

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• DNA quality. • Partial SNP profile. • Success is genealogy database dependent. Two most famous cases solved by this path breaking technique have been discussed below:

The Ekeby Man Case The technique puts forward a great challenge for low copy number degraded DNA as in the case of forensic samples. Progression in DNA sequencing methodology has enabled the probability to process low copy number degraded biological samples (Prüfer et al., 2014). One such example is to identify an unknown male remains (“the Ekeby man case”) found murdered in Sweden in 2003, where whole genome sequencing was done on a bone sample along with bioinformatics tools which generated around 1.4 million SNPs (Tillmar et al. 2020). The SNP genotypes were searched for relatives on DNA database GED match. A list of relatives was prepared to identify the unknown remains, and investigative leads were obtained.

The Golden State Killer The man known as the Golden State killer (De Angelo serial killer, 74 years now) was sentenced to multiple life sentences for dozens of crimes he committed. He was convicted for the rapes and killings which he committed from 1975 to 1986 covering a wide geographical area that was thought initially because of multiple people. He began with home burglaries before committing many rapes and killings across southern California. He often broke into people’s homes at midnight and carry out rapes and killings. The crimes mysteriously ended in 1986. The heinous crimes committed might have gone unsolved without the innovative genetic technique that was developed to reunite adoptees with their biological parents. DNA recovered from one of the crime scenes was put into the DNA database to find the relatives of the killer. A common ancestor among them was found, and family trees down to the present day were created (Phillips, 2018). De Angelo appeared as a possible suspect. Eventually an item containing De Angelo’s DNA was found by the investigators and compared with the DNA recovered from the crime scenes, and a match was obtained. He was arrested in April 2018. Dozens of victims testified before the court. He was sentenced to life imprisonment without parole on August 21, 2020.

The Rapid DNA Instrument Generating DNA profile from capillary electrophoresis involves a large number of tedious steps. The setup required for performing STR analysis includes centrifuge machines, thermal cyclers, and capillary electrophoresis instrumentation in a

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centralized laboratory consuming at least 10 hours. Advancements in instrumentation have led to automation during the last few years (Hopwood et al. 2010). Robotic platforms (Frégeau et al., 2010) have reduced hands-on time. The rapid DNA instruments combine the steps of isolation, faster amplification, denaturation, sizing, and genotyping (Fig. 14). RapidHIT ® 200 and the RapidHIT ID Systems have been developed by IntegenX for generating STR profiles from reference samples in nearly 90 minutes. This also reduces the chance of contamination between the samples (Pleasanton, CA) (Shackleton et al., 2019). One could employ such kind of a unified instrument for reference samples with the least amount of time required by the analyst (Dash et al., 2020). This kind of technique not only reduces the risk of contamination but has the added advantage of swab reusability for conventional DNA methods. Such kind of instruments can be used by the law enforcement personnel in the police stations, in the crime investigation labs for increased lab productivity, and in the field as well according to the need. The RapidHIT™ ID system (Thermo Fisher Scientific) is a fully automated system for human identification. Run information is transferred to Rapidlink™ software to process. Reagents used for each instrument can be traced at a central location. All the data is fed into computer, and any DNA profile can be matched. Quality ags/colored ags can tell about the run quality. One person at a central position can see all the data coming from all the instruments and can address the problem. Data quality is monitored at one workstation by the software. The system is cartridge based with two kinds of cartridges, viz., primary cartridge and sample cartridge: 1. Primary cartridge contains polymers, capillary, and buffers. 2. Sample cartridge are of two types the Ace GlobalFiler Express sample cartridge for single source reference samples and the Rapid INTEL™ sample cartridge for forensic samples such as bones (Buscaino et al. 2018). The developmental validation studies for this system were performed with mock casework samples according to the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines (Scientific Working Group on DNA Analysis

Fig. 14 Rapid hit system

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Methods Validation Guidelines for DNA Analysis Methods. December 2016) (User Bulletin, “RapidINTEL™ Sample Cartridge for blood and saliva samples, Thermo Fisher Scientific). The samples are analyzed on the GeneMarker ®HID STR Human Identity software on the instrument (Holland & Parson, 2011). Samples that require fast results may benefit from the rapid platform. Numerous Rapid Hit systems linked with RapidLink software can prove to be useful in criminal investigations.

Conclusion Rapid DNA examination will soon facilitate new applications (Butler 2015). Massively parallel sequencing (MPS) provides the potential to multiplex diversified forensically relevant markers and multiple samples together in a single run in comparison with traditional capillary electrophoresis method (Churchill et al., 2016). Sequence based allele frequency data with the establishment of within STR allele sequence variants, will aid forensic community to enhance the power of discrimination for human identification and mixture deconvolution by increasing the effective allele number (Gaag et al. 2016) and kinship analysis. Forensic DNA phenotyping (FDP) and bio-geographic ancestry (BGA) from an unknown crime scene sample are gaining interest of the forensic community (Xavier et al., 2020). Whole genome sequencing has been successfully used to constitute genealogy DNA databases for generating investigative leads in cold cases (Tillmar et al. 2020). DNA intelligence marks a considerable peculiar application of genetic evidence unlike the one presented in the courtroom (Kayser 2015). NGS is an up-and-coming technology and is increasingly being implemented in forensic case work examination.

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Biological Sources of DNA: The Target Materials for Forensic DNA Typing Pankaj Shrivastava, R. K. Kumawat, Pushpesh Kushwaha, and Manisha Rana

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Semen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vaginal Secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oral Fluids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sweat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Urine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fecal Matter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Among physical evidences encountered at the crime scene, biological evidences, viz., blood, semen, vaginal secretion, saliva, urine, and sweat, are the most ubiquitous in nature, and their presence aids in linking perpetrator to the victim as well as crime scene. Recognition of biological fluids as substantive evidences P. Shrivastava (*) DNA Fingerprinting Unit, State Forensic Science Laboratory, Sagar, Madhya Pradesh, India R. K. Kumawat DNA Division, State Forensic Science Laboratory, Jaipur, India P. Kushwaha Dr. APJ Abdul Kalam Institute of Forensic Science and Criminology, Bundelkhand University, Jhansi, India DNA Fingerprinting Unit, State Forensic Science Laboratory, Sagar, Madhya Pradesh, India M. Rana Department of Forensic Science, Guru Ghasidas University, Bilaspur, India DNA Fingerprinting Unit, State Forensic Science Laboratory, Sagar, Madhya Pradesh, India © Springer Nature Singapore Pte Ltd. 2022 H. R. Dash et al. (eds.), Handbook of DNA Profiling, https://doi.org/10.1007/978-981-16-4318-7_2

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is endorsed by presence of DNA in them. Advancing forensic DNA typing techniques have a great potential in characterization and individualization of biological evidences encountered during criminal investigation, but their applicability on each biological fluid for human identification varies in a great deal due to variation in the amount of nucleic acid available within the fluid. DNA concentration is relatively high in fluids such as blood that contains large number of cells while fluids such as urine and sweat possess low amount of DNA. Besides reference biological samples, forensic DNA typing can be implemented for detecting presence of traces of biological fluids on physical surfaces. The concept of “Touch DNA” or “Transfer DNA” involves analysis of low amount of DNA deposited on the surfaces that have come in human contact and can constructively help in associating evidences with perpetrator and/or victim. However, identification of sources of such materials, their collection methods, and preservation conditions can influence the quality of result. Further to this, degradation, impurity, contamination, and presence of inhibitors in such evidences demand purification and isolation of high-quality DNA. This chapter deals with various biological sources of DNA commonly encountered at the scene of crime and their evidential value, along with various factors and conditions affecting forensic DNA typing of such samples. This chapter deals with various biological samples used for forensic DNA typing along with various factors and conditions that affect forensic DNA typing. Keywords

Biological fluids · Forensic DNA typing · Touch DNA · Crime Scene · Perpetrator

Introduction Among physical evidences encountered at the crime scene, biological evidences, viz., blood, semen, vaginal secretion, saliva, urine, and sweat, are the most ubiquitous in nature, and their presence aids in linking perpetrator to the victim as well as crime scene. Recognition of biological fluids as substantive evidences is endorsed by presence of DNA in them. Advancing forensic DNA typing techniques have a great potential in characterization and individualization of biological evidences encountered during criminal investigation, but their applicability on each biological fluid for human identification varies in a great deal due to variation in the amount of nucleic acid available within the fluid. DNA concentration is relatively high in fluids such as blood that contains large number of cells while fluids such as urine and sweat possess low amount of DNA. Besides reference biological samples, forensic DNA typing can be implemented for detecting presence of traces of biological fluids on physical surfaces. The concept of “Touch DNA” or “Transfer DNA” involves analysis of low amount of DNA deposited on the surfaces that have come in human contact and can constructively help in associating evidences with perpetrator and/or victim.

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However, identification of sources of such materials, their collection methods, and preservation conditions can influence the quality of result. Further to this, degradation, impurity, contamination, and presence of inhibitors in such evidences demand purification and isolation of high-quality DNA. This chapter deals with various biological sources of DNA commonly encountered at the scene of crime and their evidential value, along with various factors and conditions affecting forensic DNA typing of such samples.

Blood Blood is one of the preeminent evidences that are alighted on at various crime scenes including cases of sexual assault, homicide, suicide, accidents, and burglary in disparate forms of blood-pool and blood stains adhered to the surfaces such as floor, walls, clothes, and the weapons involved in the crime. Whole blood is a composite of various blood components that are classified into liquid element, plasma, and cellular (or formed) elements, erythrocytes (or red blood cells), leukocytes (or white blood cells), and thrombocytes (or platelets). Discovery of ABO blood typing in 1900 instigated the scope of human blood identification that further strode toward individualization in the 1980s with the advancements of molecular techniques. Existence of individual-specific minisatellites in the human genome that can aid in human identification cases was affirmed by analyzing blood samples of 20 unrelated individuals using southern blot hybridization technique (Jeffreys et al. 1985). Thereafter several protocols and their modifications with varied incubating reagents, time frame, and techniques were designed and reported for extraction and purification of human genomic DNA (Table 1). A number of considerable pre-analytical factors influence the quantity and quality of genomic DNA extracted from the whole blood or clotted blood sample. Sample collection with zero or minimal contamination is an important consideration that affects the stability of blood sample. Use of sterilized syringe is preferred for collection of blood pools. Wet blood soaked objects are air-dried and collected; if object is inflexible, stains can be collected on sterilized cotton swabs and air-dried. Scraping or tape-lifting is employed for collection of dried blood stains. Stability of blood is influenced by other factors such as use of stabilizing agents such as heparin or anticoagulant agents such as EDTA, time difference between collection, and storage of sample (Vaught 2006). Storage period, storage conditions such as temperature of blood, as well as isolated DNA are shown in Table 2. Exposure to ultraviolet radiations, heat, light, humidity, and soil contaminations (McNally et al. 1989) have adverse effect on extraction of good quality of DNA. Blood evidence in various conditions, viz., frozen blood, clotted blood, or dried blood spots, can also be found during forensic investigation. Such samples require additional pre-analytical treatment or modified protocols for extraction of DNA. Modification in conventional proteinase K/phenol chloroform isoamyl alcohol (PCIA) protocol with pre and post-trypsination of frozen blood samples with the

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Table 1 Protocols and their modifications for DNA extraction from blood S. no 1

2

3

4

5

6

Protocol Rapid method of DNA isolation from human leukocyte Rapid method for the purification of DNA from blood Salting out procedure for extracting DNA from human nucleated cells Non-organic procedure for the isolation of genomic DNA from blood Direct PCR from whole blood, without DNA extraction

Isolation of fetal DNA from nucleated erythrocytes in maternal blood

Material used SDS for lysis, potassium acetate for precipitation

Technique used Diethylaminoethyl (DEAE) cellulose chromatography

Result 50–70 μg of DNA in 10 ml of blood sample

Reference Potter et al. (1985)

Guanidine hydrochloride, ammonium acetate, sodium sarkosyl, and proteinase K 1 ml of saturated NaCl after digestion

Southern blotting

20 μg of DNA in 1 mL of blood

Jeanpierre (1987)

Centrifugation at 2500 rpm for 15 min

DNA quantity comparable with that of phenolchloroform protocol

Miller et al. (1988)

Ice cold CLB (0.32 M sucrose, 10 mM Tris-HCl pH 7.6, 5 mM MgCl2, 1% Triton X-100) Blood introduced directly to the PCR reaction of 50 mM KCl, 10 mM TrisHCl pH 8.0, 1.5 Mm MgCl2, 0.1 mg/ml gelatin, 200 uM each dNTP Phosphate buffered saline (PBS: 0.137 M NaCl/0.002 M KCI/0.008 M Na2HPO4/ 0.0015 M KH2PO4, pH 7.4), 2% fetal calf serum, and 0.1% sodium azide on ice

Southern blotting

Isolation period less than 4 h

Grimberg et al. (1989)

3 PCR cycle of 3 min at 94  C then cooled for 3 min at 55  C

Convenient alternative to the tedious DNA extraction process

Mercier et al. (1990)

TfR Analysis

0.1–1 ng of fetal DNA present in maternal blood at 15–16 weeks of gestation

Bianchi et al. (1990)

(continued)

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Table 1 (continued) S. no 7

Protocol Alkaline extraction of Human Genomic DNA

8

Modified salting out method using laundry detergent

Material used 5 μL of sample incubated with 20 μL 0.2 M NaOH at room temperature for 5 min in case of blood and at 75  C in case of stain Additional treatment with laundry powder solution, glass beads, and NaCl prior to DNA precipitation

Technique used Centrifugation at 12000xg for 5 min

Result 30 ng of nuclear DNA per μL of blood

Reference Dissing et al. (1996)

Centrifugation at 15000 rpm for 5 min

30 mg/ml of powder yielded sufficient DNA 56.3 μg/mL

Nasiri et al. (2005)

Table 2 Storage conditions for blood samples Storage condition 45  C 23  C 30  C Dried blood spots on FTA cards Blood stains at room temperature

Maximum time 6–7 weeks 1 week 12 years 16 years 15 years

Reference Madisen et al. (1987) Chen et al. (2018) Rahikainen et al. (2016) Barbaro and Cormaci (2006)

inference that trypsination before cell lysis yielded DNA with 88.17% purity that declined to 63.23% in case of untrypsinized frozen blood (Ahmad et al. 1995). Another rapid protocol with a modified composition of cell lysis buffer and extraction buffer substituting the toxic reagents yielded DNA from frozen blood that was comparable with fresh blood (Guha et al. 2018). In case of clotted blood, mechanical shearing of clot by homogenization, scraping, or slicing (Xu et al. 2010) raised the quality of DNA. Use of nylon mesh and serum separator in various studies reports a good yield of DNA from clotted blood. The texture, quality, and absorptivity of the surface acting as blood-stain carriers also influence the yield of DNA. Prinz and Berghaus (1990) successfully isolated sufficient amount of DNA from two days old dried blood stains on eleven different stain-carrier surfaces including variety of fabrics stores at room temperature. DNA isolated from surfaces such as wool, denim, suede, and carpet was chemically contaminated possibly due to surface-specific challenges in extracting leukocyte from the carrier. A commonly encountered forensic situation is the deliberate removal of visible blood stains with cleaning agents that does not possibly obstruct the visualization of the stains but generate contamination of stain and degradation of genomic DNA (Tas 1990) (Thabet et al. 2018). Bleach has the most detrimental impact on the yield of

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DNA as compared to other chlorinated and non-chlorinated detergents (Harris et al. 2006). Bloodstains treated with fingerprint-enhancing and/or blood-enhancing reagents are another set of challenges for recovery of DNA. Fingerprint enhancement reagents such as Cyanoacrylate Fuming (Newall et al. 1996; Mutter et al. 2018), silver nitrate (Lee et al. 1989), and other bloody fingerprint enhancement chemicals such as luminol (3-aminophthalhyrazide) (Manna et al. 2000), benzidine, leucomalachite green (LMG), phenolphthalein KM, Amido Black (methanol based), Crowle’s Double Stain, and Hungarian Red (fuchsin acid) (De Almeida et al. 2011; Frégeau et al. 2000; Everson et al. 1993; Tobe et al. 2007) have deteriorating impact on the quality and quantity of DNA. Yield of DNA is also reduced by exposure to short ultraviolet rays that are used for enhancing fingerprints found in the blood (Andersen and Bramble 1997). DNA fingerprinting technique is influenced by diverse range of factors including ecological factors, improper collection techniques or mishandling during the chain of custody, as well as analytical procedure. The inference drawn from the wellknown O. J. Simpson trial (1985) (Thompson 1996) elicited the necessity of improving the diligence of evidence collection and analysis by the forensic DNA laboratories as well as upgrading the presentation of DNA evidence in the courtroom. The defense alleged on the collection and preservation of evidence more willingly than raising questions on the validity of DNA evidence by presenting evidence of negligence by Los Angeles Police Department (LAPD) in form of crosscontamination, switching of dried swatches, and premeditated planting of blood onto the evidences (Butler 2005).

Semen Sexual assault cases contribute nearly 50% of the total cases received in the Forensic DNA Laboratories. The importance of DNA in sexual assault cases was known to be long ago since the “Pitchfork Case” of Leicestershire (1988), the first trial involving DNA analysis of semen for conviction of rape and murder. Semen, a viscous, slightly yellowish or grayish fluid mainly comprises of seminal fluid (made up of water, proteins, sugars, minerals, and vitamins) and sperm cells (spermatozoa). A typical ejaculation releases 2–5 mL of semen of which spermatozoa (50 μM in length) make up approximately 5% of the total volume of semen. Swabs, clothing, vaginal slides, and bedding items are generally collected for DNA analysis. For a successful STR (Short Tandem Repeats) analysis, nucleated cells with a sufficient amount of DNA are required, and once seminal fluid is detected on samples by applying different preliminary and confirmatory tests, the next step is DNA extraction. DNA fingerprinting has two basic utilities in sexual assault cases. Firstly, the individualization of semen (when spermatozoa is less in number) and secondly is to differentiate mixed stains when the number of perpetrator is more than one. The situation becomes complex when contaminated samples are there or semen is mixed with other body fluids like blood. And here, the hemoglobin acts as an inhibitor in

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the PCR (Polymerase Chain Reaction) process. Generally, semen is found to be mixed with vaginal secretion which cartons the presence of spermatozoa as the amount of vaginal secretion is much higher. Male DNA can be separated from the DNA of other cells using differential extraction method which was firstly detailed in the publication by Gill et al. (1985) and later by Wiegand et al. (1992) and Yoshida et al. (1995) (Table 3). Another difficulty which DNA experts face is when multiple males are contributors and two or more alleles may be present for one marker and in this situation chances of allele dropout can’t be ruled out. If spermatozoa is absent in

Table 3 Differential extraction protocols and their modifications Protocol for differential extraction Sperm nuclei lysis with SDS, proteinase K, and DTT mixture Sample treated with Lysis buffer I (10 mM Tris-HCl, 0.4 M NaCl, 2 mM EDTA) with proteinase K and SDS and incubated at 37  C for 40 min followed by centrifugation at 13000 rpm for 5 min. Supernatant lysed with lysis buffer II (proteinase K, SDS, and DTT), incubated at 56  C for 1 h, centrifuged at 13000 rpm for 5 min, and DNA isolated using organic extraction protocol First incubation with TNE buffer, 1% SDS and proteinase K at 70  C for 3 h, followed by centrifugation at 15000 rpm for 5 min. Second incubation with TNE buffer with 1% SDS, 100 ml proteinase K, and 0.04 M dithiothreitol (DTT)) for more than 8 h at 56  C in a shaking water bath Tris(2-carboxyethyl)phosphine (TCEP) in Triton X-100 as lysis agent to lyse sperm cells and collect DNA on-chip, incubated for 15 min followed by addition of 40 μL of proteinase K solution (1 μg mL 1) and incubation for 4 h at 55  C. 100 μL of Buffer AL and 100 μL of ethanol were added to the samples, mixed by vortexing and run through gDNA extraction using a Qiagen spin column protocol Sample treated with extraction buffer (10 mM Tris-HCl (pH 8.0), 100 mM sodium chloride, 1 mM EDTA (pH 8.0), TNE, 1% SDS, and 0.2 mg/ml proteinase K) and incubated for 2 h at 37  C followed by centrifugation of samples in spin baskets at 18000 g for 5 min, separation of supernatant, and multiple washing of pallet. Pallet lysed with sperm extraction buffer (10 mM Tris-HCl (pH 8.0), 100 mM sodium chloride, 1 mM EDTA (pH 8.0), 2.5% sarkosyl, 0.39 M dithiothreitol, and 0.5 mg/ml proteinase K) and incubated at 37  C for 2 h. DNA purified from epithelial and sperm portion using Qiagen EZ1 Advanced XL system Sample lysis with stain extraction buffer (1 mol/L Tris–HCl, ddH2O, 5 mol/ L NaCl, 0.5 mol/L EDTA, 10% SDS, pH ¼8.0) and 15 mL of proteinase K (20 mg/mL) (Fisher Scientific, Pittsburgh, PA, USA) followed by an overnight 56 C incubation. Isolation of lysate (non-sperm fraction) using DNA IQTM spin baskets (Promega Corporation, Madison, WI, USA) at 7500 g spin for 5 min. The sperm pellet was subsequently resuspended in 200 mL of phosphate buffered saline solution (Fisher Scientific), 20 mL of Qiagen proteinase K stock solution, and 20 mL of 1 mol/L DTT (Fisher Scientific), vortexed, 200 mL of Buffer AL (Qiagen) was added, and samples were incubated at 56  C isolation with QIAamp DNA Investigator kit. DNA was eluted in final volumes of 100 mL (nonsperm fractions) or 60 mL (sperm fractions) of Buffer ATE (Qiagen)

Reference Gill et al. (1985) Wiegand et al. (1992)

Yoshida et al. (1995)

Inci et al. (2018)

Alderson et al. (2018)

Goldstein et al. (2019)

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semen (oligospermic, azoospermic, or normospermic), Positive Semelogenin (Sg) samples may be suitable. Y-chromosome identification and Sg biomarker should be thoroughly examined in the laboratories (Martínez et al. 2015). The condition of the exhibit before examination remains to be a censorious aspect for successfully detecting and analyzing semen, and for this appropriate handling parameters during drafting, collecting, packaging, storing, and transportation of samples are the fundamental strides.

Vaginal Secretion Another important evidence found in cases of sexual assault is vaginal secretion. In these cases, the identification of vaginal secretion is crucial as it can support in verifying the allegations of sexual assault. For instance, a stain is observed during investigation of a sexual assault case, and forensic DNA analysis affirms that the stain is originated from the victim, thus creating a link among the suspect and the victim. But, the litigant may contradict any criminal act by claiming that the stain is originated from sweat due to spontaneous contact. So, the evidence would have a significant value if vaginal secretion stain was found. A mixture of vaginal secretion and semen stain is generally found, and the presence of vaginal secretion confirms the incidence of sexual assault. Human vagina is composed of squamous mucosa (comprises of stratified squamous epithelial tissue), submucosa, and muscularis. The vaginal secretion basically consists of epithelial debris, tissue fluid, leukocytes, electrolytes, lactic acid, and proteins which is generally derived from the glands of the uterus, cervix, transudation of the vaginal epithelium, and Bartholin’s glands. Forensic laboratories use various methods for identification of vaginal secretion, and once it is identified, then DNA analysis is done. The main problem experts face is the ominous proportion of male to female DNA, with a surplus of the victim’s material. Differential lysis is applied in this situation to isolate male DNA from epithelial cells (Gill et al. 1985). Some other factors which should be kept in mind in order to get better results are the type of material used for collection and storage must be selected correctly. The contamination of genetic material from other sources (e.g., from the examiner and other biological evidence) should also be avoided (Butler 2005). Contamination may occur during the sexual contact (e.g., if there is more than one executioner), during collection and packaging, during transportation, during the medical examination, and in the laboratory. Proper care must be taken to restrict cross-contamination between sexual assault evidences.

Oral Fluids Oral fluids or whole saliva is a mixture of secretion produced from major specific salivary glands, numerous minor salivary glands along with secretion from non-salivary sources such as nasal secretion, gingival crevicular fluid, bronchial

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mucus, buccal cells, bacterial products, and food remains. Non-invasiveness, easy collection, and less contamination are some of the beneficial features of saliva over blood for DNA typing. Saliva are generally recovered from the scene of crime in conjunction with bite mark evidence on the skin as in cases of violent crimes, on eatables, clothing, cigarette butts, chewing gums, chewed betel quid stains, documents, postage stamps, and other objects (Anzai-Kanto et al. 2005; de Oliveira Musse et al. 2019). Despite frequent occurrence, quick drying of saliva stains makes them indiscernible, hampering their recognition and collection. The earliest DNA isolation from the saliva on cigarette butts involved PCR amplification at HLA-DQ alpha and D1S80 markers and analysis by reverse dot-blot technique and polyacrylamide gel electrophoresis (Hochmeister et al. 1991). Similar study of DNA isolation from saliva and saliva-stained samples such as buccal swabs, gags, envelopes, and cigarettes stored at different conditions demonstrated identical DNA banding patterns as obtained from blood, hair, semen, or mixed saliva (Khare et al. 2014; Walsh et al. 1992) (Table 4). Watanabe et al. (2003) reported inhibitory impact of certain dyes present in the cigarette butts on PCR amplification. Sweet et al. (1996) were constantly involved in the studies related to DNA extraction from saliva in various conditions. In 1996, they proposed modified Chelex method involving pre-analytical use of proteinase K, incubation at 56  C for 60 min, and 100  C for 8 min and subsequent microconcentration of solution (Sweet et al. 1996). Saliva deposited on the skin is present in limited amount. Double swab technique ensures maximum collection of saliva stains with

Table 4 Salivary DNA extracted from different surfaces Substrates Betel quid Food

Skin

Number/type of samples 50 20 cheese pieces

Conclusion 92% success rate for DNA isolation from 4 years old forensic BQ samples Collection of saliva from the center instead of peripheral surface yielded better results

2 surfaces of cheese

Variation in DNA concentration recovered from upper and lower surface

5

Lower DNA recovered from skin probably due to degradation during saliva deposition, collection, and extraction Double swab technique yielded better DNA quantity than filter paper and single swab technique Mixed DNA profile with minor component correlating with DNA profile of suspect

15

Cigarette butts

A body submerged in water 200 100

Deterioration in concentration of DNA with storage time, but all DNA were PCR amplifiable Inhibitory effects of dyes present in cigarette

Reference Chiou et al. (2001) de Oliveira Musse et al. (2019) Sweet and Hildebrand (1999) Anzai-Kanto et al. (2005) Sweet et al. (1997) Sweet and Shutler (1999) Hochmeister et al. (1991) Watanabe et al. (2003)

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minimal contamination (Cherian et al. 2015). It involves swabbing of the skin surface with first swab immersed in sterile water in circular motion followed by second swabbing with dry swab using same pressure and motion (Sweet et al. 1997). In a drowning case, the bite mark present on the victim’s body submerged for 5.5 h in water served as the source of salivary DNA, and DNA profile of the suspect was identified and distinguished at HUMTH01 and HUMvWA loci (Sweet and Shutler 1999). Storage of saliva at 70  C yields fair quality of DNA up to 1 month. Storage at  4 C led to bacterial growth but yields sufficient PCR product (Ng et al. 2004). Quantity and quality of DNA from saliva stored at 4  C and 20  C for up to 3 months was comparable to that of fresh saliva samples. However, gradual deterioration was observed when the storage period was extended to 5 months (Kim and Kim 2006). Trace quantity of saliva can also be transferred to the surfaces during speaking, coughing, and flipping pages of documents. Double swab technique for collection of such samples is preferred. Deposition of trace samples can also be the result of contamination at the crime scene by adventitious transfer or during investigation and analysis. Use of face mask during investigation, collection, and analysis of samples is recommended to minimize such contamination.

Sweat Sweat became unfailing evidence that are deposited unconsciously at various touched surfaces or handled objects and due to their transparent and evaporative nature, rarely engages one’s attention during deliberate cleaning of other evidences. Biologically, sweat is a watery fluid secreted from eccrine and apocrine sweat glands present throughout the body along with dissolved mineral, metabolites, and epithelial cells. Around 650 sweat glands are present in average square inch of skin resulting in primary transfer, i.e., deposition of trace amount of sweat on surfaces that come in contact with the skin. The concept of “Touch DNA” by primary transfer and “Transfer DNA” by secondary transfer of DNA relating to Locard’s Principle of exchange has gained much attention over the last few years (Kisilevsky et al. 1999; Ladd et al. 1999). Touch DNA or trace DNA are described as low levels of DNA deposited on handled, touched, or worn object without presence of detectable body fluid. Minute traces of epidermal cells along with sweat generally result in deposition of touch DNA. Transfer DNA, on the other hand, are resulted from secondary transfer and include foreign DNA present on individual’s hand from previous contact that are subsequently deposited on other surfaces (Wickenheiser 2002). This may include skin-skin-object mode of transfer or skin-object-skin mode of transfer (Burrill et al. 2019). Quality and quantity of touch DNA recovered from any surface is affected by a wide variety of factors. Shedding status is one of the factors that influences the yield of touch DNA and is described as the tendency of an individual to lose skin cells. It is reported to be higher in women compared to men due to the presence of

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thicker stratum corneum in men, making it more stable (Faleeva et al. 2018). Shedding rate also depends upon the individual’s age due to high proliferation rate and less degraded DNA in children as compared to elderly people (Poetsch et al. 2013). The yield of trace DNA is affected by the number of deposited DNA-bearing cells, nature of the surface carrying the deposited DNA, lapse of time between deposition and recovery coupled with exposure to environmental conditions, method of sample collection and DNA extraction employed and is independent of timeframe for which skin remains in contact with the surface (Alketbi 2018). Worn clothing, footwear, beddings, wallets, and bags and door handles are some of the common substrate bearing sweat stains. A study on potential transfer of touch DNA revealed that samples of sweat collected from the beddings after one night of sleep provide good DNA profile. DNA profiles of former individual in contact with the bedding can also be generated. Similar study on sweats of foot and soles of footwear inferred higher DNA amount from the top of foot than the soles. Microbial impacts, cell compressions, and presence of certain PCR inhibitors at the underside of foot and sole justify the loss of DNA on the sole. Synthetic sport shoes yielded better amount of DNA than the leather shoes. The areas as well as techniques of sample collection were also found to affect DNA recovery (Bright and Petricevic 2004). Adhesive tape lifting, dry swab, and cutting out are some of the commonly employed techniques of sample collection. Double swabbing technique for collection of sweat stains, with first wet swab and second dry swab, yields greater DNA recovery from dry swabs than the wet swabs. Minimal amount of available touch DNA necessitates maximum sample collection and extraction of DNA in shortest possible time period. Zhou et al. (2016) described use of 96-well centrifugal filtration plate and automated DNA extraction on liquid workstation from swabs from door handles, gloves, beverage bottles, cigarette butts, tools, etc. resulting in 54.43% successful profile rate. Recovery of touch DNA from metal surfaces such as ammunition, door handles, and furniture is affected by interactive nature of DNA with metal cations as well enzymatic actions of certain metals on degradation of DNA. Combination of collection method and buffer specific to the metal surface carrying DNA resulted in improved recovery of DNA (Tucker 2015). Latent fingerprints resulting from deposition of sweat, skin cells, and particulate matters, frequently occurring in any crime scene, are also an efficient source of samples for DNA profiling. Recovery of DNA from fingerprints depends on the substrate, standard pressure, frequency of hand washing, and exposure to fingerprintenhancing chemicals (Table 5).

Urine The importance of bodily fluids as sources of DNA for identification purpose has been known since a longer period of time (Hilhorst et al. 2013). Urine, being a useful tool as a source of genetic material, has not been densely applied as a

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Table 5 Effect of fingerprint-enhancement chemicals on DNA profiling Fingerprint-enhancement techniques White powders Black powders Metal powders Black magnetic powders

Surface Glass and wood

UV light, DFO, ninhydrin White powders, black powder, magnetic powder Cyanoacrylate fuming, Cyanoacrylate + rhodamine 6G, Cyanoacrylate + VMD Ninhydrin

Paper Glass

Vacuum metal deposition (VMD) Cyanoacrylate fuming

Plastic

Ninhydrin

Paper

1,8-diaza-9-fluorenone (DFO)

Paper

Black fingerprint powder

Glass

Magnetic latent print powder Luminescent cyanoacrylate fuming

Glass Plastic

Effect of DNA profiling Better DNA yield from wood surface than glass. BVDA white, Faurot white, magnetic black, special black fingerprint powders had no impact on DNA and yielded a good profile Increased inhibitory effects observed No effect on DNA yield

Plastic

Rhodamine stained latent fingerprint resulted in better DNA profile

Paper

Less amount of DNA but sufficient for DNA profiling without any inhibitory effects No effect on DNA quality and quantity No effect on DNA quality and quantity Degradation and contamination of DNA Degradation and contamination of DNA Acceptable decline of DNA quality and quantity Less efficient genetic analysis Observable extent of DNA degradation

Plastic

Reference Van Hoofstat et al. (1999) Raymond et al. (2008)

Schulz et al. (2004) Bhoelai et al. (2011)

Alem et al. (2017) Khuu et al. (2018)

potential source of DNA for identification purposes in Forensic Sciences (Junge et al. 2002). Urine may be submitted as forensic evidence in violent crimes, hanging, and illicit drug screening tests (Ng et al. 2018). The DNA is contained in epithelial cells of human urine, such as renal tubular, squamous cells, transitional urothelial, leukocytes epithelial cells (renal tubular, squamous cells and transitional urothelial), and malignant cells. There are a lot of factors on which the quantity of extractable DNA depends such as the extraction procedure, storage condition, gender, bacterial contamination, and release of nucleases from cells. As the concentration of DNA in urine is low and the instability of DNA in urine preservation, personal identification using urine samples becomes difficult. Healthy individuals, especially males, contain very less nucleated cells and also shelter bacteria that can act as inhibitor during amplification of DNA. The major urine component, i.e., urea, also acts as an inhibitor and can affect the yield of DNA (Aoki et al. 2017). The yield of DNA is dependent on many factors like the gender of the urine sample, temperature and storage conditions, quality, quantity, and age of the sample.

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Generally, yield is greater in females than males and in fresh urine sample rather than stored urine sample (Aoki et al. 2017; Ng et al. 2018). In cases of urine stains, sample should always be collected from the largest stains that are available. The collection and storage of samples should be properly done in order to avoid degradation so that maximum DNA could be extracted (Yokota et al.1998).

Fecal Matter Fecal matter identification is helpful in providing relevant clue during a criminal investigation, and the individual peculiarities of a fecal sample can be adequately determined by DNA analysis. Fecal as a forensic evidence can be found in many cases where crimes with cruelty are done like sexual assault, sodomy, vandalism, and burglary during which the executioner defecated at the scene of crime. In cases related to animals/wildlife species, genetic evidence from animals, plants, bacteria, and viruses has been used in criminal investigations as forensic tool for identification and individualization purpose (Forgacs et al. 2019). Feces are a type of waste matter formed in the intestines during the last phase of digestion as a direct result of food. Its composition includes a complex mixture of undigested foodstuffs, intestinal bacteria, intestinal epithelial cells, electrolytes, bile pigments, soluble and insoluble gastrointestinal tract products, mucus, and water. Due to low number of cells and co-extraction of various unwanted substances (due to presence of several PCR inhibitors from digestive system, soil, and foodstuffs), DNA analysis from fecal matter is a challenging aspect. Although the quality and quantity of DNA from fecal matter is comparatively lower than traditional sources of DNA (such as blood, semen, saliva, etc.), few studies have suggested fecal samples as a crucial and valid source of genetic material by comparing the results from the same individuals to high-quality DNA samples (Forgacs et al. 2019). So, DNA analysis from fecal matter can be highly important in criminal investigation. After three and half decades, due to technologocal advancements, forensic DNA technology has become a potential tool in the court of law, resulted into increasing the rate of conviction. Nowadays, reliable and sensitive techniques for DNA isolation from a variety of biological samples have been developed which attained through gradual technological advancement which shown in Fig. 1.

Fig. 1 Timeline of DNA extraction protocols

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Collection, Preservation, and Transportation of Biological Evidences Hirak Ranjan Dash and Kamayani Vajpayee

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biological Evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA Content in Biological Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Collection of Biological Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methodology for the Collection and Preservation of Biological Evidence . . . . . . . . . . . . . . . . . . Collection, Preservation, Transportation, and Storage Procedures for Commonly Encountered Biological Evidences at the Crime Scene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Liquid Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Liquid Blood and Wet Bloodstains from Crime Scene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dried Blood Stains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Semen and Seminal Stains and Evidences from Sexual Assault Cases . . . . . . . . . . . . . . . . . . . . . Soft Tissues and Organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Teeth and Bone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Touch DNA Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chain of Custody of Forensic Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

DNA fingerprinting technique is considered to be one of the most irrefutable evidence in the criminal justice system. To obtain a proper result to be produced in the court of law, proper collection, preservation, and transportation of H. R. Dash (*) School of Forensic Science, National Forensic Sciences University, Delhi Campus, New Delhi, India K. Vajpayee DNA Fingerprinting Unit, Forensic Science Laboratory, Bhopal, Madhya Pradesh, India School of Arts and Sciences, Ahmedabad University, Ahmedabad, India Biological and Life Sciences, School of Arts and Sciences, Ahmedabad University, Ahmedabad, Gujarat, India © Springer Nature Singapore Pte Ltd. 2022 H. R. Dash et al. (eds.), Handbook of DNA Profiling, https://doi.org/10.1007/978-981-16-4318-7_3

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biological evidences play an imperative role. To conduct a DNA test, the biological samples can be divided into two types, the questioned exhibits and the reference exhibits. The results obtained from both the exhibits are analyzed and compared to draw a conclusion. Questioned sample may be obtained from varied sources such as bone, teeth, hair, vaginal swab, clothing, knife, or any other article suspected to contain any biological material. However, the reference samples are always the peripheral blood or the buccal swab of the suspected individual or the survivor. In this regard, the present chapter describes the various nature of the biological exhibits along with their proper collection, preservation, and transportation to the laboratory to obtain a proper DNA profiling result. Keywords

Biological exhibits · DNA profiling · Collection · Preservation · Transportation

Introduction In the past few decades, DNA profiling technique has gained a huge attention among the investigators in solving criminal and civil cases of varied nature such as paternity dispute, identification of mutilated bodies, sexual assault, and murder. Courts have appraised physical witnesses such as DNA technology to be one of the most irrefutable evidences in the criminal justice system. DNA profiling-based physical evidences take advantage of trace DNA technologies and link between suspect/ victim to a crime independently and objectively (Lee and Ladd 2001). Over the years, the technology has undergone a huge transformation in terms of its sensitivity and discrimination power. After the invention of STR-based PCR amplification technique, biological evidences that were not useful in earlier days has also become of huge importance. Further advancement of this technique also allows the amplification and detection of DNA fragments in previously unsuitable evidences such as samples containing PCR inhibitors and samples with degraded DNA. Over the time, the nature of collection, preservation, and transportation of biological evidences has been modified as per the technological advancements. Generating a quality DNA profile is highly dependent on the proper collection, preservation, and transportation of biological evidences. DNA fingerprinting technique relies on the analysis of DNA found inside every biological cell. Such biological samples are perishable in nature and need appropriate storage conditions with the use of suitable preservatives. Every forensic technology is different and we should not assume that the same biological samples should be stored in the same preservative for different analyses. For example, blood samples are examined for the presence of alcohol or other intoxicants by toxicological analyses. In such instances, 1–5% sodium or potassium uoride is used as a preservative (DinisOliveira et al. 2016). Whereas, for DNA analysis, liquid blood sample should be collected in EDTA.

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Crime scenes contain evidences which are ad rem to any investigation. Investigators recon mostly on the samples that have been collected from the crime scene to use them as a proof or evidence in a trial. These evidences play an important role either in exonerating a person or to prove him guilty (incrimination) in front of jury. Evidences, on the basis of their nature, can be classified broadly into various categories, viz., physical evidence (glass, soil, paint, fiber, etc.) and biological evidence (blood, semen, saliva, etc.). Among all, biological evidence has gained much of the attention in the recent years. The advent of new and sensitive technologies has aided in the processing and analysis of these trace evidences. Earlier, the biological evidences especially blood was used for blood typing to discriminate the suspect and to establish the link between the crime, victim, and criminal. Later with the invention of the polymerase chain reaction (PCR) technology, the use of short tandem repeat (STR) markers, the genetic analysis of the biological evidences became possible and has revolutionized the field of forensic genetics. Therefore, the technology initially was applied to the cases where abundant and a good quality of biological evidences could be recovered such as in the case of sexual assault, murder, and homicide. However, the field of genetics developed further and allowed the invention of more sensitive and reliable technologies which could handle the analysis of trace evidences (sample) having negotiated quality such as cold, degraded, and contaminated samples. The basic principle behind forensic genetics relies on a fact that every biological substance (cell) contains genetic material and that it can be isolated to generate a profile identical to an individual (individualization). Thus, this very concept allowed investigators to collect all the possible biological exhibits from the scene of crime. These include a wide range of biological samples like blood, semen, saliva, bone, teeth, or even the samples which may have the possibility of containing touch DNA like clothing (Hess and Haas 2017). The technological advancements in the field of forensic genetics or the DNA profiling methodology has opened the field for the array of crime scenes for which the DNA profiling can now be performed such as theft, burglaries, motor vehicle crimes, terrorism, etc. Investigators are now collecting and submitting wide array of samples for their DNA analysis. While dealing with trace evidences and sensitive instruments, it has now become more important to formulate and carefully follow the protocols regarding collection, preservation, and storage of the exhibits. The cautious use of collection and preservation methods will protect the samples from further degradation occurring due to environmental, physical, and chemical factors, cross contamination. There are specific protocols and procedures laid down by several accrediting agencies that are being recommended to professionals and crime scene investigators. A thorough training of officers on importance of handling different types of evidences, proper collection, and preservation procedures should be mandatory. Attention should also be made on identifying the different types of evidences, their evidentiary value, and proper packaging and transportation procedures. Thus, realizing the importance of proper collection, preservation, and transportation of the exhibits encountered at crime scene, this chapter aims at laying out the standardized guidelines expected to be used by investigators at the scene of crime.

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Biological Evidence Forensic science deals with the application of well-established scientific techniques on the biological exhibits to draw a conclusion which is accepted in the criminal justice system. As it is a science of comparison, the biological exhibits can be divided into two types, i.e., the questioned exhibits and the reference exhibits. Firstly, the examination is conducted on the questioned sample and the results obtained from the questioned sample are compared with the reference sample. Based on the observation and matching the results between the questioned and the reference samples, conclusion is drawn in a case. In a simple case of paternity dispute, the mother is considered to be known, child is questioned, and the father is the accused/alleged. Similarly in a case of identification, the unknown/questioned sample may be of either bone, teeth, fetus, hair, or any other biological object. Here, the reference samples can be considered of the biological samples obtained from the putative father, mother, child, wife, or any other patrilineal/matrilineal relatives. The questioned sample in case of a murder may involve the clothing, blood stained soil, any murder weapon of any other sample that presumably contains biological uids of either deceased or the accused which has the potential to link the deceased, accused, crime scene, and the murder weapon. Additionally, the questioned samples in a case of sexual assault involve any article collected from the sexual assault survivor’s body or its belongings. Here the Locard’s exchange principle comes into play, where the body uid of the culprit and the survivor gets exchanged during the course of sexual assault (Sammons 2014). Reference sample for forensic DNA analysis can be defined as a sample with high quantity and quality DNA and is collected in front of witnesses to be accepted in the court of law (“Glossary for Crime Scene Investigation: Guides for Law Enforcement” 2021). Hence, irrespective of all type of cases, the widely used reference samples include the peripheral blood collected either in a K3EDTA vial or in a FTA ® card. Alternatively, buccal swab can also be collected as an alternative to the liquid blood sample as a useful noninvasive sample (Ghatak et al. 2013). However, there exists certain limitations; at many instances, the sample donor is a habitual tobacco chewer. In such cases, buccal swab samples may not yield an optimum DNA result as tobacco acts as a potential PCR inhibitor (Adamowicz et al. 2014); hence, peripheral blood samples should be treated as a reference sample.

DNA Content in Biological Samples Various types of biological samples are being collected from the crime scene and are sent to forensic laboratory for their analysis. The DNA content in these samples varies significantly (Andréasson et al. 2006). There are samples like liquid blood where a sufficient amount of DNA can be extracted, and on the other hand, there are

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Table 1 Summary of DNA content in common biological evidences Type of biological material Blood

DNA content 30–40 μg/mL

Bone Hair Buccal swab Teeth

100 ng/gram 0.4 ng per strand 18 ng/μl – 433 ng/μl 247.807
23.8 ng/μl

References (“How much DNA and RNA can be expected from human blood cells? – QIAGEN” 2021) (Iwamura et al. 2004) (Heywood et al. 2003) (Livy et al. 2011) (Rubio et al. 2018)

samples like hair from which a very small amount of DNA can be recovered. Table 1 summarizes the amount of DNA that can be recovered from various biological samples. While analyzing the crime scene, an investigator must have a prior knowledge about the genetic content in the biological materials so that materials with higher DNA content can carefully be collected for better interpretation and conclusion. Further this would also help in preserving the only samples collected from the crime scene with little DNA content. • Blood: Blood is an excellent source of DNA. Blood contains erythrocytes, leucocytes, platelets, and plasma. Among all, leucocytes are the sources of genetic material since mature erythrocytes lack nuclei. Approximately 30–40 μg of DNA is found in per milli liters of blood (“How much DNA and RNA can be expected from human blood cells? – QIAGEN” 2021). • Bone: Bone is the choice as the DNA source only in case of decomposed body. Bones can also be taken for consideration in case of ancient DNA analysis. DNA can be isolated from the demineralized long bones since calcium is a potential PCR inhibitor. Usually bones may contain 100 ng of DNA per gram of bone (Iwamura et al. 2004). • Teeth: Similar to bones, teeth also act as a DNA source in case of decomposed, burnt, and charred body. The tooth pulp is known to be the richest source of DNA. Studies have shown that more DNA is retrieved from multi-rooted teeth than single-rooted teeth. Usually teeth may contain 247.807
23.8 ng of DNA per micro liters of demineralized tooth (Rubio et al. 2018). • Hair: Shed hairs are mostly found in crime scenes although plucked hair samples are also collected sometimes as reference material and/or in the sexual assault cases. In hair, the sufficient quantity of nuclear DNA is only found in the root sheath whereas mitochondrial DNA is abundantly found in hair shaft. Approximately 0.4 ng of nuclear DNA can be isolated from a single hair. It is difficult to standardize the amount of DNA that can be recovered from the hair since every hair from a single source differ in their DNA content (Heywood et al. 2003).

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• Buccal Swab: Buccal swabs are alternate to invasive sample collection method. When blood could not be taken as reference sample, buccal swabs are considered as second option. Buccal swabs are prone to contamination, and thus, it is recommended to use mouth wash before sample collection. Buccal swabs contain epithelial cells and saliva as the source of DNA. Researchers have estimated that a buccal swab may contain 18 ng/μl – 433 ng/μl of DNA (Livy et al. 2011).

Collection of Biological Samples Once the investigating officer reaches the scene of crime, he must first secure the crime scene so as to maintain its integrity (Pepper 2010). He must observe the scene of crime with utmost care and look for the best method of examining it. Moreover, a brief observation is required to locate all the possible evidences. The expert must try to collect fragile evidences in first place so that degradation due to environmental factors could be avoided. It is the primary duty of the crime scene investigator to ensure that all the materials required for collection, preservation of the evidences from the crime scene is available with him. Table 2 enlists the materials that are required for the proper handling of the evidences at the crime scene. Although there might be the cases where some specialized materials, instruments or reagents would be required apart from those listed in the table. Depending upon the type of crime committed (homicide, sexual assault, murder, theft, motor vehicle accidents, etc.), a range of biological evidences can be found in a crime scene. These may include blood, semen, saliva, sweat, hair, bone, urine, etc. Before collection, it is necessary to carefully observe and recognize all the evidences present at the scene of crime. There are samples like blood, bone, tissue which are visible to the naked eye and can thus be detected easily. Detection of the samples like semen, saliva, fingerprints, etc. can be difficult owing to their color and quantity. Such samples are made visible by using either alternative light sources or chemicals. For example, Polilight is used to detect seminal and blood stains whereas Luminol can be used to detect trace amounts of blood. The areas with a doubt of being touched can be analyzed for the presence of latent fingerprints by using enhancement powders (Miranda et al. 2014; Sterzik et al. 2016; van Oorschot et al. 2010). Apart from collection, investigating officer must take some precautionary measures to ensure proper preservation of the exhibits. The preservation methods are purely dependent upon the type of sample to be preserved. Table 3 outlines some important measures that are to be taken care of while collecting, preserving, and transporting the biological evidences. As regards to the storage conditions, every sample has its own optimum temperature under which the sample integrity is maintained. Usually short-term and long-term storage conditions vary for the samples. Tables 4 and 5 defines the short-term and the long-term storage conditions for the biological evidences, respectively.

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Table 2 List of materials required at the crime scene (Modified from Mozayani and Parish-Fisher 2018) Items Protective personal equipment (PPE) Gloves (latex, nitrile, cotton) Facemasks Hair cap Disposable overshoes Safety goggles Sterile sealed swabs Sterile tweezers Plastic tubes Sterile disposable plastic pipettes Disposable sterile scalpels or razor blades Biological hazard bags and sharp bins Polythene bags Brown paper sacks Cardboard boxes ( at pack) Scissors Adhesive tape Stapler and pins Evidence tape and tags Scene of crime barrier tape Identification labels Pens and markers Ruler and tape measure Plain paper (A4 and A3) Thermometer Magnifying glass Torch Fingerprint brushes and fingerprint powders Lifting tape with acetate sheet Alternative light source (ALS) Digital and video camera Clipboard Crime scene investigation forms and necessary laboratory documents Reagents Deionized/ultrapure water Ethanol Bleach Reagents for presumptive testing of biological stains Fingerprint enhancement reagents

Methodology for the Collection and Preservation of Biological Evidence An investigator encounters a wide range of biological evidences at the crime scene. For their better recovery, it is essential to follow standard procedures of collection. There are several methods like double swabbing method, cutting method, etc. that allow the collection of biological evidences while preserving their integrity. These methods can be used at the scene of crime as well as in the laboratory.

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Table 3 Measures to be taken while collecting, preserving, and transporting the biological evidences While attending the scene of crime, investigator must ensure that he/she is carrying all the necessary items required for collection and preservation of the exhibits. Investigating officers along with the other officers dealing with the crime scene must wear proper safety garments which include gloves, shoe cover, hair cap, mask, and safety suit. To avoid any contamination and to maintain the integrity of the evidences, touching the anything bare hands must be avoided. A proper documentation of the crime scene along with the exhibits before collection should be prepared that may include notes, sketching, photography, and videography. Attention must be paid to avoid any kind of cross contamination among the biological evidences. Further individual packaging of the exhibits should be practiced. Liquid samples like blood must be dried in air before packaging. Similarly, swabs and slides should be allowed to dry before packaging. In the case of collection of the stain, a controlled sample must also be collected and sent to laboratory. This controlled sample has to be collected from unstained portion. Paper bags and card board boxes should be used to pack biological stains. Plastic bags have the tendency to retain moisture and therefore must be avoided. After collection and packaging, the package needs to be sealed, labelled with brief description about the type of evidence, date of collection, location, name, etc. the package may also include appropriate storage conditions. Investigator must prepare a checklist of all the items collected from the scene of crime. After collection, all the evidences must be sent promptly to the laboratory for analysis. Care must be taken while transporting the exhibits to the laboratory. Samples liable to temperature and light must be kept in ice box so as to avoid further degradation or damage. Chain of custody is important; hence, a proper documentation regarding chain of custody must be prepared before transporting the exhibits to the laboratory.

Scraping Method The scrapping method is used to recover dry materials on porous surface. The method is followed under controlled environment so as to avoid any kind of contamination. Following steps are to be taken while working with this method: • • • • • • •

Locate the stain of interest. Check if the stain is dry before performing further steps. Carefully scrape the stain from the object on to a clean, sterile paper piece. Avoid using bare hands; always wear hand gloves before recovering the stain. Secure the scrapings and place the paper in an envelope. Seal and label the envelope. Store as per physiological conditions (Mozayani and Parish-Fisher 2018).

Double Swabbing Method Dry samples on nonporous surface (glass, metal, etc.) are collected using double swabbing method (Pang and Cheung 2007). This method can also be used to collect evidences for touch DNA (Pang and Cheung 2007). The steps are as follows:

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Table 4 Recommended physiological conditions for short-term storage of biological evidences. (Modified from Ballou et al. 2013)

Type of evidence

Liquid blood

Urine

Frozen

Refrigerated

Never

Best

Bone

Best

Vaginal Slide/Smear Buccal Swab

• • • • • •

hrs

Less than 24 hrs Best

Acceptable

Less than 24 Best

Acceptable

Acceptable

Hair Swab

Room Temperature

Less than 24

Dry Stains Wet Stains

Temperature Controlled

Best (wet)

hrs Acceptable

Acceptable

Best

Acceptable

Best (dried)

Best

Less than 24 Best

hrs

Locate the stain on a surface. Moisten the swab with normal saline water. Wet the dry stain using the moisten swab which will soften and rehydrate the cells. Using another moist swab recover the stain from the surface. Allow the swab to dry in air and place it in an envelope. Seal, label, and store the envelope (Mozayani and Parish-Fisher 2018).

Cutting Method When stains are found on large objects like sofa, carpet, cutting method is used to recover them. However, if stains are found on small objects like clothing, cigarette butts, experts use this method for sample collection. The steps taken are as follows:

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Table 5 Recommended physiological conditions for long-term storage of biological evidences. (Modified from Ballou et al. 2013)

Type of evidence

Liquid blood Urine

Frozen

Never

Refrigerated

Best Best Best (Liquid)

Acceptable

Best

Hair

Best

Swab

Best (Dry)

Slide/Smear Buccal Swab

• • • • •

Acceptable

Bone

Vaginal

Room Temperature

Best

Dry Stains DNA Extracts

Temperature Controlled

Acceptable

Best

Best

Locate the stain on a surface. Allow the stain to dry if wet. Cut around the stain with care. Pick the cut pieces and place them in an envelope. Seal, label, and store the envelope (Mozayani and Parish-Fisher 2018).

Tape Lifting Method Dry stains on nonabsorbent materials can be recovered using this method. Tape lifting method is majorly used to lift fingerprints and the areas suspected to be touched for the collection of touch DNA. The steps taken are as follows: • Locate the stain on a surface. • Place the adhesive tape over stain and press gently. • Carefully lift the tape off the object and secure the adhesive side with acetate paper. • Place the tape in an envelope. • Seal, label, and store the envelope (Mozayani and Parish-Fisher 2018).

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Picking Method Picking method is used to collect solid items found at scene of crime like hair, bone, etc. The steps followed are as follows: • • • •

Locate the evidence. With the help of sterile forceps/tweezers, pick the solid biological materials. Transfer the material into an envelope or box. Seal, label, and store the envelope/box (Mozayani and Parish-Fisher 2018).

Collection, Preservation, Transportation, and Storage Procedures for Commonly Encountered Biological Evidences at the Crime Scene Liquid Blood For DNA typing technology, liquid blood is the most suitable evidence. Liquid blood is usually collected as reference sample from an individual may be victim or suspect. It is also at times encountered by the investigating officer at the crime scene (Dash et al. 2020). Liquid blood is collected by an experienced medical practitioner only after taking a written consent from the person. All the medical concerns have to be documented along with the sample such as organ or blood transfusion, etc. Approximately 2 ml of blood sample in duplicates is collected in EDTA vial. Blood sample can also be taken in a vacutainers containing 5 mM sodium citrate, heparin as anticoagulant. Blood from the deceased body can also be collected in a similar way. Moreover, it must be collected from areas like heart or major internal blood vessels and within 24 h of death. After collection, the vial should be labelled well including the name of the person, name of the collector, exhibit number, date, location, and time. The package must be sealed properly and should be stored at low temperatures before transporting it to the laboratory. The liquid blood can also be collected on Whatman FTA ® cards by dropping approximately 125 μl of blood per inch inside the marked area. The card should be labelled well and dried in air before packaging in individual envelopes. These cards can be transported to laboratories at room temperature. The blood samples are stored at low temperatures but are not freezed.

Liquid Blood and Wet Bloodstains from Crime Scene Liquid blood can be found in a crime scene along with the blood clots. Blood from the blood pool can be collected through a syringe and transferred either to EDTA vial or vacutainers containing 5 mM sodium citrate, heparin as anticoagulant. In a similar way, the blood clot can be collected using sterile tweezers or spatula.

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Blood serum and the blood around the clot can be collected by soaking a sterile cotton swab. The exhibits can then be labelled with name of the collector, exhibit number, date, location, and time, sealed, and packaged. The samples are refrigerated (not freezed) and transported to the laboratory at the earliest. Sometimes liquid blood stains can be found on the surface of the objects. In such cases, the stains can be cleaned using a sterile cotton swab. The swab is allowed to dry in air followed by packaging in an envelope with proper markings.

Dried Blood Stains Dried blood stains can be found either on large immovable objects or on removable objects in a crime scene. If the blood stain is on immovable object, the blood stain pattern must be documented before collection. Methods of collection like tape lifting and scrapping can be used to collect the stain. Moreover, the blood stain can also be collected using double swabbing method where a moist cotton swab is used to wipe out the stain. Prior to packaging, the swabs must be allowed to dry in air so as to avoid any fungal growth and degradation. In both the cases, the sample must be packed separately to avoid cross contamination. Also the source could be different for the different stains; thus, packaging of stains in separate envelopes becomes important. On the other hand, if the blood stain is found on removable objects like cloths, knife, etc., the whole object can be collected, packed, labelled, and sent to the laboratory. In addition, if the blood stain is found on the object which can be cut, then cutting method of evidence collection can be utilized to recover the stain. These objects can be transported to the laboratory at normal room temperature (Dash et al. 2020).

Semen and Seminal Stains and Evidences from Sexual Assault Cases In sexual assault cases, semen, seminal stains on objects like bedsheet, pillows, clothing, etc. can easily be found. Before recovering the seminal stains, it is always practiced to document the stain either by sketching, photography, or videography. If the encountered stain is dry, it can be collected using scrapping method, cutting method, or double swabbing method. Alternatively, if the stain is wet, it must be allowed to dry in air before collection so as to avoid any contamination. The swabs are to be packed in an envelope and stored in lower temperatures. Liquid semen sample can be transferred to a tube either by using sterile disposable pipette or syringe. In both the cases, there must be a proper labelling and sealing. In sexual assault cases, medical examination of the victim remains priority and must be carried out at the earliest. Since samples like seminal uid starts

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degrading faster in female body due to the presence of enzymes and acidic pH, other samples like buccal swab, anal swab, vaginal swab, etc. must be collected based upon the case. The slides, swabs prepared must be dried before packaging. The package must be properly labelled with name of the victim, date of collection, name of the medical expert, exhibit number, etc. Similarly, clothing of victim and the suspect should also be recovered, dried, and properly packed with all the relevant details. All these samples collected are sent to the laboratory in normal temperature. Samples like vaginal slides, seminal slides are to be stored in lower temperatures but are not to be freezed (Dash et al. 2020).

Soft Tissues and Organs Soft organs or tissues must be collected with the help sterile forceps. They should be packed in a clean, dry, sterile, and airtight glass jar. The tissues/organs should be preserved in normal saline solution. Additionally, antibiotics could be added to the solution to prevent the growth of microbes. The tissues should be packed separately and labelled well. The jars should be sealed and sent to laboratory in ice boxes. In case where the organs are collected from a deceased body having advanced decomposition, a deep muscle along with few other organs should be collected in said manner and sent to laboratory at the earliest. It is always recommended to preserve heart or brain for DNA analysis. Generally, liver and kidneys are not recovered in such cases. It is important to note that these tissue/organ samples are never preserved in formalin or paraffin wax (Dash et al. 2020).

Teeth and Bone Teeth and bones are often collected from the deceased skeletonized body. It is the duty of the investigator to the source of the sample from any skilled medical practitioner prior to packaging and transporting to the laboratory. If possible, intact and rooted molars are recommended for collection. In case of bone, long bones especially femur should be collected for DNA analysis. The samples are picked using sterile tweezers and are wrapped cautiously with paper. The package must contain all the information like name of the collector, exhibit number, date, location, and time. The package is stored at room temperature and is sent to the laboratory for further analysis (Dash et al. 2020).

Hair Nuclear as well as mitochondrial DNA can be isolated from the hair samples. For nuclear DNA analysis, hair needs to have root sheath, whereas mitochondrial DNA

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can be isolated from hair shaft samples. If hairs are found in the crime scene, it should be picked up using sterile tweezers and must be packed in a ziplock polybag or envelope. The package needs to be transported to laboratory and stored in room temperature. In case of reference sample collection, approximately 20–25 hair samples have to be submitted since it may require for additional microscopic examination (Dash et al. 2020).

Touch DNA Samples With the advent of DNA profiling technique from RFLP method to PCR-based STR analysis, the amount of DNA required for the examination is getting decreased day by day. Most of the currently available commercial STR kits require 05–1.0 ng of genomic DNA to generate a complete DNA profile (Cisana et al. 2017; Ludeman et al. 2018). Hence, the analysis of transfer of trace DNA from an object or a person due to mere physical contact has been explored widely recently. Touch DNA samples do not attribute to a particular body uid, rather, the source of touch DNA may include the shed corneocytes, endogenous/transferred nucleated epithelial cells, fragmented cells and nuclei, and cell-free DNA (Tang et al. 2020). Various factors such as physical activity of the donor, sex, age, substrate, temperature, and humidity contribute to the amount of touch DNA contributed by a donor. In most of the crime scene evidences, wet/dry swabbing technique is commonly applied. However, a study revealed the usefulness of scraping or tape lift method during collection of touch DNA samples (Williamson 2012). Before collection of a trace DNA evidence from the crime scene, the primary aim should be to locate the possible target surfaces harboring the trace DNA evidence. In suspected homicide cases, in addition to the deceased’s body uid, touch DNA can be explored from surface of murder weapons such as revolver, knife, or pistol. In sexual assault cases, touch DNA sample of the perpetrator can be targeted from the skin or clothing of the survivor (Sowmyya 2016). Looking at the crime scene, the investigator and the forensic expert can ensure the presence of any trace DNA evidence to be collected for a DNA testing.

Chain of Custody of Forensic Samples Chain of custody is the most important step of evidence documentation to assure the court of law regarding the authenticity of the evidence. Proper documentation of the continuity of possession of evidence, and its movement and location from the point of discovery, recovery, or collection till laboratory examination and admission in the court collectively accounts for the chain of custody of an evidence. In this regard, a record of chain of custody must be maintained and established in the court before assigning it as an exhibit. The lack of which may make the evidence inadmissible in the court and its legitimacy, integrity, and

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related examinations become questionable. The chain of custody document ensures that none other than the authorized person has access to the exhibits. The document should be prepared in a comprehensive manner citing information regarding the evidence collection, people handling the evidence, period of possession of the evidence, safekeeping conditions, and other information. Thus, maintaining chain of custody of a DNA evidence becomes utmost important in addition to the other evidences. As the biological samples require proper preservatives and storage conditions, hence, the appropriate storage conditions need to be mentioned in the chain of custody form for any biological exhibit.

References Adamowicz M, Stasulli D, Sobestanovich E, Bille T (2014) Evaluation of methods to improve the extraction and recovery of DNA from cotton swabs for forensic analysis. PLoS One 9(12): e116351. https://doi.org/10.1371/journal.pone.0116351 Andréasson H, Nilsson M, Budowle B, Lundberg H, Allen M (2006) Nuclear and mitochondrial DNA quantification of various forensic materials. Forensic Sci Int 164(1):56–64. https://doi.org/ 10.1016/j.forsciint.2005.11.024 Ballou S, Stolorow M, Taylor M, Bamberger P, Brown L, Brown R et al (2013) The biological evidence preservation handbook: best practices for evidence handlers; technical working group on biological evidence preservation. https://doi.org/10.6028/nist.ir.7928 Cisana S, Cerri N, Bosetti A, Verzeletti A, Cortellini V (2017) PowerPlex ® fusion 6C system: evaluation study for analysis of casework and database samples. Croat Med J 58:26–33. https:// doi.org/10.3325/cmj.2017.58.26 Dash HR, Shrivastava P, Das S (2020) Collection, transportation, and preservation of biological evidences for DNA analysis. In: Principles and practices of DNA analysis: a laboratory manual for forensic DNA typing. Springer Protocols Handbooks, Humana/New York. https://doi.org/ 10.1007/978-1-0716-0274-4_3 Dinis-Oliveira RJ, Vieira DN, Magalhaes T (2016) Guidelines for collection of biological samples for clinical and forensic toxicological analysis. Forensic Sci Res 1:4251 Ghatak S, Muthukumaran R, Nachimuthu S (2013) A simple method of genomic DNA extraction from human samples for PCR-RFLP analysis. J Biomol Tech jbt.13-2404-001. https://doi.org/ 10.7171/jbt.13-2404-001 Glossary for Crime Scene Investigation: guides for law enforcement. National Institute of Justice. (2021). Retrieved 13 April 2021, from https://nij.ojp.gov/topics/articles/glossary-crime-sceneinvestigation-guides-law-enforcement Hess S, Haas C (2017) Recovery of trace DNA on clothing: a comparison of Mini-tape lifting and three other forensic evidence collection techniques. J Forensic Sci 62(1):187–191. https://doi. org/10.1111/1556-4029.13246 Heywood D, Skinner R, Cornwell P (2003) Analysis of DNA in hair fibers. J Cosmet Sci 54:21–27 How much DNA and RNA can be expected from human blood cells? – QIAGEN. Qiagen.com. (2021). Retrieved 13 April 2021, from https://www.qiagen.com/be/resources/faq? id¼01070e31-3a4c-42d7-870c-e8005285889f&lang¼en&Print¼1#:~:text¼Blood%20of%20a %20healthy%20individual,10x%20more%20DNA%20than%20RNA Iwamura E, Soares-Vieira J, Muñoz D (2004) Human identification and analysis of DNA in bones. Revista Do Hospital Das Clínicas 59(6):383–388. https://doi.org/10.1590/ s0041-87812004000600012 Lee HC, Ladd C (2001) Preservation and collection of biological evidence. Croat Med J 42: 225–228

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Livy A, Lye S, Jagdish C, Hanis N, Sharmila V, Ler L, Pramod B (2011) Evaluation of quality of DNA extracted from buccal swabs for microarray based genotyping. Indian J Clin Biochem 27 (1):28–33. https://doi.org/10.1007/s12291-011-0154-y Ludeman MJ, Zhong C, Mulero JJ, Lagacé RE, Hennessy LK, Short ML, Wang DY (2018) Developmental validation of GlobalFiler™ PCR amplification kit: a 6-dye multiplex assay designed for amplification of casework samples. Int J Legal Med 132:1555–1573. https://doi. org/10.1007/s00414-018-1817-5 Miranda G, Prado F, Delwing F, Daruge E (2014) Analysis of the uorescence of body uids on different surfaces and times. Sci Justice 54(6):427–431. https://doi.org/10.1016/j.scijus.2014. 10.002 Mozayani A, Parish-Fisher C (2018) Forensic evidence management, 1st edn. CRC Press, pp 29–41 Pang B, Cheung B (2007) Double swab technique for collecting touched evidence. Legal Med 9(4): 181–184. https://doi.org/10.1016/j.legalmed.2006.12.003 Pepper IK (2010) Crime scene investigation: methods and procedures, 2nd edn. Open University Press Rubio L, Sioli J, Gaitán M, Martin-de-las-Heras S (2018) Dental color measurement to predict DNA concentration in incinerated teeth for human identification. PLoS One 13(4):e0196305. https:// doi.org/10.1371/journal.pone.0196305 Sammons J (2014) Digital forensics. Introd Inf Sec:275–302. https://doi.org/10.1016/b978-159749-969-9.00013-4 Sowmyya T (2016) Touch DNA: an investigative tool in forensic science. Int J Curr Res 8: 26093–26097 Sterzik V, Panzer S, Apfelbacher M, Bohnert M (2016) Searching for biological traces on different materials using a forensic light source and infrared photography. Int J Legal Med 130(3):599– 605. https://doi.org/10.1007/s00414-015-1283-2 Tang J, Ostrander J, Wickenheiser R, Hall A (2020) Touch DNA in forensic science: the use of laboratory-created eccrine fingerprints to quantify DNA loss. Forensic Sci Int 2:1–16. https:// doi.org/10.1016/j.fsisyn.2019.10.004 van Oorschot R, Ballantyne K, Mitchell R (2010) Forensic trace DNA: a review. Investig Genet 1 (1):14. https://doi.org/10.1186/2041-2223-1-14 Williamson AL (2012) Touch DNA: forensic collection and application to investigations. J Assoc Crime Scene Reconstr 18(1):1–5

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Forensic DNA: From New Approaches for the Bio-stain Identification to the Evaluation of the Genetics Evidence in Courtroom E. D’Orio, P. Montagna, M. Mangione, and G. Francione

Contents Introduction About the Bio-Stain Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . New Approaches for the Bio-Stain Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Analysis Techniques in Forensic Genetics and New Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic Traces in a Fire Scene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic Traces in a Fire Scenario . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Biological Traces Evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . From Scientific Proof to “Beyond Reasonable Doubt” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA and Fire . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Final Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The New Popperian Epistemology of the Criminal Process: Strong Scientific Evidence and Reduction of the Clues to Conjectures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Revolution of the Justice Method: Popper’s Way for Strong Proofs . . . . . . . . . . . . . . . . . . The Neutrality of the Researcher and the Rigorous Acquisition of the Proofs . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

86 89 93 98 99 101 102 105 111 112 113 115 116

Abstract

The correct application of DNA analysis in forensic field is the final objective of every forensic biologist who carries out their work to support justice by providing technical-scientific information. In recent years, the advancement of technology significantly contributed to achieve higher levels of accuracy and precision in analysis. This chapter will examine the various aspects with regard to new areas of development of forensic biology, covering both new approaches to improve the pre-analytical investigations related to the search and identification of traces and new procedure for genotyping. Lastly, the topic of the correct methodology for presenting genetic evidence to the judge will be investigated. The aim of this chapter is to allow scientific data to be appreciated in a balanced and accurate way E. D’Orio (*) · P. Montagna · M. Mangione · G. Francione Bio Forensics Research Center, Angri, Italy e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 H. R. Dash et al. (eds.), Handbook of DNA Profiling, https://doi.org/10.1007/978-981-16-4318-7_58

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by judges, preventing any potential form of error caused by excessive or limiting evaluations relating to genetic data offered by forensic scientists. Keywords

Forensic DNA · New approaches · Biological stain identification · Forensic biotechnology · DNA in Court

Introduction About the Bio-Stain Identification In the field of Forensic Science, the identification of biological traces is a critical point. Forensic genetics technologies allow scientists to determine the identity of the person who released a trace on a place or object of investigative interest. Although forensic genetics techniques have extremely high levels of accuracy, techniques for the detection of latent biological traces do not have such high margins of accuracy. It should be noted that genetic analysis is considerably conditioned by pre-analytics, i.e., those operations that take place before the typical procedures of genetic investigations. Especially in a context where biological traces (Gardner and Krouskup 2018) are hardly visible, the methods of exaltation of the traces are very important; this makes it possible to identify the different traces present on an object (or on a place) with precision and further allows the operators to carry out the sampling with accuracy. If the biological traces are not visible, then there is a strong risk that operators will sample inaccurately – known as random sampling – which can generate the following conditions: 1. A failure to acquire biological trace evidence 2. The acquisition of several distinct biological traces in the same sample (generating a mixed genetic “false”) Figure 1 shows evidence coming from a crime scene with potential biologicalforensic interest. Figure 2 shows a latent biological macro-trace. Figure 3 shows the latent biological trace enhanced with the use of special forensic lights. There are many techniques for the detection of latent biological traces evidence. Among the tools available are as follows: – Forensic lights – Wood’s lamp – Luminol Forensic lights are light sources that emit variable wavelengths of light. They generally emit light in the visible spectrum, between 350 nm and 480 nm. They enhance biological traces that are not visible to the naked eye. However, observation

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Fig. 1 Shows a find coming from a crime scene with potential biological-forensic interest

Fig. 2 Shows a latent biological macro-trace

by the technician can only occur correctly if he or she uses specific visual filters for light (goggles). Several scientific industries produce a variety of forensic lights: some with specific emission, while others with variable emission from the same instrument. Thanks to the particular light emission, these instruments enhance the latent biological traces due to the uorescence of some biological tissues (such as semen, saliva and urine), as well as to the absorbency, which is most typical of blood tissue.

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Fig. 3 Shows the latent biological trace enhanced with the use of special forensic lights

Wood’s lamp (Al Aboud and Gossman 2020)is a light source that emits electromagnetic radiation mainly in the ultraviolet range, to a negligible extent, in the field of visible light. The use of Wood’s lamp, even for long periods of time, is not toxic for humans; in fact, this use involves the emission of ultraviolet radiation called UV-A, which does not generate phenomena of DNA alteration (unlike UV-B and UV-C). Wood’s lamp produces light that is not directly visible to the human eye; however, it can be used to illuminate materials on which ultraviolet radiation induces effects of orescence. In the biological-forensic field, Wood’s lamp is used to search for organic traces that are not visible to the naked eye. Many other applications of Wood’s lamp are possible: from the fight against banknote forgery (documentary forgery) to medical, microbiological, commercial, and paleographic applications. Luminol (Barni et al. 2007) (also known as IUPAC 5-amino-2,3-dihydro-1,4phthalazindione) is a chemical compound often used by the Judicial Police to detect latent blood. Luminol is a liquid compound, which is sprayed onto the surface of interest directly on-site. It is a very versatile substance which, when mixed with an appropriate oxidizing agent, develops blue luminescence. Luminol is particularly useful to operators when looking for traces of “washed” blood. In fact, once it is sprayed on the surface of investigative interest in dark conditions, the luminol generates luminescence. This reaction occurs due to the chemical reaction and, more specifically, to the presence of hydrogen peroxide. In the presence of iron, the reaction is activated and luminescence is developed (for about 40–60 s). Iron is one of the typical components of hemoglobin, a protein present in the blood that functions as an oxygen carrier. However, the luminol reacts in a non-specific way with everything that contains iron, which is why it can generate

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multiple “false negatives.” The most classic of these is the reaction of luminol with bleach. It should be noted that all technologies for the search of latent biological traces are presumptive, never confirmatory. It should also be noted that, unlike what happens in DNA analysis for forensic purposes, the analysis aimed at identifying latent biological traces is still without fixed protocols. Only generic guidelines are presently available. Obviously, this generates a condition of subjectivity in the search for latent biological trace evidence. It is therefore appropriate that a highly specific research activity is carried out, dedicated to the validation of protocols for the use of these technologies and in order to ensure reproducibility and homogeneity of the analysis results.

New Approaches for the Bio-Stain Identification The potential of screening technologies for latent biological trace evidence is strong, but it still requires further validation. The definition of an operational protocol would, in fact, ensure the controlled and controllable performance of the acts of biological trace evidence research at every stage. Currently, partly due to the presence of the guidelines, and partly due to the presence on the market of multiple types of technological tools useful for the research of biological traces, it is not possible to conduct this preliminary phase of genetic analysis in an extremely accurate and standard way. It should also be preliminarily considered that the subject of “biological trace evidence research” has taken a back seat to DNA studies for forensic purposes. Yet, a successful outcome of the trace research phase directly affects the success of the genetic analysis that will result, and as such, it is therefore deserving of greater scientific attention. In addition, there are many publications (Baranowska 2016) and case reports that demonstrate the validity of the application of technologies for the screening of biological traces, as well as how this research, done in a targeted way, can lead to excellent investigative results. Considering the potential application of the technologies for the biological evidence detection in the forensic field, certainly not inferior to those of DNA analysis, there are still active research projects aimed at the validation of standard operating protocols for the development of the “bio-stain identification” phase. A first study was carried out at the University of Copenhagen’s Faculty of Forensic Medicine, Forensic Genetics Section, Crime Unit group; this study has brought to light some very important data upon which increasingly accurate and refined research projects in the field can begin to develop (D’Orio et al. 2018). Firstly, these studies have generated a “reference sample,” thanks to which the ability of the human eye to see biological traces of the size of 50 μl was calculated. To carry out this evaluation, different types of substrates were used, and the biological trace

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was adhered to. The data analysis showed that the human eye is able to reveal only about 15% of all existing biological traces under the standard conditions considered. Figure 4 shows the detection increase (in %) using “the human eye,” an ALS protocol, and a second ALS protocol. This data fully confirms that, for a high-quality forensic investigation, it is essential that operators use specific tools for the identification of biological traces. The study also led to the definition of new parameters that were previously unconsidered, but which, however, must necessarily be taken into account: 1. The color of the substrate – find – on which the forensic operator conducts the search for biological trace evidence 2. Minimum Quantity Detectable (MQD), i.e., the minimum amount of biological trace that can still be detected The study concluded that there is a direct correlation between substrate color and MQD. This is a necessity that forensic practitioners must take into account in two stages: 1. At the moment of research concerning the biological traces on the findings 2. At the moment of presentation of the data in the technical report/in court. In fact, the forensic scientist must describe not only the number of latent biological traces found but also the maximum degree of accuracy that can be reached starting from the examination of that specific type of evidence/result (you choose). All this is necessary to ensure the correctness in the communication of scientific data to judges and lawyers and to avoid that the weight of the scientific data that arises may be overestimated or underestimated.

Fig. 4 Shows the detection increase (in %) using “the human eye,” an ALS protocol, and a second ALS protocol

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In detail, the study also showed that there is a need for at least two different protocols for the use of detection technologies that, when used in synergy, allow forensic biologists/investigators (you choose) to achieve an operational efficiency of more than 95%. Figure 5 shows that the use of a single protocol, in fact, did not allow the efficiency to exceed 65%. A detailed study of the data identifies the cause of this limitation. In particular, textile substrates with dark and medium-dark colors, examined with this particular type of screening protocol, and did not provide positive results. Figure 6 shows the particular textile fibers, used as substrates, from which it was impossible to make a successful identification of biological traces. For these reasons, a different protocol of use of the same instrument used for the search of biological traces has been standardized, and the samples found to be negative at the first examination have been reanalyzed. Figure 7 shows the data related to this synergistic screening. In terms of absolute efficiency, with the synergistic combination of the two experimental protocols, latent biological traces are detected in 19 samples out of 20 (all in triplicate, to confirm the reproducibility of the data), with a relative efficiency of 95%. Data were also collected in mixed latent biological trace contexts. In these cases, the data showed that the relative efficiency is even more accurate. Figure 8 shows the efficiency of technological tools for the detection of latent biological traces composed of two different biological tissues in equal concentrations. Figure 9 shows the efficiency of technological tools for the detection of latent biological traces composed of two different biological tissues in different concentrations.

Fig. 5 Shows that the use of a single protocol, in fact, did not allow the efficiency to exceed 65%

92 Fig. 6 Shows the particular textile fibers, used as substrates, from which it was impossible to make a successful identification of biological traces

Fig. 7 Shows the data related to this synergistic screening

Fig. 8 Shows the efficiency of technological tools for the detection of latent biological traces composed of two different biological tissues in equal concentrations

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Fig. 9 Shows the efficiency of technological tools for the detection of latent biological traces composed of two different biological tissues in different concentrations

The experimental study also showed that, in terms of absolute latent trace, under certain conditions, with protocol 1, the forensic operator is able to detect and correctly document biological traces consisting of 0.78 μl. Nevertheless, it should be pointed out that although this technology has presently shown most interesting data in terms of identification of the biological trace evidence, it is not yet able to lead to information on the histological tissue from which the biological trace under examination derives. Further research activities are currently in progress; these activities are precisely aimed at the definition of an operational protocol that will allow the following: 1. To identify latent biological traces 2. To facilitate homogeneity of procedures and data 3. To identify the histological tissue from which the biological trace evidence under examination is generated (e.g., blood, semen, saliva, etc.) Other recent experimental activities have been focused on the analysis of biological traces exposed in critical contexts, such as fire conditions – fire scene (D’Orio et al. 2020).

Analysis Techniques in Forensic Genetics and New Technologies Since the publication of the article “The forensic use of the genetic fingerprint” by Alec Jeffreys, Peter Gill, and David Werrett in 1985, the application of genetics in the field of forensic science has undergone an exponential development due to the rapid evolution of methods of analysis, such as the discovery of the polymerase

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chain reaction (PCR). This is a widely used method to produce thousands of miles of copies of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it to a suitable amount to study it in detail. PCR was developed in 1984 by the American biochemist Kary Mullis (1987). At that time, a large sample of biological material was needed in order to generate a full DNA profile for forensic analysis. In fact, before the introduction of this technique, human identification was carried out through the characterization of human protein polymorphisms. In particular, the ABO, Rh, and HLA systems were analyzed using immunological and electrophoretic techniques (Spinella et al. 1997). However, this method gave poor results, relating to the type and size of the evidence. The analysis of protein polymorphisms has been progressively replaced by new molecular techniques, which analyze DNA polymorphisms. The sequencing of the human genome showed that only 1.5% of DNA is composed from coding regions, while the remaining 98.5% is represented by non-coding sequences. These sequences can be present both in single and in multiple copies. In total, the repeated DNA make up more than 50% of the whole genome. Unlike coding sequences, which are highly conserved, non-coding sequences are subjected to mutation events that lead to an increase in inter-individual differences that often do not affect the phenotype (Fowler et al. 1988). However, it is useful to point out that most of the human genetic material (more than 99.5%) does not vary between individuals. Therefore, only a small fraction of our genome, less than 0.5%, is subject to variability. This minimal dose of variability makes each individual unique, and through its analysis it is possible to use this information for personal identification. DNA variability can be explained by the concept of polymorphism: a DNA sequence is defined as polymorphic when at least two allelic forms are present in the reference population. The less frequent of these allelic forms is present with a frequency greater than or equal to 1%. In the case that the frequency of an allele in the population is less than 1%, this form is defined as a rare variant. Forensic genetics uses DNA polymorphisms for personal identification. The power of discrimination of the polymorphisms used for this analysis is based on their ability to distinguish two individuals in a population. The ability to discriminate between genetically unrelated individuals depends on the number of alleles characterizing the locus and their distribution in the reference population. Polymorphic regions are presumed on both autosomal and sex chromosomes. There are two categories of DNA polymorphisms, based on the molecular mechanism that gives rise to such variability: sequence polymorphism and length polymorphism. The first polymorphism, also known as SNP (single nucleotide polymorphism), arises from a single nucleotide substitution determining differences between two homologous DNA sequences. The SNPs basically produce biallelic polymorphisms for this they can provide limited information for personal identification. In order to obtain sufficient discriminatory power for personal identification, it would be necessary to analyze a set composed of fifty Snps (Sanchez et al. 2006).

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Because SNPs are smaller and more abundant in each cell than STRs, SNP analysis can be useful when DNA is highly degraded. The VNTRs (variable number of tandem repeats) are length polymorphisms consisting of a variable number of DNA sequences repeated in tandem. Each characterized by multiallelic forms, each defined by the variable length of the sequence and the number of repetitions. VNTRs are divided into two categories, based on the size of the repeating sequence: minisatellites which are characterized by repetitions in an interval ranging from 16 to 70 bp and microsatellites, or STRs (Short Tandem Repeat) with a repetition interval that ranges from 2 to 6 bp (Weber and May 1989; Beckman and Weber 1992; Hearne et al. 1992). STRs are the markers used in forensic genetics. For the high degree of polymorphism and the high rate of heterozygosity, they can be used as genetic markers to generate a DNA profile that is extremely rare in a population of unrelated individuals (Biondo et al. 2001). Typically, markers are examined at a minimum of 16 loci plus a sex marker in an individual’s DNA. These are the European Standard Set (ESS) (set out from the European Network of Forensic Science Institute (ENFSI) and the European DNA Profiling Group (EDNAP) (Welch et al. 2012). These are visualized as a series of peaks on a graph and the position of which corresponds to the length of the STR and is recorded as a number. Each location has two STRs (one from our mother and one from our father), which means that an individual’s genetic profile can be represented as a series of numbers. Each pair of digits always corresponds to a specific location on a chromosome. Commercial kits that amplify more than 20 STR loci have now been adopted by many laboratories worldwide. These new kits enable more international sharing of DNA data with increased compatibility between STR data, and as more loci are examined, the probability of two individuals having an exact match decreases rapidly. In fact, when a DNA profile has been generated from a crime scene sample, it can then be compared to other profiles, such as DNA from other crime scenes, the suspect’s DNA, the victim’s DNA, or DNA profiles within a National DNA Database. If two profiles are identical, this is called a full match; if only parts of the profiles match, this is a partial match. Once a match has been declared, the strength of evidence supporting the identification of an individual can be calculated. It is important to calculate the probability that the DNA on the evidence collected will match that of someone else. It depends on how many loci in the DNA we look at. Although the chances of two full DNA profiles from two different unrelated individuals matching are extremely rare, DNA profiles from crime scenes are rarely perfect, and they may not contain information about every genetic marker analyzed resulting in a partial DNA profile or in a mixture of DNA from two or more people. The smaller the number of genetic markers the DNA profile is composed of, the greater the risk of a false match occurring. Once a DNA sample has been analyzed, two kinds of statistics may be reported. The simplest is the match probability, which addresses the question of how rare a DNA profile is in a population of random unrelated individuals. This must not be

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confused with how likely the person is to be innocent of the crime. These match probabilities are fine if we are dealing with full DNA profiles from a single individual, or we have a partial DNA profile from one individual as well. However, this statistical approach cannot be used in more complex cases, such as mixtures of two or more individuals. Under these contexts, a second kind of analysis is applied: a likelihood ratio. This weighs the evidence in favor of competing hypotheses, one from the perspective of the prosecution and one that of the defense. It compares how probable the observed evidence is under each argument. Even if statistical experts agree that likelihood ratios are the best approach for complex DNA profiles, their adoption by many labs has proven to be slow. This is partially done to fear that courts may misunderstand the results. In fact, due to the complexity of the calculations, particularly once several people’s DNA gets mixed together, forensic genetics now uses specially designed computer programs. Several such programs are employed around the labs, but because different programs are prepared using different mathematical approaches and assumptions about the data, they could give different values for the likelihood ratio. There have been cases where the prosecution has used one program and the defense another, meaning that the court had been presented with two different answers about the strength of the DNA evidence. Usually the difference is slight, but cases where one program favors the prosecution and another favors the defense are obviously important and need further investigation. To assist in the appropriate use of one of the commonly used mixture interpretation approaches, some rules for the combined probability of inclusion (CPI) were spelled out (Bieber et al. 2016). The most popular probabilistic genotyping software (PGS) to assist DNA mixture interpretation is generally systems use a “semi-continuous” model that uses the presence or absence of peaks along with probabilities of allele drop-out or drop-in or a “continuous” model that takes peak heights into account as well as the presence or absence of peaks along with probabilities of allele drop-out or drop-in. Several validation studies were published for the PGS system STRmix including the challenges of estimating the number of contributors with low levels of DNA were explored (Norsworthy et al. 2018). The variation of results with four different continuous PGS models was studied (Swaminathan et al. 2018), and responses to court admissibility challenges with STRmix were provided (Buckleton et al. 2019). However, as forensic techniques have improved, their ability to detect smaller and smaller amounts of DNA has increased. This means that tiny, invisible traces of DNA can now be recovered and analyzed. The analysis of such limited quantities of DNA is called low copy number (LCN) (Gill et al. 2000) or LT DNA typing. Under these conditions, stochastic substitutes in different amplification reactions (of the same sample) that produce different replication result are defined unstable genetic profiles (Whitaker et al. 2001). In fact, these apparently con icting results, resulting from polymerization artifacts, the univocal determination of the genetic profile of a biological sample is not obtained (Gill et al. 2007). With LCN sample, the electropherogram does not re ect the real DNA profile composition. This is due to the formation of stochastic variations (drop in, drop out, allelic imbalance and stutters) during sample amplification. These stochastic effects

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introduce a high degree of uncertainty and difficulty in the interpretation of LCN genetic profiles. A method to check the accuracy of genetic profiles obtained from complex forensic samples consists in the preparation of replicate assays, aimed at investigating the repeatability of the result in distinct PCR reactions. To do this, at least one duplicate of independent amplifications is required (Graham 2008; Buckleton 2009). In this way, multiple amplifications of the same extracted DNA can be compared in order to obtain a “consensus profile.” As such, alleles that occur more than once are considered “reliable” as they are reproduced by separate assays (Cowen et al. 2011). In addition, the Y-chromosome DNA testing is important for forensic evidence. It can be crucial in sexual assault cases, making the examination of a male perpetrator’s profile possible even in a mixed sample with high levels of female DNA concentration. It makes use of genetic information on the Y chromosome (which only males have). The Y chromosome is inherited from father to son only, which means that all male relatives on the paternal side of the family will normally share the same Y chromosome. Consequently, this type of analysis is not identifying, because the male individuals of the same family have the same Y DNA profile. The new kit containing a large battery of Y-STR loci is expected to be very helpful to increase the power of discrimination and help differentiate male individuals within the same family in a higher percentage of cases (Ottaviani et al. 2014). Mitochondrial DNA analysis uses DNA from mitochondria. Mitochondrial DNA (mtDNA) is more abundant than other types of DNA and can be useful in cases where biological material is limited or damaged by environmental exposure, such as heat, light, or water, which breaks up the DNA strand. MtDNA is inherited by a child from its mother, so all relatives on the maternal line of the family will share the same mtDNA. Like the Y-DNA analysis, it is not identifying but can be useful for exclusion. Next-generation sequencing (NGS) opens potential new applications including biogeographical ancestry, phenotyping of externally visible characteristics, and finer details on STR alleles to possibly improve mixture component resolution. In fact, NGS describes a suite of emerging DNA sequencing technologies, where sensitive tests can be done simultaneously and STR profiling, biogeographic ancestry testing, and phenotyping tests at the same time. Its use in forensic science is still starting out, but a special issue of the journal Electrophoresis on novel applications of MPS in forensic DNA analysis was published in “November 2018” (McCord and Lee 2018). The current limits for implementation of NGS were identified as lack of consistent nomenclature and reporting standards, lack of compatibility with an existing national DNA database, lack of population data to support statistical calculations, and lack of an adequate legislative framework. In May 2019, the NDIS Board of the FBI Laboratory began accepting data from approved NGS kits for upload to the US national DNA database. Biogeographic ancestry testing is a technique that enables an individual’s broad geographic origins (Africa, Asia, Europe) to be estimated based on genetic differences in their DNA. This method uses DNA markers that are common in different parts of the world and can help narrow down a pool of suspects when no match in a national DNA database has been found.

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A new application is the development of forensic DNA tests that can predict aspects of someone’s physical appearance. This approach is called forensic DNA phenotyping (Kayser 2015). This technique uses DNA to make predictions about someone’s appearance (hair and eye color). It is another way of narrowing down a pool of suspects using DNA markers found in the genes that determine aspects of human appearance. As it is a new technique, it has been used in a very small number of forensic cases. In fact, not all externally visible characteristics are equally predictable from DNA information at present. Predicting eye color is difficult, due to the fact that it is in uenced by many genes, of which six are currently used in forensic DNA phenotyping tests. Predicting other externally visible traits such as height is equally as difficult and currently not yet possible because they are determined by large numbers of genes, many of which remain unknown. They are also in uenced by environmental factors which cannot be predicted from DNA. Phenotyping can be used as an investigative tool, to reduce the number of potential suspects when the suspect pool is very large and to help prioritize who to focus on first or next. But it cannot be used as final evidence in court. It is currently possible to predict eye and hair color from a DNA sample, although none of these tests are 100% accurate. Some of these tests have been forensically validated (Chaitanya et al. 2018). Skin color is likely to be the next appearance trait that forensic scientists will be able to predict from DNA with tests that are currently being developed and validated. Forensic DNA phenotyping raises some ethical issues too. Whereas standard forensic DNA profiling involves genetic markers found in parts of the human genome that are non-coding regions, the markers used in forensic DNA phenotyping are located within or close to genes involved in coding regions. If forensic DNA phenotyping techniques were extended to also include non-visible characteristics, such as genetic risk of disease, they could reveal sensitive and private information. This can be avoided through national regulation. With the goal of generating faster DNA results, Rapid DNA instruments have been developed that can produce a DNA profile in less than 2 h. These instruments offer a full automation of the STR typing process consisting of DNA extraction, amplification, separation, detection, and allele calling from reference oral swabs. Two rapid DNA instruments are currently available: the ANDE 6C (6-color) Rapid DNA System from ANDE (Longmont, CO) and the Rapid HIT ID instruments by Thermo Fisher Scientific. These instruments can be operated at police booking stations and border crossings and in traditional forensic laboratories. Currently, these instruments would appear to be used successfully to process single-source reference samples and not crime scene evidence containing mixtures (SWGDAM; NDAA).

Genetic Traces in a Fire Scene In an investigative-forensic context, genetic traces have a central importance in reconstructing the dynamics of the events that occurred in the fire scene to identify the persons responsible for the crime.

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However, often with the express purpose of erasing the traces, many criminals use fire as a tool to weaken the investigative process, making the biological traces potentially present on a given crime scene or on finds less usable and available. One of the greatest merits of Salvatore Ottolenghi, father of the Italian school of forensic sciences, was to understand how the police investigation should be traced back to the more general area of natural sciences. The biological traces present on the scene of the crime are, in fact, “natural signs.” It is no coincidence that English criminologists speak of physical evidence to mean circumstantial evidence. Therefore, the places and things that relate to a crime, if properly examined, have much to reveal, identity of the victim, on that of the aggressor, on their relationships, and more generally on the most eeting or deep interrelations between human action and the environment.

Genetic Traces in a Fire Scenario An investigative context of a fire scene with the presence of genetic/biological traces is certainly complex, as it requires the experience and skills of different professionals: on the one hand, forensic biologists and, on the other, forensic engineers (Augenti et al. 2011; Mangione et al. 2015). In fact, for a suitable investigative analysis in these cases, only the synergistic work between these two professional figures can, in fact, trigger a forensic investigation that has excellent objective chances of success. Specifically, in these investigations, there is a synergy between the forensic biologist, who is a specialist in biological trace evidence, and the forensic engineer (fire investigator), who is a specialist in scenarios of confined fires. There is a synergy between the forensic biologist, who is a specialist in biological trace evidence, and the forensic engineer (fire investigator), who is a specialist in scenarios of confined fires. The scientific data on this topic present but not in considerable quantities. However, it is known that, even following a fire, the biological traces have a fair chance of being preserved and suitable for investigative use. In fact, the degradation of biological traces is “outbreak-dependent”; in the place where the spread of the fire and/or possibly a subsequent explosion occurs, biological traces, being particularly exposed to the phenomenon of fire, suffer considerable structural damage. Such damage that biological cells receive cause cellular and genetic lysis phenomena, often leading to the impossibility of obtaining a suitable genetic profile for future investigative use. Furthermore, some studies describe how, in the event of a fire, the biological traces have a “substrate-dependent,” that is, the material on which they deeply affects is deeply affecting the state of the biological traces. It has been well described that biological traces present on substrates such as “nylon” have a very considerable degradation in the event of a fire.

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Furthermore, it is also highlighted that blood traces assume, following a fire, different phenotypic (i.e., visible) characteristics depending on the body tissue considered. It has been shown that traces of blood take on a color ranging between dark brown and black, following a fire; on the contrary, traces of sperm remain unchanged in their color. This occurs due to the particular protein composition of the blood. In the blood, cells are rich in the protein “hemoglobin,” which contains a red pigment. In the event, or prolonged exposure over time to high temperatures, a colorimetric change occurs; the red pigment of hemoglobin, due to heat-induced denaturation, goes toward darker colors, such as dark brown. These colorimetric characteristics are sometimes detectable with the naked eye, but sometimes they are detectable thanks to the use of special forensic lights that are able to enhance the latent traces that are no longer visible to the human eye due to their small size or high degradation. Furthermore, other sector studies have simulated a fire scenario in a multi-room house. A known series of biological traces have been placed in different rooms. Following the fire, it was possible to see that in the ignition room, the one from which the ames originated, the biological traces still usable, and identifiable amounted to only 25% of the total traces deposited before the fire. Otherwise, in the other rooms, also affected by the ames, the biological traces still identifiable and usable for investigations amounted to 80% of the total traces. This scientific data give considerable confidence to the investigators, as it is highlighted that the total compromising of biological traces does not occur and that in the relevant environments, even if affected by the ames, will be considerable probability of still finding traces using biological products. This data, from an investigative-scientific point of view, is quite satisfactory and above all confirms that, even if the perpetrators of a crime try to erase the biological traces on a crime scene, even through the use of fire, there is a good chance of finding biological trace evidence of the perpetrators despite the fire scenario used to weaken the investigation. It should also be emphasized that the degradation of biological traces due to fire damage, from a biological point of view, can always be assessed in two ways: the biological cell is composed, structurally speaking, of nucleus and other intracellular organelles. In the nucleus, as well as in the mitochondria, there is genetic material which, by practice, is analyzed for investigative purposes. However, it must be specified that the genetic material of the nucleus and the mitochondrion is profoundly different. Indeed, nuclear DNA is the only one of the two that allows, through the use of particular molecular genetic techniques, to define a genotypic profile. The DNA present in the mitochondrion does not have this peculiarity because it is transmitted intact, from generation to generation, from mother to child.

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For this transmission phenomenon, the mitochondrial DNA, speaking, can only give information about the maternal family strain of the subject that released the trace. Another important difference between these two DNAs, which certainly is applicable in the assessment of the progressive biological damage caused by fire, is the structure of the two DNAs. Nuclear DNA is a molecule composed of 46 chromosomes, a double-stranded helix of nitrogenous bases. Instead, mitochondrial DNA is a single circular molecule. The spatial conformation, circularity of the molecule in the case of mitochondrial DNA, ensures greater resistance to all external degradation phenomena, including fire or heat. It should also be added that nuclear DNA is present in a single copy within a biological cell, while mitochondrial DNA is present from one hundred to one thousand copies per biological cell. Therefore, thanks to the numerical presence and conformation factors of the molecule, the damage that is progressively produced by the heat of the fire can be assessed by referring to the nuclear DNA/mitochondrial DNA degradation index. The following figure represents the biological finds most present on crime (Fig. 10).

The Biological Traces Evidence Biological evidence in fire investigations can be of various natures. In order to be able to catalog them, they must be part of a standardized investigative methodology that represents a potential aid for the search of evidence in fire investigations activities applicable in all cases of confined fires. The need to code the operations is strongly felt in the environments of the scientific police force, where the user, who is interested in conducting investigations, often does not have a clear overall picture of the operations and controls to be carried out on the scene to by-pass certain checks and therefore not to find specific traces in the forensic field. Fig. 10 Overview of the main biological found on the crime scene

The most common places to find DNA evidence are: Blood Saliva Hair Semen Also found in: Urine Bone Skin cells

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Fig. 11 Colorimetric modification of biological traces on the same substrate following a fire

Of considerable importance is therefore the collection of significant events of the scene to be performed immediately after the fire, in order to allow the scene to be frozen at the time of collection and when the state of the crime scene has not yet been compromised by third parties. On occasion, some details may escape even with the most attentive investigator and, subsequently, become a relevant unexpected in the continuation of the investigation. In these terms, for example, the intervention of the Fire Brigade, the presumed duration of the fire, photography supplemented by descriptive reports, etc. can assume proof value at a forensic level. There are therefore various critical issues in fire scenarios in the search for biological evidence. By way of example, the phenotypic characteristics induced by fire on biological traces are presented in the following way (Fig. 11). During the evidence, it is necessary to be attentive to those events that could involve alterations of the scene and clues of a biological nature. For example, the fingerprints present in a malicious scene can often be compromised due to the necessary shutdown of the fire.

From Scientific Proof to “Beyond Reasonable Doubt” The investigation presupposes the occurrence of a criminal event and starts with the news crime, with the completion of direct and indirect investigations (Mangione 2017). Direct investigations are also called objective probative acquisition investigations, since they are carried out directly on things, places, or situations relevant to the crime and involve an analysis of the elements found on the scene. For example, these investigations include planimetric and photographic surveys, laboratory analyzes on finds, and so on (Mangione 2018). Indirect investigations, on the other hand, or investigations of subjective probative are subsequently carried out and in tandem with direct ones carried out subsequently and in parallel with direct ones.

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A different but related problem is whether the judge is able, not being an “expert in the field,” to understand if the resulting evidence is altered or is able to perceive any changes. In reality, the investigations carried out are called into question only in the crossexamination between the parties, and it is at this moment that the issues emerge that will then form the topics for the evaluation of the evidence. In summary, the judge’s control must be exercised both at the beginning, at the moment of admissibility of the test, and at the end, in the evaluation of the result. This judicial control is enacted to ensure a new line of thought in legal doctrine, carried out by judge G. Francione, suggests the establishment of figures of active support in these preliminary stages (Francione 2019). Specifically, we are talking about the figure of the “pro-unknown consultant,” employed by the judicial judiciary who supports the PG in the investigations and has the task of ensuring the quality of the work and submitting observations and requests aimed at guaranteeing maximum efficiency and compliance with all scientific and legal procedures in carrying out the first technical investigative acts. The causal relationship cannot be considered to exist on the basis of the statistical probability coefficient alone, but must be verified in the same way as a judgment of high logical probability. The reasonable term indicates insufficiency, contradiction, and probative uncertainty; therefore, the plausible and reasonable doubt is based on specific elements which, based on the available evidence, corroborate it in the specific case. The search for traces is mainly aimed at identifying, documenting, and also removing fragments of papillary prints, which at the crime scene can be of two types: visible and latent prints. The visible footprints are those: – Which are produced by contact of digital surfaces soiled with substances of various kinds (blood, ink, stain, etc.) on rigid surfaces, generating fingerprints by overlapping – Produced by the pressure or sinking of the papillary ridges on malleable substances, such as wax and so on, generating impressions for modeling. Generally these types of imprints concern surfaces that can be removed with the entire substrate on which they are impressed and must therefore be photographed with the appropriate technical devices, such as filters, polarized light, in order to enhance the contrast with the surface, same on which they are located. Requests for intervention aimed at enhancing fragments of footprints on the dashboards of motor vehicles, made of plastic material, are increasingly frequent. Figure 12 shows a technique for removing soot in order to identify the presence of papillary prints and traces of blood left on the surface of a door before the fire. Figure 13 shows instead some impressions on ceramic, highlighted by vacuum metal deposition (VMD), after the surfaces have been respectively exposed to the following:

104 Fig. 12 Extrapolation of footprints from a door

Fig. 13 Imprints on ceramic material as the temperature varies

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Even in the extreme conditions of a fire, it is therefore possible to identify traces of interest for forensic purposes. In this regard, the work carried out by Harper in 1938 showed that there is the possibility of identifying papillary traces on objects exposed to a temperature between 100 and 200  C more clearly demonstrated in Fig. 14.

DNA and Fire The effects of heat on DNA do not simply just create damage. Scientific research on DNA evidence and fire investigation is undergoing enormous growth, especially abroad. Currently, scientific research in the field of forensic genetics is tending more and more frequently toward the accuracy and assurance of the robustness of analytical data. In this case, studies on compromised DNA and studies on the interpretation of mixed profiles are currently underway in many universities. The study of the methodologies for the analysis and interpretation of degraded DNA also closely concern the fire investigation. In fact, with the implementation of analytical methodologies, genetic information from fire scenarios will certainly have a greater yield and, consequently, also greater application in the investigative field. In other words, these methodologies are aimed at recovering, and making it usable for analysis, the genetic traces that today are “lost” because they are too degraded. Figure 15 shows heat denaturation process of nuclear DNA. It is noted how double-stranded DNA can be denatured. Physiologically speaking, DNA is a molecule composed of a double helix held together by non-covalent bonds between the nitrogenous bases that make the genetic structure. In order to carry out normal vital functions, the cell continuously “packs” and “unpacks” the DNA, thanks to the use of particular enzymes and a highly controlled biochemical process of reactions. In fact, just think that biological cells normally replicate.

Fig. 14 Papillary traces as the temperature changes

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Fig. 15 Heat denaturation process of nuclear DNA

When cell replication occurs, the DNA double helix must open (and it does so, thanks to specific enzymes) and must replicate bi-directionally. This phase, the cd, called “DNA synthesis,” is the basic phase of the cell division process. Thanks to this base, the two daughter cells (which will be equal to each other and equal to the mother cell that generated them) will have the same DNA. The modern technologies of molecular and genetic studies have led to the discovery, years ago, of the famous PCR (Polymerase Chain Reaction) technique, according to which DNA, due to controlled heat administration, first denatures and later realigns itself later. This process was used for research in the health diagnostics field. It was later seen that the same technique was also useful in applications in an investigative-forensic context. DNA can denature at different temperatures based on its internal composition (A-T; C-G) (the nitrogenous bases that make up the DNA are four, namely adenine, thymine, cytosine, and guanine); these comprise the DNA structure by pairing with each other through the formation of non-covalent bonds. Specifically, adenine binds with thymine, and cytosine binds with guanine. Other bonds between these molecules are not chemically possible. Figure 16 below shows how A-T-rich DNA denatures at a medium temperature compared to C-G-rich DNA. This can be explained, thanks to elementary principles of biophysics; in fact the pair of nitrogenous bases C-G is united by three non-covalent bonds, while the pair A-T is united only by two bonds. This is why heat-induced denaturation is relatively simpler for A-T-rich DNA stretches. Obviously, all this confirms the principle according to which heat, simulated or coming from a fire, has effects on the DNA structure and, having reached certain

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Fig. 16 Average temperatures at which the denaturation of the nitrogenous bases making up the DNA double helix takes place

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levels, causes alterations of the genetic structure that are initially repairable, then irreparable depending on the time and temperature reached. There are two types of DNA in nature: nuclear and mitochondrial. These types of DNA are found in practically all cells of the human body (with the exception of erythrocytes, which have no nucleus). These types of DNA have very different characteristics and peculiarities. Physiologically, nuclear DNA is present in duplicate within the nucleus of human cells (this is why the human being is genetically called “diploid”); the nuclear DNA is inherited directly from the biological parents of the individual with equal distribution (50% of the genetic material comes from the biological father, and 50% comes from the biological mother); nuclear DNA is present in a “double helix” conformation. Mitochondrial DNA, on the other hand, is present in the cellular cytoplasm; it is present, depending on the cell type, in a number ranging from 100 to over a thousand copies per cell; it has a totally circular conformation; the mitochondrial DNA is inherited by the individual solely through the mother (this happens because, at the time of fertilization, meaning the fusion between the paternal sperm cell with that of the maternal oocyte, the sperm nucleus enters the maternal egg cell, while the paternal mitochondria, present in the sperm tail, remain outside the new cell that is formed by fusion). From the point of view of the peculiarities of analysis, nuclear DNA and mitochondrial DNA are profoundly different.

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Due to the nuclear DNA physiological characteristics and particular analytical kits, is able to be typed (or analyzed) in a highly specific way. In other words, nuclear DNA allows us to identify a particular individual in a unique and univocal way and to discriminate it from all the others present in the human population. Mithocondrial DNA, due to its physiological process that occurs at the time of fertilization (brie y illustrated above), does not allow a specific individual to be typified in a unique and univocal way, but allows to go back only to the maternal family of origin (what is referred to as “maternal stock of biological origin”) (Fig. 17) Mitochondrial DNA is the most resistant. Often, it represents the DNA object of forensic investigation if the biological material is particularly compromise. Due to the characteristics of the conformation of the genetic molecules and their relative number of presence that is weird within each single biological cell, nuclear DNA is much more “fragile” than mitochondrial DNA (Fig. 18). The textile substrate affects the degradation of DNA The following table shows the results from a study which analyzed the damage caused to biological/genetic material following a fire. In this study, different biological uids were compared to evaluate whether the biological cells of different tissues (in this case blood and sperm cells) are equally damaged by heat or have a non-homogeneous denaturation. The study provides a chemically evaluation, while the chemically different substrates with the aim of assessing the composition of the substrate can affect biological/genetic damage. Genetic markersa widely used, such as CSF and TH01 were used to evaluate the genetic damage; of these two markers, the reciprocal relationship was used for the evaluation of genetic damage (Fig. 19). The ratio of the selected genetic loci does not significantly change between blood and sperm following high temperatures. The following histograms (Fig. 20) show the relationship between specific gene loci (or markers, to be understood) called “CSF” and “TH01,” specially chosen for Fig. 17 The biological cell. Relationship between nuclear and mitochondrial DNA

Nuclear 2 copies/cell inherited form both parents unique to individual Mitochondrial >100 copies/cell maternally inherited not unique to individual

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Fig. 18 Comparison of the characteristics of nuclear and mitochondrial DNA

the study of the comparative degradation of genetic material. It is shown that, following damage from fire, biological traces from blood or semen suffer genetic damage in equal measure. If we associate this result with the fact that we are sometimes in a condition of minimal trace, such as in the case of traces of semen, this information regarding environmental damage is particularly useful because it ensures that the trace undergoes a “controlled” degradation that is not different from what occurs with traces from other biological tissues. This, from an investigative point of view, strengthens the probability of carrying out successful genetic sampling, even in critical environmental conditions due to fire. The comparison and discussion of the above data, of both the tabular and histogram examples, confirms that the genetic damage that the cells suffer as a result of heat/fire is “homogeneous,” meaning that the biological traces are all denatured at the same way. In this case, it is emphasized that biological traces from different biological tissues (in this case, blood and seminal uid) have the same degree of degradation calculated through the ratio between the selected genetic markers CSF and TH01. Again, this data clearly demonstrates that, for what has been studied, cell denaturation and genetic damage are absolutely not “biological-dependent tissue.” Moreover, this data show how much the chemical nature of the substrate on which the biological traces are adhered in uences the degradation.

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52

53

54

55

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Blood

Blood

Blood

Semen

Semen

Semen

Blood

Blood

Nylon

Nylon

Nylon

Nylon

Nylon

Nylon

Nylon

Nylon

1

1

1

1

1

1

1

1

CSF

33.26

30.79

N/A

32.03

1.24

TH01

49.15

45.08

N/A

47.12

2.04

CSF

31.77

26.25

N/A

29.01

2.76

TH01

48.77

42.19

N/A

45.48

3.29

CSF

35.22

29.37

N/A

32.3

2.93

TH01

53.91

43.25

N/A

48.58

5.33

CSF

156.23

133.95

N/A

145.09

11.14

TH01

270.76

227.3

N/A

249.03

21.73

CSF

105.38

97.95

N/A

101.67

3.72

TH01

183.36

182.93

N/A

183.15

0.22

CSF

143.25

134.7

N/A

138.98

4.28

TH01

288.57

279.23

N/A

283.9

4.67

CSF

15.58

12.23

N/A

13.91

1.67

TH01

12.56

10.84

N/A

11.7

0.86

CSF

1.51

1.46

N/A

1.49

0.03

TH01

0.73

0.74

N/A

0.74

0.01

0.68

0.638

0.665

0.583

0.555

0.49

1.188

2.015

Fig. 19 Summary data of biological/genetic damage from fire (CSF; TH01-target) Fig. 20 Relationship between CSF and TH01 gene markers following fire/heatinduced damage in blood and sperm cells

Specifically, it is evident that all the biological traces present on the “nylon” substrate occur in greater denaturation than those present on the “polyester” substrate. In other words, this shows that the biological/genetic damage is absolutely dependent on the type of chemical composition of the substrate on which the biological traces are adhered (Fig. 21).

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t

tcrit

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Sig. Dif. More Degraded

Blood vs. Semen Blood vs. Semen

0.11

2.021

No

N/A

Donor 1 Blood vs. Semen

0.347

2.131

N/A

Donor 2 Blood vs. Semen Nylon Blood vs. Semen

0.359 1.497

2.069 2.08

No No No

N/A

Polyester Blood vs. Semen

1.899

2.11

No

N/A

Nylon vs. Polyester

Nylon vs. Polyester 2.06 3.191

Yes

Nylon

Blood Nylon vs. Polyester Semen Nylon vs. Polyester

2.503 2.411

N/A

2.131

Yes

Nylon

Yes

Nylon

No Yes

Nylon

Donor 1 Nylon vs. Polyester

1.725

2.306 2.201

Donor 2 Nylon vs. Polyester

2.761

2.179

Donor 1 vs. Donor 2

Donor 1 vs. Donor 2 1.02 2.026

No

Blood Donor 1 vs. Donor 2

0.421

2.131

No

N/A N/A

Semen Donor 1 vs. Donor 2

0.712

2.08

No

N/A

Nylon Donor 1 vs. Donor 2 Polyester Donor 1 vs. Donor 2

1.429 0.069

2.08

No

N/A

2.131

No

N/A

N/A

Fig. 21 comparison of the denaturation of biological traces placed on different textile substrates

For the sake of completeness, it is important to specify that, when we talk about the evaluation of genetic damage following a fire, the standard to which we refer to in the evaluations is the electropherogram that arises from the typing of DNA through the use of a number of markers between 16 and 24. Below is an example of an electropherogram over 23 loci (Fig. 22) :

Final Remarks With the advent of sophisticated technologies, nowadays scientific proof becomes certain proof; this is by virtue of both modern methodologies and the control figures in charge of checking compliance with the rules, procedures, and scientific protocols. In science, therefore, as well as in the investigation and in the process of investigation, the method is mainly aimed at discovering the error that leads to the truth. The presence of genetic traces in a fire scene certainly makes it more complex, but the investigation, if well planned, leads to a solid conclusion. While, in fact, the inquisitorial system proceeded from the claim to already know the whole truth by lowering it from above in the process in the form of already established evidence, the accusatory system takes into consideration the evidence regularly formed (i.e., made less uncertain) in the cross-examination between the

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Fig. 22 Example of a genetic electropherogram for forensic purposes

parties before a third judge; therefore the investigation, conducted according to precise rules, constitutes one of the founding ideas of the new criminal trial. Under these circumstances therefore, there is no investigation that is not naturally included in a system of checks, weights, and counterweights. The examination of forensic trace evidence in a fire scenario is currently the subject of scientific research and undoubtedly deserves targeted studies for the resolution of numerous judicial cases (Stella 2000).

The New Popperian Epistemology of the Criminal Process: Strong Scientific Evidence and Reduction of the Clues to Conjectures With the movement for the new renaissance of Justice, we go all over Italy holding conferences on various topics exposed by the avant-gardes to improve the applied law. What are the remedies for a truly fair new justice (My design of a new justice led me to found the MOVEMENT FOR THE NEW RENAISSANCE OF JUSTICE (MOV.RIN.GIU). The early Renaissance was represented by the Enlightenment, which virtually crushed the inhumane righteousness of the Inquisitors. The seeds for a revolution of Themes are still waiting to be realized with our second Renaissance. Even today, the indicative process is underway with the risk of condemning innocent people, subverting Voltaire’s maxim: “It is better to run the risk of not condemning a guilty person than of condemning an innocent person.” The HUMANIST AND FRATERNAL JURIST is the final target of the movement in order to realize a RIGHT JUSTICE.)?

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In this period, some striking judicial cases (e.g., Meredith Kercher, Melania Rea, Elena Ceste, Guerina Piscaglia, Roberta Ragusa, Yara Gambirasio, Sara Scazzi, Chiara Poggi, etc.) have brought to the limelight the suspects who continue, although arrested, to proclaim their innocence. The deficiency of certain evidence and the founding of processes on purely clues have generated on the network and social media opposing groups of innocentists and guilty parties. The peninsular tour has implemented a series of conferences (in Rome, Caserta, Crotone, Viterbo, Palaia-Agliati, Naples, Milan, Verona, Torre Annunziata), and others are planned. There have been interventions of insiders, emblematic characters (such as Raffaele Sollecito, attorney Piero Tony, lawyer Giuseppe Lipera defender of Contrada), and finally institutions particularly interested in the construction of a fair justice (Basilicata Region, Province of Crotone, Municipalities of Caserta, Palaia, Corsico) (https://www.bbc.com/news/world-europe-21938080). Conferences tend to verify the problems related to clues, preferably having investigators go in search of very strong and crossed proofs, which only establish a fair process to be sure of putting in prison the guilty and not the innocent.

The Revolution of the Justice Method: Popper’s Way for Strong Proofs The first investigation that must be carried out by a fair judge in the search for a procedural truth is that on the method used and on its effectiveness. Here modern epistemology, in particular, the philosophy of Karl Popper, helps us (https://www. simplypsychology.org/Karl-Popper.html). In science, conjectures based on clues are valid to create a scientific thesis, but this must be submitted to the scientists to experiment in the laboratory. The thesis is valid only if all the scientists reach the same conclusion. Mutatis mutandis also applies to judges. If a conjecture leads to different results on the part of the analyzers, then that conjecture is fallacious or at least it is not known to what extent it is true. The judge in the analysis of evidence must merge with the traditional criterion of verification, based on the search for data confirming the incriminatory conjecture, the most modern devised by Popper in the epistemology of falsification, i.e., going to research, even beyond the evidence sometimes, facts that could contradict the main statement. “The criterion of falsifiability maintains that an assertion, to be empirically informative, that is to say scientific, must be falsified principally and not denied in fact, despite the most severe attempts to make it fall.” We must abandon the lethalness principle of the “free conviction of the judge.” It is necessary, therefore, that the magistracy models a new scientific methodology, avoiding confusion as it has sometimes happened in the past. Only by distinguishing legal science as a conjecture (based on clues) and legal science as a result, based only on strong evidence of proofs, can we have a real guarantee of a criminal justice free from prejudice and truly egalitarian. Using these principles, as a judge of the Court of Rome, on June 13, 2000, I raised, in vain, the question of unconstitutionality of the process based on the clues, but the Constitutional Court with Ordinance no. 302 of 2001 rejected my request in a

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brusque way. A noted journalist, Gigi Trilemma, wrote in his article “The Constitutional Court has lost an opportunity to abandon permanently the literary processes and give definitive space to the scientific process based on certain evidence and not on clues. I am sorry the hasty system with which the Constitutional Court has solved the epistemological question, avoiding to tackle the crucial matter about the so called war on the proof versus the clues. The criminal judge, on the other hand, demanded just to do this, that is to decide not with the tautological criteria of legal formalism but based on the principles of modern epistemology, which can only define what is certain and what is false in any proceedings to collect evidence on facts.” Trials are made for strong proofs not for clues that only serve to create conjectures, invalidated if no evidence is found. This is the Popperian scientific process, not a mediaeval novel. The clues only serve to open investigative tracks, but then if there is no strong evidence, the process falls. A thousand clues do not form a single proof not like 1000 rabbits which form a warren and certainly not a lion! Discovering the authors of the crimes is anything but simple. Detective stories say that no crime is perfect. Indeed perfect crime does exist! A big number! And justice enjoys finding culprits at all costs to show that it works. To limit the judicial freedom of the judges in a scientifically way, together with the late professor Imposimato, we came up with a list of legal evidence to be followed. In this regard, the judges must demand not only confession and/or smoking pistol but also unequivocal telephone tapping, crisscrossed testimonies, reconstructed paths with CCTV cameras, post delictum markings with bugs, applications antistalking as Mytutela, and scientific surveys done properly and 100% safe. Certainly not as in the cases of Cogne, Melania Rea, Meredith, Bossetti, not to mention the case of Ceste where you do not even know how the woman died, or Guerina Piscaglia and Roberta Ragusa whose bodies were not even found not knowing if they died or not, if they were killed, and how and by whom. If you do not go through strong proofs, all you can do is trigger indictment trials against alleged perpetrators, keeping them out of jail anyway. If then the clues do not lead to proofs, this serious, precise, and concordant process has failed. Now trial based on clues is required by law, but it is irrational because in itself it always creates reasonable doubt so much so that these striking cases create the faction of the guilty and that of the innocentists, thus lacking upstream certainly of the final verdict. We continue to fight to make the declaration process unconstitutional. Also because against the expression of the norm, what was supposed to be an exceptional process has become the rule by putting the weaker subject in jail and setting him up as a scapegoat. According to statistics, 90% of the processes today on a clue basis would be wiped out, remaining only 10% of processes to be carried out until the possible sentence. A quick but right way to dispose of the backlog.

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The Neutrality of the Researcher and the Rigorous Acquisition of the Proofs After the examination of judicial science as a method, we pass to that of science strictu sensu, asking ourselves the question of who and how to collect the traces of a crime and examine them. We use DNA as a model of study, which is even considered in some striking processes as a proof, being, instead, a simple clue. Both the facts of the media and the positions of various “insiders” show us that the genetic test is not infallible as is believed. With the rigorous Popperian method, the first notation is that DNA sampling and analysis must be guaranteed by the creation de iure condendo of a national sampling service and investigations with super partes experts, depending on the magistracy (we believe to exhume the investigating judge) and not of the prosecutor. At the time, those delicate acts of investigation must be guaranteed by the presence of a defense counselor also for the unknown murderer; otherwise all is nill (article 111 of the Constitution). It is necessary to provide a legal defender and a legal advisor for the unknown to avoid the formal aw of the control and verification procedure. It is not pure theory given the problems created by the scientific police in the Meredith Kercher case, which ended with the acquittal of Amanda Knox and Raffaele Sollecito. Besides the criterion of detector neutrality, it is necessary to guarantee a supervisor in the key stages of the collection of exhibits, the correct chain of custody, the laboratory analysis to ensure the right assumption (procedures, instruments, etc.), and conservation and analysis of data. The currently dominant static criminology is Aristotelian and apodictic and is aligned with the clue process. The dogmatic omnipotence of DNA is part of it. Dynamic criminology, on the other hand, requires a rigorous answer to the questions: “Quis quid ubi quibus auxiliis cur quomodo.” This is of course a Latin phrase, which literally means “who, what, where, by what means, why, how, when?” It is a hexameter elaborated by Cicero (quoted by St. Thomas Aquinas) which contains the criteria to be observed in the conduct of a literary composition: to consider the person acting (quis); the action, what he does (quid); the place where it happens (ubi); the means that he uses in executing it (quibus auxiliis); the purpose it has (cur); the way it is done (quomodo); and the time it takes him to execute it (when) (https://crimsonpublishers.com/fsar/pdf/FSAR.000521.pdf). So, we use the brocardo with the addition of the “quantum” to implement the reconstructive sequence of a crime in a criminal key. Therefore, we have built a complete sequence of a crime in terms of dynamic criminology and strict response to every single question in verification and falsification of data according to the teachings of Popper. The scheme described above in the Bossetti case is admitted and not granted that the DNA is his (https://www.theguardian.com/world/2016/jul/02/yara-gamirasiomurder-massimo-bossetti-dna-evidence-italy-guilty-verdict). This element is not

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enough to attribute the crime to him. It is necessary to establish precisely “how,” but it must be considered that with the possible homicidal action, it cannot rule out accidental or artful contamination. It is possible that the suspect has left traces not because he is the murderer himself but because he has touched the corpse post delictum accidentally or concealed the dead body. Before wrapping up, the fair trials are done by science and strong proofs, certainly not by fictional clues.

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Hearne CM, Ghosh S, Todd JA (1992) Microsatellites for linkage analysis of genetic traits. Trends Genet 8:288–294 Kayser M (2015) Forensic DNA phenotyping: predicting human appearance from crime scene material for investigative purposes. Forensic Sci Int Genetics 18:33–48 Mangione M (2017) Dalla progettazione antincendio all’investigazione sugli incendi. Rivista Antincendio Mangione M (2018) Investigazione su una scena d’incendio: aspetti forensi. EPC Rivista Antincendio Mangione M, Bontempi F, Crosti C (2015) Atti del convegno IF CRASC’15. Structural Fire Investigation e Ingegneria Forense. Università La Sapienza, Roma McCord B, Lee SB (2018) Novel applications of massively parallel sequencing (MPS) in forensic analysis. Electrophoresis 39(21):2639–2641 Mullis K (1987): US patent 4,683,202 process for amplifying nucleic acid sequences NDAA Position Statement on Rapid DNA, January 30, 2018. https://dps.alaska.gov/getmedia/ fb933229-8e52-4cf8-8fe0-cb72d5e039e3/NDAA%20Statement-on-Use-of-Rapid-DNATechnology.pdf Norsworthy S, Lun DS, Grgicak CM (2018) Determining the number of contributors to DNA mixtures in the low-template regime: exploring the impacts of sampling and detection effects. Legal Med 32:1–8 Ottaviani E, Vernarecci S, Asili P, Agostino A, Montagna P (2014) Preliminary assessment of the prototype Yfiler ® plus kit in a population study of Northern Italian males. Int J Legal Med 9:1078–1079 Sanchez JJ, Phillips C, Børsting C, Balogh K, Bogus M, Fondevila M, Harrison CD, MusgraveBrown E, Salas A, Syndercombe-Court D, Schneider PM, Carracedo A, Morling N (2006) A multiplex assay with 52 single nucleotide polymorphisms for human identification. Electrophoresis 27:1713–1724 Spinella A, Marsala P, Biondo R, Montagna P (1997) Italian population allele and genotype frequencies for the AmpliType PM and HLA-DQ-alpha loci. J For Sci 42:514–518 Stella F (2000) Leggi scientifiche e spiegazione causale nel diritto penale. Giuffrè Editore Swaminathan H, Qureshi MO, Grgicak CM, Duffy K, Lun DS (2018) Four model variants within a continuous forensic DNA mixture interpretation framework: effects on evidential inference and reporting. PLoS One 13 SWGDAM position statement on rapid DNA, October 23, 2017. https://docs.wixstatic.com/ugd/ 4344b0_f84df0465a2243218757fac1a1ccffea.pdf Weber JL, May PE (1989) Abundant class of human DNA polymorphisms which can be typed using polymerase chain reaction. Am J Hum Genet 44:388–396 Welch LA, Gill P, Phillips C, Ansell R, Morling N, Parson W, Palo JU, Bastisch I (2012) European network of forensic science institutes (ENFSI): evaluation of new commercial STR multiplexes that include the European standard set (ESS) of markers. Forensic Sci Int Genet 6:819–826 Whitaker JP, Cotton EA, Gill P (2001) A comparison of the characteristics of profiles produced with the AMPFISTR SGM plus multiplex system for both standard and low copy number (LCN) DNA analysis. Forensic Sci Int 123:215–223

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Tools and Techniques Used in Forensic DNA Typing Akanksha Behl, Amarnath Mishra, and Indresh Kumar Mishra

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Basic Principles of DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA Typing Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Blood Group Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Forensic Protein Profiling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . RFLP-Based DNA Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PCR-Based Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Y Chromosome DNA Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . STR Typing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondrial DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X Chromosome Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microbial and Animal Forensics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Next-Generation Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

The chapter starts with a basic overview of the history of DNA and its use in human identification. Then, it discusses the fundamental information about DNA, its structure, and its function. Further sections of this chapter explain the various techniques used for forensic DNA analysis. It covers the processes involved in the preparation of the samples for DNA amplification through the polymerase chain reaction or PCR methods. Separate sections are dedicated to the explanation of commonly used STR markers, Y chromosome markers for specifically A. Behl · A. Mishra (*) Amity Institute of Forensic Sciences, Amity University Uttar Pradesh, Noida, Uttar Pradesh, India e-mail: [email protected]; [email protected] I. K. Mishra Amity Institute of Forensic Sciences, Amity University Uttar Pradesh, Noida, Uttar Pradesh, India Forensic Science Laboratory, Rohini, Government of NCT, Delhi, India © Springer Nature Singapore Pte Ltd. 2022 H. R. Dash et al. (eds.), Handbook of DNA Profiling, https://doi.org/10.1007/978-981-16-4318-7_4

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identifying the male contributor of a sample, and mitochondrial DNA, which is maternally inherited and utilized in cases where highly degraded DNA is found. Finally, in the last section, the topic of nonhuman DNA is touched, which describes how the “other” DNA can aid forensic investigations. This DNA examination constitutes the investigation of animal, plant, and microbial DNA testing. The technology portion of the chapter includes the description of the separation of DNA molecules using slab gel and capillary electrophoresis. Fluorescent detection methods are generally used. It also mentions the use of digital DNA databases to solve crimes and identify suspects. It likewise explores issues that are one of a kind to the scientific DNA analysis such as sample mixtures, in particular combinations, debased DNA tests, PCR restraint, and tainted, all of which sway criminological casework since numerous examples don’t come from a sterile, controlled climate. The national DNA information bases that consist of the genetic data will profit the law requirement by connecting various crimes. Keywords

Forensic DNA typing · Polymerase chain reaction · Restriction fragment length polymorphism · Forensic protein profiling · STR markers

Introduction DNA typing is also known as DNA fingerprinting or DNA profiling. It is a widely used technique in forensic DNA analysis. It was first introduced during the 1980s; initially, highly polymorphic regions were discovered in human DNA by Wyman and White. This particular discovery has changed forensic science forever and empowered law enforcement services to match criminals with crime scenes. It became the foundation for the study of banding patterns which was specific to an individual, and it was studied after restriction fragment length polymorphism or RFLP analysis of repeated DNA sequences This study was conducted by Professor Sir Alec Jeffrey at the University of Leicester. He observed that certain regions of DNA contained DNA sequences that were repeated over and over again next to each other. Along with that, he also observed that the number of repeated sequences present in a sample could differ among different individuals. These particular “repeated” regions are called as variable number of tandem repeats or VNTR. restriction fragment length polymorphism (RFLP) technique was used by Dr. Jeffreys to examine the VNTRs. This technique employs the use of a restriction enzyme, which is used to cut the specific regions of DNA surrounding the VNTRs. In the RFLP technique, at first, the DNA is extracted and then purified. RFLP technique consists of dividing the DNA strands into wanted lengths utilizing limitation chemicals, isolating the DNA sections through the gel medium as per their sizes (atomic loads), moving the parts on to the strong backings (nylon or cellulose film) hybridizing the particular DNA parts

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with reciprocal DNA pieces (tests labeled with radioactive or nonradioactive material) and taking the pictures or impressions of the ideal sections. RFLP methods were first used for DNA typing, after that they were subsequently replaced by PCR methods (Jamieson and Bader 2016).

Basic Principles of DNA DNA is a deoxyribose nucleic acids are composed of nucleotide units. DNA is a large, polymeric molecule. These units consist of three parts: a nucleotide base, a sugar, and a phosphate. The nucleobase is responsible for the variation among each nucleotide unit; the phosphate (PO4) and sugar units, meanwhile, form the backbone structure of a DNA molecule. It consists of four nucleobases: A (adenine), T (thymine), C (cytosine), and G (guanine). The structure is of a DNA double helix. The sides of the ladder are a linked chain of 5-carbon sugars and phosphate groups. The rungs connected to the 5-carbon sugars are known as bases. In all living beings, numerous combinations of these four bases lead to diversity in biological characteristics. Humans have approximately 3 billion nucleotide positions in their genomic DNA. Thus, with four possibilities (A, T, C, or G) at each position, innumerable combinations are possible. In a cell, DNA (deoxyribonucleic acid) is within the nucleus of chromosomes. Gene is the basic hereditary unit and it’s defined as a segment of DNA molecule of a chromosome. Genes are responsible for the physical characteristics of an organism. All inherited characteristics are dependent on one or several genes (Fig. 1).

Fig. 1 DNA structure

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DNA Typing Methods A variety of techniques have been discovered and employed for the forensic DNA typing such as single-locus probe and multi-locus probe restriction fragment length polymorphism (RFLP) methods and polymerase chain reaction (PCR)-based assays. Numerous advances have been made in the last quarter of a century in terms of sample processing speed and sensitivity. Various advances have been made in the last quarter of a century as far as to test preparing pace and affectability. Rather than requiring enormous bloodstains with very much protected DNA, minuscule measures of the sample, as meager as a couple of cells in some criminological cases, can yield a helpful DNA profile (Alamoudi et al. 2018) (Fig. 2).

Blood Group Testing Karl Landsteiner was a researcher at the University of Vienna, Austria. In 1900, he discovered that blood agglutination, which happens when blood belonging to different people, was mixed. He identified four blood types: O, A, B, and AB. According to his research, type O was noticed 43%, type A 42%, type B 12%, and type AB 3%. For the blood transfusion process to work, the donor and recipient require compatible ABO blood types. Otherwise, transfusion reactions will occur, such as agglutination of incompatible cells, and it can also cause death.

1980 - First RFLP polymorphic marker

1985 Multilocus VNTR probes discovered

1985 - PCR research published

1988 - FBI initiates DNA Analysis

1991 - STR research published

1995 - UK DNA DATABASE was formed

1998 - FBI formed the DNA Databse "CODIS"

Fig. 2 Short bullet points depicting the history of DNA typing

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Table 1 Blood types, antigens, and antibodies

Blood groups :

Blood Group A

Blood Group B

Blood Group AB Blood Group O

Blood types : A

B

AB

O

Anti-A, Anti-B

Antibodies : AntiB

AntiA

None

A-

BAntigen

A and B Antigen

Antigens :

Antigen

None

Blood groups are identified with antigen polymorphisms present on the outside of red platelets. These antigens may be protein, carbohydrate, glycoprotein, or glycolipid differences that exist between people. The antigens are acquired from the parents of a person and thus can be utilized to check the paternity. Antibody-based blood tests are employed for the detection of various blood group antigenic alleles (Butler 2010) (Table 1).

Forensic Protein Profiling In forensic science presently, data about the potential biological origin of forensic samples is generally ascertained by utilizing protein-based possible testing. As of late, mRNA-profiling or messenger RNA profiling has arisen as a system to analyze the natural origin. The improvement of a solitary multiplex mRNA-based framework is explained for the separation of the most well-known serological body liquids and skin cells. A DNA/RNA co-segregation convention was set up that outcomes in DNA yields comparable to our norm in-house approved DNA extraction system which utilizes silica-based sections. An endpoint RT-PCR test was built up that at the same time duplicates 19 (m)RNA markers. In forensic biology and genetics, mRNAs have progressively obtained fame with respect to their capabilities to recognize human body liquids and other forensically important tissues. Alternative strategies for cell-profiling incorporate miRNAs that are tissue-specific, DNA methylation, and

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microbial marker. miRNAs are known as 20–24 nucleotides which are small in size, and they are regulatory in nature. They are unequivocally connected with the class of proteins called Argonautes, which makes them entirely steady and beneficial when debased forensic samples are considered. Likewise, epigenetic DNA methylation markers have been introduced that can be separated between some tissue types. Both miRNA and DNA methylation markers appear to be encouraging, yet in their early stages with respect to the occurrence, more markers are expected to segregate the measurable forensic range of body liquids. The utilization of microbial markers has been proposed for the identification of the vaginal mucosa. Notwithstanding, it isn’t yet settled whether similar organisms likewise happen on skin surfaces that are in the nearness of, or contact with the vaginal microbial vegetation, for example, skin surfaces of the hands, crotch, or penis. Thus, we see tissue-explicit mRNA examination as the most adaptable cell-profiling approach. mRNA-profiling has developed from a singleplex PCR procedure to a multiplex RT-PCR stage, giving articulation of information on numerous qualities all the while. mRNA-profiling is promptly joined with DNA-genotyping since RNA and DNA can be gotten from precisely the same examples. The diverse multiplexes that have been created incorporate markers for venous blood, salivation, semen, vaginal epithelia, and feminine emission, and their determination was essentially founded on the capacity portrayed in writing or the tissue-explicit articulation as detailed in articulation information bases. Devoted entire genome articulation exhibit investigation in examples from forensically significant body liquids that were put away for different time stretches has recently appeared to convey stable mRNA markers valuable for legal tissue distinguishing proof. Skin is an extra forensically significant cell type. As of late three mRNA records (LOR, CDSN, and KRT9) were accounted for to show high articulation in skin tests comparative with other forensically pertinent cell types. The expansion of skin markers to an RNA-based cell-typing multiplex would expand the scientific estimation of the test for two reasons: (1) a more complete view on all cell types present in an evidentiary follow is set up which is significant in light of the fact that skin cells are required to happen in numerous wrongdoing scene tests, and (2) a sign for the presence of contact DNA can be gotten. The regularly utilized strategy to show the presence of contact follows is through dactyloscopy unique mark examination, yet additionally other microscopical and immunohistological strategies have been depicted which can recognize skin cells. Unique mark representation techniques don’t make a difference to a wide range of substrates, and some can effectively affect the nucleic acids in the skin cells, while others can present pollution. To effectively and unbiasedly evaluate the organic starting point of a measurable evidentiary follow, a solitary profiling examination was built up that tests both the forensically pertinent body liquids and skin cells (Butler et al. 2001). The amino acid sequences of some proteins vary from person to person. The utilization of multi-protein polymorphisms can lead to some differentiation when there is a coincidence probability of two unattached persons one of the hundreds of hundreds. Before DNA testing became available, protein profiling was the basic technique that was performed in forensic laboratories as a raw way to differentiate

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Fig. 3 Overview of basic steps involved in DNA Analysis

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Collection and storage of samples

Extraction of DNA

Quantitation

Amplification

Separation

Detection

between the samples (Q and K), and the serum contains several isozymes. These isozymes are the different types of a protein enzyme that can catalyze the same biochemical reaction even though it has slightly different amino acids acid sequences. However, there are usually only two or three forms of each isoenzyme, which make them quite weak at distinguishing humans. Electrophoresis of starch gel, agarose gel, and polyacrylamide gel divide these proteins into differential alleles. In the early 1980s, many laboratories have started isoelectric-focused polyacrylamide gel electrophoresis (IEF), which has a higher resolution than protein electrophoresis because it creates smarter stripes (Fig. 3).

RFLP-Based DNA Testing When the RFLP process was first introduced, it was lengthy and took several weeks to complete. For DNA extraction, a biological sample was collected such as blood. The extraction of the DNA was done from the cells by disintegrating the cell membranes and removing the protein layers around DNA. To slice the to cut the long, extracted DNA molecules into smaller pieces, a restriction enzyme was utilized. These enzymes possess the capability of discovering and cleaving specific DNA sequences. In the next step, agarose gel is used to separate e these DNA fragments. These fragments are put into the deep areas called, “wells” in a gel that

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oats in a buffer solution. This solution is placed in a chamber between two electrodes, and an electric current is passed through it, which causes the movement of negatively charged fragments toward the positive end. DNA fragments of smaller size (shorter fragments) move faster than the bigger ones. This is how the separation of DNA fragments is done into bands, on the basis of their size. Southern blotting was done after the separation of these fragments, on the basis of their size. In this technique, the DNA fragments that were separated before were transferred to a nylon membrane, after bringing the membrane in contact with the gel. These DNA strands were turned into single-stranded strands by the action of alkaline solution. UV light is then fixed onto the membrane of one of the strands by cross-linking the DNA onto the membrane with UV light. This “UV light” is also called a radioactive or chemiluminescent probe. These probes consist of a VNTR sequence, which hybridizes the DNA attached to the nylon membrane (Butler 2015a). Hard binding occurred at the appropriate hybridization temperature and ionic strength, allowing the classified probe to detect its complementary set with complete accuracy. Strong binding occurred at acceptable hybridization temperatures and ionic strength, allowing the labeled probe to search for its complementary sequence with complete accuracy. The additional probe is then removed after repeated washing of the membrane. In the last step, the position of the probe was determined by inserting a membrane-bound to an X-ray film. Relative molecular analysis in the human genome and sets of sequences of bases are repeated varied times. Such repeated sequences are known as minisatellites. Minisatellites show a really high degree of cistron variation within the range of repeat units and consequently in their length. Hence, they type the idea of differentiating or distinctive people supported this length polymorphism at the molecular level by mistreatment either multi-locus or single-locus probes. The use of just one multi-locus probe will offer adequate numbers of variable bands that establish the positive identification of an individual. Thus, it’s one powerful take a look at positive matching of body tissues and determination of parentage. The chance of getting identical patterns of bands from the deoxyribonucleic acid of two people mistreatment multi-locus probe is of the order of one in 1014 to 1030 that is over 5X109, the overall world population. Thus, the DNA prints obtained from multi-locus probes are extremely unique. Single-locus tests or a combination of at least two tests are utilized for distinguishing variety at a particular minisatellite locus. A solitary locus test uncovers an example of up to two bands though a combination of a few tests uncovers multiple bands. These tests are profoundly delicate and thus can be utilized for little and even incompletely debased examples of DNA. These are helpful in distinguishing proof of blended examples as in instances of various assaults.

PCR-Based Tests PCR or polymerase chain reaction is an enzymatic process in which a specific piece of DNA is simulated at several times to obtain multiple copies of the specified

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sequence. PCR was first described by Kary Mullis in 1985. PCR is a molecular process, and it consists of “photocopying” which refers to heating and cooling of the sample to the exact thermal cycle pattern for ~30 cycles. One copy of the target DNA sequence is generated for each cycle, and the target sequence contains the molecule. It proved to be more suitable than the previously used MLP and SLP systems. There was a time when SLP and PCR techniques were used in parallel to identify a person. PCR technology has been used to enter most of the VNTR region for forensic purposes. Several polymorphic loci were used for analysis, such as D1S80 and HLADQ (Butler 2015) (Fig. 4). DNA process technology took a dramatic modification in the early 1990s. With the arrival of a new technique the polymerase chain reaction (PCR), the tiny amounts of template DNA fragment got amplified. The strategy was quick, dependable, and was proficient to work even with deteriorated examples In the PCR-based procedure the time needed to investigate an example was essential. For the recognition of various molecules from one another, separation needs to be performed so as to pull the fragments on the basis of their sizes. Electrophoresis is the most commonly used separation method, and it is conducted in a gel or capillary manner. The word “electrophoresis” originates from the Latin word “phore” which means bearer and the Greek word electron which means charge. In this manner, the cycle of electrophoresis alludes to electrical charges (positive and negative ions) transported by the molecules. On account of In the DNA structure, the phosphate groups are basically the backbone and consist of the negative charge. In nucleic acids, the phosphate groups donate H+ ions, and it turns them into negatively charged acid in buffers. When an electric field is applied to it, these DNA molecules will move away from the negatively charged electrode (a cathode), and it will move toward a

Fig. 4 Steps in PCR process

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positively charged electrode (an anode). The speed of movement of DNA fragments depends upon the voltage. If it’s high, then the fragments will move faster as they will feel more force. The migration of particles in an electric field leads to the production of heat. This heat that is generated must be dispersed or it will be consumed by the system. Extreme heat can cause a gel to soften and self-destruct. Two kinds of gels are regularly utilized in molecular and forensic DNA analysis for the purpose of separation of DNA. Agarose gels are used as they have generally huge pore sizes and are utilized for isolating bigger DNA particles, while polyacrylamide gels are preferred to be utilized to acquire high separation of smaller DNA particles, generally under 500 or 1000 bp. In Forensic DNA typing, usually techniques the two kinds of gels are utilized. Restriction fragment polymorphism (RFLP) techniques use agarose gels to isolate DNA pieces going in size from ~600 bp to ~23,000 bp. Low-atomic-weight DNA particles are not all well isolated with agarose gels. Then again, PCR-amplified STR alleles, which range in size from ~100 bp to ~400 bp, are better served by polyacrylamide gels. On account of some STR loci that contain micro variants, the high-goal capacity of polyacrylamide gels is fundamental for isolating firmly measured DNA atoms that may just contrast by a solitary nucleotide (Carracedo Angel 2005). In multiplex PCR process, more than two primer units designed for amplification of different objectives are protected inside the equal PCR reaction. Using this approach, more than one goal sequence in a medical specimen can be amplified in a single tube. As an extension to the realistic use of PCR, this technique can save effort and time. The primers used in multiplex reactions ought to be decided on cautiously to have similar annealing temperatures and should be no longer complementary to every different. The amplicon sizes must be able to form bands that can be seen through gel electrophoresis. Multiplex PCR can be employed in two different ways, one being a single-template PCR response that uses several sets of primers to make bigger specific areas within a template, and the second one multipletemplate PCR response, which uses more than one templates and several primer sets inside the identical reaction tube. Although the usage of multiplex PCR can lessen charges and time to concurrently hit upon two, three, or more pathogens in a specimen, multiplex PCR is greater complex to expand and frequently is much less sensitive than single-primer-set PCR. The benefit of multiplex PCR is that a set of primers can be used so that false positives or negatives should not be there. Multiple regions can be copied through the polymerase chain reaction as it lets in concurrently by truly adding one or more primer set to the response aggregate. This simultaneous amplification of DNA and its units is generally referred to as multiplexing or multiplex PCR. The primers should be similar so that a multiplex response should work properly. The primer-annealing temperatures should be similar, and excessive areas of complementarity must be avoided so that the primers bind to each other and not to the template DNA. Any addition of a new primer in a multiplex PCR reaction leads to an increase in the complexity of feasible primer interactions (Butler et al. 2001). Practically, multiplex PCR optimization is better than singleplex reactions due to the fact such a lot of primer-annealing events should occur without interference with each other. For the purpose of achieving a balance

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among the amplicons, various loci should be amplified. Primer sequences and concentrations alongside magnesium concentrations are normally the maximum essential to multiplex PCR. Obtaining successful co-amplification with nicely-balanced PCR product helps in identification (Cavanaugh and Bathrick 2018) (Table 2).

Capillary Electrophoresis Capillaries used for separation can generate results quickly in minutes as opposed to hours. This is because of the fact that higher voltages are allowed with better dissipation of the heat from the vessels. One of the major advantages of CE is that the quantitative data is promptly accessible in an electronic configuration following the finish of a run. No additional means such as filtering the gel or snapping a photo of it are required. Path following isn’t important since the example is contained inside the narrow, nor is there dread of the traverse from contiguous wells with CE. The one significant disservice of CE instruments is throughput. Because of the reality that examples are investigated successively each in turn, single fine instruments are not effectively fit for preparing high quantities of tests or test throughputs. Capillary electrophoresis (CE) is one of the most prominent improvements in the measurable DNA profiling work process, maybe second just to the creation of the polymerase chain response (PCR). The event of locales containing rehash successions inside the DNA particle and their polymorphic nature was found in the mid. In the 1990s, highly polymorphic (STR) markers supplanted VNTRs. At first, intensified STR parts were isolated utilizing piece gel electrophoresis and distinguished by silver recoloring. Creations in uorescent colors, uorescence location, and multiplex PCR made ready to the as of now utilized STR composing conventions. A CCT trio framework was effectively evolved in the mid-1990s for synchronous enhancement of CSF1PO, TPOX, and THO1 loci. Multiplexing permitted concurrent partition and location of numerous intensified pieces in a single path and now in one capillary (Sanger et al. 1977). The main features of CE are its adaptability; inorganic particles, natural atoms, and macromolecules can be isolated on a similar instrument – and much of the time a Table 2 Depiction of contrasts between the two main DNA typing methods, i.e., RFLP and PCR methods Properties and conditions DNA sample form DNA amount required Analysis time taken Sample mixtures DNA condition

PCR method Double or single-stranded DNA can work 0.1–1 ng 1–2 days Can be analyzed Degraded DNA can be analyzed

RFLP method Only double stranded DNA will work 50–500 ng Generally longer, about 1 week or 6 weeks (depending on different probes) Can be analyzed Only intact DNA can be analyzed

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similar slender – while changing just the piece of the running support and detachment medium. Consequently, CE is the most generally utilized diagnostic technique supplanting gel electrophoresis, elite uid chromatography (HPLC), gas chromatography, and other partition strategies. CE has a very high settling ability because of its fitting stream and insignificant dissemination. CE offers various focal points over section gel electrophoresis in scientific DNA investigation applications. Automated software for the purpose of data collection and its handling for a mechanized assortment of information is accessible. Real-time information can be conceivable as well as quantitative data can be inferred. Separation is always highresolution. Results are exceptionally reproducible and quite precise with this technology. In this method, only a little segment of an example is utilized, which can be, so it tends to be retested/reinjected as well, if necessary. And deleterious end waste products are much lesser. It is essential to take note that quantitative contrasts seen in the CE information may not actually relate to the quantitative contrasts in the first examples. Particular amplification may in uence the extents of enhanced DNA identified on CE, explicitly with respect to the minor supporter extent. The CE results include height and peak which are used for improvement of multiplex PCR responses (Giltay and Maiburg 2010). In theory, about a billion copies of the target areas on the DNA template are generated after 30 cycles. This PCR product is referred to as “amplicon” and therefore insufficient quantity which can be easily measured by various methods. PCR is usually performed with a sample volume of 5 to 100 μl. With such small volumes, evaporation can be problematic and result in precise dosing components that react can become problematic. There was a period when both SLP- and PCR-based techniques were utilized to distinguish an individual. PCR innovation was used to type numerous VNTRs locale for measurable purposes. PCR innovation was utilized to kind a few VNTRs district for logical purposes. Numerous polymorphic loci like D1S80 and HLADQ_ were utilized for investigation. The PCR reaction is prepared by combining various specific components. Purified water was added to it to obtain the desired volume and concentration of all ingredients. Commercial kits containing premixed ingredients are also utilized for PCR. Such kits are easy to use for forensic purposes. The two most important components of a PCR reaction are short DNA sequences that precede or “spin” the copied region. The primer is used to identify or “target” a portion of the DNA template used. It is a chemically synthesized oligonucleotide concentration versus DNA template for PCR induction. Some VNTR loci have moderately short size alleles, and they can be PCR-intensified. Locus D1S80 was utilized in a criminological arrangement for the Amp-FLP investigation. In this locus, the fragments are in the scope of 14–42 recurrent units (16 bp per unit). The enhanced sections were isolated by size utilizing polyacrylamide gel electrophoresis and distinguished utilizing silver stain. The discrete alleles are contrasted in a direct manner. The Amp-FLP procedure requires less DNA than the RFLP strategy and functioned admirably for corrupted examples. This locus can be investigated in multiplex style with an amelogenin locus for sex determination. Short tandem repeat (STR) a subclass of VNTR is a locale of human

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DNA containing 2–7 base sets (bps) rehash unit. These STRs are simple tandem repeats, or they are also referred to as microsatellites. A total of 105 STRs exist in the human genome out of which some STRs have been described explicitly for scientific DNA profiling. Priorly the STR profiling was completed utilizing polyacrylamide gel electrophoresis of the PCR item and recoloring the gel with silver stain. Economically accessible nylon strips to which DNA tests are appended are treated with PCR items. Consistently, longer expansion times can increment the yield of longer PCR items since less items are synthesized in long range PCR. The preliminaries (test) utilized in the response are named with an organic tag called biotin. On responding with a protein streptavidin/horseradish peroxidase (HRP) and tetramethylbenzidine (TMB) with hydrogen peroxide, blue shading spots show up at the site of an official of DNA succession with the test. These dot patterns correspond to alleles of the sample. The outcomes are recorded photographically. PCR amplification is done, either of the whole sample or a part of it with different primer sets. Residual dNTPs and primers are removed from PCR through turn filtration or enzymatic assimilation. After that PCR amount is calculated. DNA sequencing is executed in response to consolidate uorescent ddNTPs with every response containing an alternate primer to direct which stand is sequenced. Expulsion of unincorporated uorescent dye eliminators is done after the finished sequencing response. Partition through capillary electrophoresis instrument was carried out, and sequence analysis of every reaction is done. Single-nucleotide polymorphisms (SNPs) are utilized for forensic identification of humans, paternity testing, hair tone, and eye color ID in scientific analysis. Throughout the long term, various strategies have been created for SNP examination, for example, estimation of uorescence, iridescence, and subatomic mass. Most measures are done in arrangements or on strong framework backing, for example, glass slide, chip, or dab. Autosomal SNPs can be utilized for some sort of scientific testing including investigation of corrupted examples. SNP loci on the Y chromosome are an additional likely marker for paternity testing due to the low transformation rate (Harbison and Fleming 2016).

Y Chromosome DNA Testing Y chromosome DNA testing is significant for various utilizations of human hereditary qualities including scientific proof assessment, paternity testing, and verifiable examinations. It is important in terms of contemplating human movement patterns from the beginning of time and genealogical exploration. There are preferences and restrictions relating to Y chromosome testing when it comes to forensic examination and application. The essential estimation of the Y chromosome in legal DNA testing is that it is discovered as it were in guys. For the identification and determination of “male,” the SRY (sex-deciding area of the Y) quality is considered. Since a great number of violations and crimes where DNA proof is useful, cases such as rapes, include men as the culprits, DNA tests intended to just analyze the male part can be significant (Fig. 5).

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Fig. 5 Human genome and its classification

To completely use the Y chromosome for scientific purposes, it is important to see decisively what makes it quite a remarkable chromosome. The standard unavailability of Y chromosomes in females permits the utilization of the Y chromosome as a marker for human sex recognition, which can add accommodating data in forensic examinations. The precise male-specific inheritance of the NRY gives to explicitly investigate DNA segments that were only given by men and separate them from those given by females, which can be profoundly significant in blended stain examination in legal sciences, for example, in instances of rape. Simultaneously, inheritance free from recombination, from fathers to children, joined with low to direct change paces of most NRY-DNA (non-recombing portion of Y) polymorphisms, implies that male family members typically share similar NRY polymorphisms. This component has both favorable properties and drawbacks for legal applications of Y chromosome DNA. Impediments come in the manner that resolutions from Y chromosome DNA examination ordinarily can’t be made on an individual level, as wanted in the criminological examination. This is on the grounds that in case of a matching DNA profile between exhibits from a suspect and a crime scene the speculations that either the suspect or on the other hand, any of his paternal male family members, has left the crime scene sample to have the estimated possibility. Benefits are credited to mutual Y-DNA profiles between male family members; a close male relative (paternal) of an expired alleged father can be utilized

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to replace the father in paternity testing of a male posterity utilizing Y-DNA investigation in inadequacy cases, where autosomal DNA profiling regularly isn’t enlightening. A similar rule can likewise be utilized in disaster casualty ID of men utilizing close or father’s side male family members in situations where autosomal DNA profiling doesn’t work. The haploid quality of the NRY likewise prompts the Y chromosome to have a lower populace size than the autosomes, with four duplicates of autosomal loci comparative with each Y locus. This lower compelling populace size results in the Y chromosome showing the least hereditary variety of any chromosome. As a result of the lower compelling populace size, Y polymorphisms can be all the more unequivocally in uenced by hereditary oat or populace level occasions, for example, bottlenecks or author impacts than autosomal loci. Moreover, the uneven spread of unmistakable polymorphisms is supported by the patrilineal transmission of the Y re ecting certain social practices, for example, patrilocality (where guys hold their familial lands, with females migrating) or polygyny (low quantities of guys having the most noteworthy conceptive achievement) (Hedman et al. 2009). Drawbacks come within the way that conclusions from Y-chromosome DNA examination more often than not cannot be made on an individual level, as required in forensics. A larger part of the Y chromosome is moved legitimately from father to child without recombination to rearrange its qualities and give a more noteworthy hereditary assortment to people in the future. Arbitrary transformations are the main systems for variety after some time between paternal relations. Subsequently, while rejections in Y chromosome DNA testing results can help in measurable examinations, a match between a suspect and proof just implies that the person being referred to might have contributed the measurable stain, could it be a sibling, father, child, uncle, fatherly cousin, or a long distant cousin from his fatherly genealogy. Y-STRs change all the more quickly contrasted with Y-SNPs; Y-STR results show greater changeability and accordingly have more prominent use in legal applications. Commonly Y-STRs are portrayed as characterizing haplotypes, while Y-SNP characterizes haplogroups. As will be examined toward the finish of the part, Y-SNPs can be helpful in DNA parentage considers. With heredity markers, the lineage data from every marker is alluded to as a haplotype instead of a genotype on the grounds that there is typically just a solitary allele for each person. Since Y chromosome markers are connected on a similar chromosome and are not rearranged with every generation, the statistical measures for an arbitrary match likelihood can’t include the product rule. Hence, haplotypes acquired from genealogy markers can never be as successful in separating between two people as genotypes from autosomal markers. These autosomal markers are unlinked and isolate independently from one generation to another. Then again, the presence of family members having a similar ChrY grows the number of possible reference tests in missing people identification and mass calamity casualty ID proof. ChrY testing additionally helps in finding the family. Insufficient paternity tests where the father is dead or not available for the examination, can be conducted if ChrY markers are utilized. In any case, an autosomal DNA test is generally favored whenever possible since it gives a higher intensity of separation. The Y chromosome

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has additionally become a well-known technique for following recorded human movement patterns through male genealogies. YHRD is also known as the Y-STR Haplotype Reference Database is the largest and most widely used forensic and general population genetics Y-STR database; it was developed by Lutz Roewer and colleagues at Humboldt University in Berlin, Germany. US Y-STR database which is a population-specific Y-STR database (US Y-STR) was launched in December 2007 to enable haplotype frequency estimates on five different US groups using the 11 SWGDAM recommended loci. The original version of US Y-STR contained 4796 African American profiles, 820 Asian, 5047 Caucasians, 2260 Hispanics, and 983 Native Americans. In the rape cases where there are mixtures of DNA samples (both male and female), where the DNA levels of a female are high, male-specific testing can help in the assessment of a male culprit’s profile. Extra mixtures may perhaps be examined (e.g., fingernail scrapings, salivation on the skin, and so on) by this technique. Transmission through the paternal line from a father to the entirety of his male children expands possible reference exhibit suppliers and, furthermore, helps in finding the family ancestries. Since patrilineal family members are indistinguishable, Y-STR composing can’t be utilized to recognize among siblings or even far off male paternal family members. The product rule can only be used with the recombination between loci; without it, subsequently, the separation power of Y-STRs is restricted by the size of the populace information base utilized. Chromosomal duplication and deletions can muddle the examination (Kumar et al. 2016).

STR Typing STR profiling is a widely utilized technique for human identification in forensic applications such as criminal profiling, paternity testing, mass catastrophes survivors’ identification, legal casework forensic investigation, distinguishing proof of missing people, etc. According to this methodology, DNA is amplified from its sources for various human-specific polymorphic STR loci in a solitary PCR utilizing uorescently marked preliminaries. These amplified regions are then isolated by electrophoresis, the crude information is examined utilizing programming that decides the size of each enhanced section, and genotypes by correlation with alleles in an allelic ladder that are run on a similar plate (Fig. 6). For STR loci amplification, the primers in the multiplex frameworks are carefully planned to accustom the alleles of the loci amplified with the primers labeled with a similar dye and prevent their covering over one another. Utilizing primers marked with various dyes, it is conceivable to investigate loci creating amplified alleles with similar sizes of the fragments. STR sequence groupings are named on the basis of the length of the recurrent units. In dinucleotides, the two nucleotides are repeated; similarly, in trinucleotides, three nucleotides are repeated and so on. Tetranucleotide repeats have become the major STR markers for personal identification proof. STR repeats differ in the length

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DNA Extraction for biological materials such as blood, semen, saliva, hair, bones teeth etc. DNA quantification by using RT-PCR machine

DNA Amplification by using PCR machine.

Capillary Electrophoresis by using genetic analyzer

Data Analysis and Interpretation.

Preparation of DNA databse for future uses Fig. 6 STR profiling process

of the recurrent unit and the quantity of the. STRs are categorized further on the basis of their pattern of repetition. Basic STR units consist of units that have identical sequences and the same length. In compound repeats, two or more simple repeats are included, whereas, in complex repeats, various repeat units of variable length are included (Lindenbergh et al. 2012). Tetranucleotide STR loci are preferred over VNTR minisatellites for a variety of reasons. It provides a narrow allele size range which allows multiplexing, and it also lessens the allelic dropout. With the use of tetranucleotide STR loci, stutter product formation lessens as well. As of 2017, the CODIS loci consist of the following: CSF1PO, FGA, THO1, TPOX, VWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, D1S1656, D2S441, D2S1338, D10S1248, D12S391, D19S433, and D22S1045. Of the first 13 CODIS STR loci, the 3 most polymorphic markers are FGA, D18S51, and D21S11. TPOX, CSF1PO, and TH01 normally display a minimal measure of variation between people. CODIS was designed to examine a target DNA record against the DNA facts contained within the database. Once a suit is diagnosed via the CODIS software program, the laboratories exchange records to affirm the match and set up coordination among their two companies. The comparison of the forensic DNA file against the DNA document in the database can be used to establish probable cause to acquire an evidentiary DNA pattern from the suspect. The regulation enforcement organization can use this documentation to gain a court order authorizing the gathering of a regarded biological reference sample from the culprit. The casework laboratory can then perform a DNA evaluation at the known organic pattern so that this evaluation can be supplied as proof in court. An average STR profiling kit comprises of the main five parts such as:

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1. A primer mixture for PCR which consists of oligonucleotides intended to enhance or amplify STR loci set. These primers are labeled with the one of each pair of uorescent color. 2. A buffer mixture for PCR which consists of deoxynucleotide triphosphates, MgCl2, and different reagents that are required to perform PCR reaction. 3. An enzyme is known as DNA polymerase, which is usually premixed with the buffer mixture of PCR. 4. An allelic ladder consisting of the basic alleles for the amplification of STR loci for the calibration of allele repeat size. 5. A positive control DNA test to check that the unit reagents are working appropriately. Various packs are accessible for single or multiplex PCR enhancement of STR markers utilized in DNA composing. An allelic ladder basically refers to the artificial amalgamation of the regular alleles present in the human populace for a specific STR. They are produced with the equivalent primers as tested exhibits and hence give a reference DNA size to every allele in the ladder. Allelic ladders are significant for exact genotype determination. These allelic ladders act like a ruler for every STR locus. They are important to fit the various estimations achieved from various instruments and conditions utilized by different research facilities. Allelic ladders are made by the union of genomic DNA or locus-specific PCR items from various people in a populace, which has alleles that represent the variation of a specific STR marker. The tests are then co-enhanced to create an artificial exhibit containing the basic alleles for the STR marker. The amount of these alleles is adjusted by changing the information measure of each part with the goal that the alleles are represented equally in the ladder (McElfresh 1991). According to various researches, miniSTRs are highly sensitive and strong for the evaluation of low template and degraded DNA. These miniSTRs are extremely useful for the analysis of forensic samples and show the proportion of samples that advantage from evaluation with additional miniSTR loci in terms of ensuing in a user profile. Degradation of DNA leads to the fragmentation of DNA into smaller units known as template fragments. The smaller amplicon length of miniSTRs results in a better amplification of fragmented DNA. In addition to the abovementioned advantages, analysis of additional (mini)STR loci affords additional discriminative capability. Analysis of miniSTRs in forensic DNA samples has major advantages over the bigger-sized STRs. DNA that is degraded to fragments smaller than the mediumand massive-sized STR amplicons could nonetheless be detected with the aid of miniSTR analysis. Forensic DNA exhibits contain DNA that remains when someone comes into touch with an object. Typically, those samples comprise handiest minute contents of DNA. Many of those samples include degraded DNA due to publicity to the surroundings. For those motives, forensic DNA exhibits are few of the toughest samples to get usable DNA profiles. Its predicted that the utilization of miniSTRs for the examination of touch DNA samples outcomes in a better percent of DNA profiles due to the advantages of miniSTRs. The amplification of smaller amplicons typically

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produces greater efficient, growing the signal and sensitivity of the PCR technique. The impacts of the miniSTR evaluation implementation of can be studied by several methods (Menotti-Raymond et al. 2003).

Mitochondrial DNA Mitochondrial DNA or mtDNA refers to the circular genome found inside the mitochondria which are different from the nuclear DNA. Researches have uncovered that mtDNA is a round genome and it’s around 16,569 nucleotides long. A very much characterized, noncoding part of the genome endures the aggregation of mutations, which can be questioned to build up a forensic mtDNA profile. The profile data will frequently be useful when distinguishing missing people also unraveling various crimes. For instance, a correlation of mtDNA characterizations permits the relation of biological proof with one individual in a criminal case, while barring people who are not related in a similar case as the origin of DNA obtained from the scene of the crime. Although the maternal inheritance of the mitochondrial genome lessens the general separation capability of the mtDNA testing framework, family members from a similar maternal genealogy usually have the same profiles. In any case, the genome’s higher mutation pace can bring about the incidents of heterogeneous pools of mtDNA types that can fundamentally expand the intensity of segregation and can be communicated in contrasting proportions across maternal lines. The material introduced in this section gives a review of the forensically important attributes of the mtDNA genome and how these qualities can be utilized to respond to questions brought up in legal examinations. Recovery of DNA data from destructed DNA is possible in some cases with mitochondrial DNA or mtDNA. While a nuclear DNA test is normally more significant, a mtDNA result is superior to no outcome by any means. There are around hundreds of duplicates of mtDNA in every cell; the likelihood of getting a DNA composing result from mtDNA is higher than that of polymorphic markers found in nuclear DNA, especially in situations where the measure of extricated DNA is minuscule, as in tissues, for example, bone, teeth, and hair. At the point when remains are very old or seriously desecrated, bone, teeth, and hair are the main organic sources left from which DNA can be extracted. The essential trademark that leads to the recovery of mitochondrial DNA (mtDNA) from damaged examples is the higher duplicate number of mtDNA in cells comparative with the nuclear DNA from which STRs are enhanced. To put it plainly, however atomic DNA contains substantially more data, there are just two duplicates of it in every cell (one maternal and one fatherly), while mtDNA gives a touch of helpful hereditary data many occasions per cell. As a result of their higher numbers, some mtDNA atoms are bound to get by than nuclear DNA (Prinz and Lessig 2014). DNA molecules are situated inside the cell core, as chromosomes are one of the sources of DNA in present-day eukaryotic cells. Mitochondria found in the cytoplasm of most cell types contain a second intracellular DNA genome. As per the broadly acknowledged endosymbiotic hypothesis of mitochondrial emergence,

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mitochondria were gotten from α-Proteobacteria that lived around 2 billion years prior inside pre-eukaryotic cells looking like old protists. Throughout the course of evolution, the endosymbiont lost its capacity to live outside the eukaryotic cell, and parts of its related DNA were retained. The endosymbiont is converted into the mitochondria, and its genome is turned into mtDNA. The mitochondrion went on with the essential function of the creation of cell energy by methods for oxidative phosphorylation and the union of molecules of ATP. As the evolution process continued, the mitochondrion preserved various qualities associated with this significant biochemical cycle, while losing huge numbers of the tasks expected to stay alive as a totally independent solitary organism. The strands which are complementary, belonging to the mtDNA sequence, are precisely unique in their structure. The “heavy strand” consists of purine nucleosides (adenosine and guanosine), though the “light strand” consists of the wealthy in pyrimidine nucleosides (thymidine and cytidine). The coding locale includes roughly 93% of the genome, with the quality successions thickly orchestrated. There are 37 qualities found in the coding area grouping: 22 qualities for transport RNAs (tRNA), 2 qualities for ribosomal RNAs (rRNA, the 12S and 16S subunits), and 13 qualities for catalysts in the respiratory chain associated with the cycle of oxidative phosphorylation and, also, ATP creation. By far most of the human genome is situated inside the core of every cell. In any case, there is a little, roundabout genome found inside the mitochondria, the energydelivering cell organelle living in the cytoplasm. The number of mtDNA atoms inside a cell can go from hundreds to thousands. Normally there are 4–5 duplicates of mtDNA atoms per mitochondrion with a scope of 1–15. Since every cell can contain several mitochondria, there can be up to a few thousand mtDNA particles in every cell as on account of ovum or egg cells. The usual number has been assessed at around 500 in the majority of cells. It is this enormous number of mtDNA particles in every cell that leads to more prominent success as compared to the nuclear DNA markers, with the examples that may have been harmed or degraded with heat or moistness. Mitochondrial DNA consists of around 16,569 base pairs with the complete number of nucleotides in a particular mtDNA genome shifting because of little insertions or deletions. For instance, there is a dinucleotide rehash at positions 514 to 524, which in most people is ACACACACAC or (AC)5, however has been seen to change from (AC)3 to (AC)7. Note that with two duplicates of atomic DNA (3.2 billion bp from each parent) and in any event, expecting that there are 1000 duplicates of mtDNA or 16,569 bp per mtDNA in a cell, mtDNA makes up just about 0.25% of the all-out DNA content of a cell. The majority of the concentration in scientific DNA studies to date has included two hypervariable locales inside the control district generally alluded to as HVI (HV1) and HVII (HV2) (Slatko et al. 2018). Every so often a third part of the control area, known as HV3, is analyzed to give more data concerning tried example human mitochondrial DNA is only inherited from the maternal side. During the process of conception, just the sperm’s core enters the egg and joins with the egg’s core. When the zygote cell partitions and a blastocyst is created, the cytoplasm and other cell parts spare the nucleus with the mother’s unique egg cell. Mitochondria with their mtDNA cells are passed straightforwardly to all posterity free of any male impact.

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Subsequently, the mutation does not occur, a mother passes along her mtDNA type to her children, and so the siblings and the maternal family members have an indistinguishable mtDNA sequencing. Henceforth, a person’s mtDNA type isn’t one of a kind to the person in question. Mitochondrial DNA variety is broadly studied in different branches of science. Clinical researchers have connected various sicknesses to changes and mutations in mtDNA. Evolutionary scientists look at human mtDNA arrangement variety comparative with different species with an end goal to decide connections. A genuine illustration of this application is the assurance that Neanderthals are not the immediate predecessors of current people dependent on control area groupings decided from antiquated bones. Anthropologists study contrasts in mtDNA groupings from different worldwide populace gatherings to look at inquiries of the family line and movement of people groups all through history. Several papers have been distributed in these fields over the recent many years. Hereditary genealogists are currently utilizing mtDNA and Y chromosome markers while trying to follow lineage where paper trails run cold. In the previous few years, various intriguing recorded IDs have been performed with the guide of mtDNA testing. Steps in mitochondrial DNA analysis are as follows: The mtDNA is extracted from the sample, and then it is amplified by PCR and HV1 and HV2 regions. These HV1 and HV2 amplicons are sequenced, and this sequence is confirmed with both forward and reverse strands. Differences are noted from the reference sequence. Meanwhile, another process must be carried out separately which includes the extraction of DNA from the reference sample. The same abovementioned steps are repeated, and the differences are noted from the Anderson or reference sequence. These sequences that are obtained by both analyses are then compared with the database to determine the haplotype frequency estimate. Steps for assessment of mtDNA exhibits are as follows : The evidence or questioned exhibit comes from a crime scene or a mass calamity. The reference or known exhibit might be a maternal family member or the suspect in a criminal examination. In a criminal forensic examination, the victim may likewise be examined and compared with the questioned and known outcomes (Sullivan 1994). PCR duplication of mtDNA is normally completed with 34–38 cycles. Conventions for profoundly desecrated DNA examples even call for 42 cycles. In some cases, more Taq is added to beat PCR inhibitors, for example, melanin. It is significant to remember that sensitivity is boosted with mtDNA testing since it is the last attempt to acquire DNA results from an exhibit. The higher the affectability of any assay, the more prominent the possibility for spoiling and consequently more care and consideration are normally needed with mtDNA work than with regular STR composing. Major mtDNA varieties between the human populace are found inside the control locale or displacement loop (D-loop). Two points inside the D-loop are called hypervariable points I (HV1, HVI, or HVS-I) and hypervariable locale II (HV2, HVII, or HVS-II) which are usually analyzed by PCR enhancement followed by sequence examination. Roughly 610 bp are ordinarily assessed – 342 bp from HV1 and, furthermore, 268 bp from HV2. For DNA sequencing, the Sanger technique was introduced more than 30 years back (Sanger et al. 1977). This Nobel Prize-winning sequencing procedure is still

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broadly utilized, and the technique includes the polymerase fusion of dideoxyribonucleotide triphosphates (ddNTPs) as chain terminators and then by a separation step equipped for single-nucleotide resolution. There is no hydroxyl bunch at the 3’finish of the DNA nucleotide with a ddNTP, and, subsequently, chain development ends when the polymerase joins a ddNTP into the synthesized strand. Extendable dNTPs and ddNTP eliminators are both present in the response blend so that a few bits of the DNA particles are broadened. Toward the finish of the sequencing response, a progression of molecules is available that vary by one base from each other. In the Sanger sequencing measure, every DNA strand is sequenced in independent responses with a solitary groundwork. Frequently either the forward or reverse PCR primers are utilized for this reason. Four diverse shaded uorescent colors are connected to the four distinctive ddNTPs (Yang et al. 2014). Heteroplasmy is characterized as a combination of more than one mtDNA genome sequence inside a cell or between cells of a solitary individual. As they gather, these heteroplasmic variations can prompt illnesses and can profoundly affect the process of aging. In a scientific analysis procedure, these variations can give extra layers of segregation potential to help distinguish human remaining parts and have become a standard for the examination procedure when investigation on exhibits is conducted in forensic case analysis. Accordingly, in light of the fact that the presence of heteroplasmy can affect the evaluation of mtDNA examinations among evidentiary and exhibits that are known experienced in criminological casework, it is imperative to comprehend the biological premise of heteroplasmy, how regularly it is noticed comparable to the sequence identification strategy being utilized, and how best to decipher the results. Since most eukaryotic cells contain hundreds to thousands of duplicates of mtDNA, it is conceivable and without a doubt likely that irregular mutations exist in various duplicates of the mitochondrial genome all through a person. The mtDNA has a higher rate of mutation than the nuclear DNA genome. Despite the fact that it is regularly estimated that the high rate of mutation is because of lesser fidelity in the γ-polymerase utilized for the genome replication, it ends up that this adaptation of the replicative DNA polymerase is very loyal. All things considered, the probable origin of mutation is from DNA destruction instigated by responsive oxygen species (ROSs) produced during oxidative phosphorylation, trailed by the activities of a not exactly satisfactory fix framework.

X Chromosome Analysis The X chromosome (ChrX) has potential criminological scientific and human identification applications because of its pattern of inheritance as compared with other hereditary markers. Usually, males have one X chromosome and one Y chromosome, while females have two X chromosomes despite the fact that there are infrequently some sporadic karyotypes, for example, XXY (Klinefelter disorder; Giltay and Maiburg 2010), XXX, and XYY. More than 40 STR markers have been described from the X chromosome, and populace examinations have been performed with huge numbers of these X chromosome STRs (X STRs). X chromosome STR

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composing can be useful in some familial relationships examination circumstances especially with insufficient paternity situations where a DNA exhibit from one of the guardians isn’t accessible for testing. For instance, if a father/girl child parentage relationship is being referred to, X STRs might be useful because of the 100% transmission of the dad’s X chromosome to his little girl. Then again, in a father/boy child parentage question, Y chromosome results would be useful records a few applications for X chromosome DNA testing. ChrX testing can be particularly useful in some missing people or catastrophe casualty distinguishing proof circumstances where direct reference tests are not accessible; what’s more, organic family members must be tried to help human identification proof. Autosomal short tandem repeat or STR examination has become the foundation of forensic personal identity testing since the 1990s when the first uorescently marked STR markers were portrayed. From that point forward, a large number of polymorphic STR markers have been explained, and at present, a number of combinations of autosomal markers permit the strict ID of people to the level of one out of trillions. In missing people and insufficient paternity cases, Y chromosomal STRs grow the pool of family reference tests that can be utilized to affirm character and have demonstrated valuable in circumstances where the male DNA is mixed with female DNA and female DNA is present in much larger quantity as compared to male one as would be the situation with a vaginal swab from a rape case (Weedn 2007). In the past few years, STR markers situated on the X chromosome have arisen as an extra tool in this forensic scientific analysis. X chromosomal STRs can be utilized to enhance STR profiling due to their inheritance pattern, and, correspondingly, the expansiveness of distributed writing regarding the matter has extended incredibly lately. STR markers on the X chromosome might be valuable for forensic purposes. To start, missing people cases generally require the examination of family members due to an absence of direct reference material. Regularly, mitochondrial DNA (mtDNA) composing can be utilized to address the potential for destructed or damaged exhibits, for example, skeletal remaining parts, especially in shut populaces also, when a direct maternal reference is accessible, as a result of its moderately high duplicate number and ensured area inside the mitochondria of the cell. Nonetheless, mtDNA is maternally acquired; accordingly, where maternal references are inaccessible or where the unidentified individual matches one of the most wellknown mtDNA haplotypes, mtDNA testing alone might be lacking. In such cases, markers on the X chromosome may give extra data. X chromosomal STRs can be especially helpful for any parent-offspring relationship that includes one female or more (e.g., father-little girl, mother-child, or motherlittle girl), for instance, the related family of parents with one son who is the father of a little girl. In this situation, if that son and his wife are unable to come for testing, it very well might be important to utilize the grandparents’ DNA profiles to reassociate their granddaughter. In this particular situation, autosomal STRs by and large give a low probability proportion of a relationship since there is on normal just one-quarter sharing of alleles between a grandparent and a grandkid. X chromosomal STRs, then again, end up being more helpful since the X chromosome of the girl child was

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acquired altogether from his mom’s genome with no commitment from his dad. This X chromosome was then passed in full to the granddaughter. In this model, one would anticipate seeing one allele from every X STR marker of the grandma present in the granddaughter’s X STR profile; hence, X chromosomal STRs will no doubt beat autosomal STRs. Other maternally related situations, for example, distinguishing cousins or auntie niece connections utilizing X chromosomal STRs to replace or expand autosomal STR testing, have been proposed. Mutations generally happen because of strand slippage during DNA replication, and they are responsible for the significant system of the serious level of polymorphism found in human microsatellites. Single-step mutations such as deletions or insertions of one recurrent unit are most occurring, and they in uence longer alleles more often than shorter ones. This pattern is noticed for markers on the Y chromosome, and no distinction is normal on the X chromosome. Mutation rates can be varied and in the comparison, among men and women, with one approximation of the proportion of mutations from paternal to maternal changes at 17:3. Mutation rates can likewise uctuate with the populace. As the quantity of new markers depicted in the writing expands, the potential for classification contrasts additionally increments. There are 36 X STRs that are ordinarily utilized by distinctive scientific research centers, and, sometimes, contrasts in allele terminology make correlations of distributed populace information between the research centers, best case scenario monotonous and to say the least unimaginable (Yang et al. 2014).

Microbial and Animal Forensics Forensic DNA profiling is usually done for the investigation of perpetrators which basically involves human DNA and that DNA is important in proving the innocence or guilt of the suspect. But there can be other DNA also, apart from the human ones that might be valuable in exhibiting the innocence or guilt of an individual associated with a crime. Pets and domestic animals’, for example, felines (cats) and canines (dogs), hair might be utilized to establish a link between the suspect and a crime scene. Showing that an herbal exhibit belongs to a particular plant from a specific plant can help in linking of a suspect to the scene of the crime. It may also help in showing that the body of an expired casualty may have been moved from the homicide site. DNA testing would now be able to be utilized to connect the origins of cannabis. One of the major applications of forensic DNA typing in the future scenario would include the examination of materials such as anthrax (bacterium Bacillus anthracis) which can be used for bioterrorism activities (Slatko et al. 2018). Such an attack of bioterrorism happened in the United States. Quite simply, Bacillus anthracis bacterium spores were sent to media and offices, through the mail. It caused about 22 contaminations and, also, 5 deaths. As the aftereffect of this assault, the world got mindful of a weakness in which numerous in the field of counterterrorism was very discerning. Bioterrorism is a genuine danger. The utilization of the US mail as a dispersal vehicle uplifted concerns since it showed that a mail delivery could be utilized to uncover individuals to a fatal microbe (an infection

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causing specialist. The Federal Bureau of Investigation (FBI) of USA, is the organization with the essential duty to explore the crimes, were not well set up to attempt the criminological examinations identified with the examination of the Bacillus anthracis assault; supporting criminological science examination of microbial proof was amazingly restricted at that time. Animal and plant testing in the forensic analysis is relatively new as compared to conventional analysis. Illegal trafficking and trade of endangered species have led to the realization that such crimes are not of any less important than the others. A cat has 18 sets of autosomes (nonsex chromosomes) and the sex chromosomes X and Y. For each of the Felis catus chromosomes, genetic markers have been established. MeowPlex is a group of STR markers, and it consists of 11 STRs on 9 distinct autosomes. In this assay, an extra marker was added for the identification of the gender by the augmentation of PCR primers. These primers are specific for the SRY gene on the feline (cat) Y chromosome. For the process of amplification, the PCR items are in between the size scope of 100 bp to 400 bp and utilize three dye color tones. Cat STR allele frequencies from pet felines have been studied to show the distinctive feature of DNA profiles in forensic examination. An ongoing quantitative polymerase chain response (PCR) test (see ▶ Chap. 6, “Tools and Techniques Used in Forensic DNA Typing”) for assessing the DNA yield extricated from homegrown feline examples has been created (Menotti-Raymond et al. 2003). This test is fit for distinguishing down to 10 femtograms of cat genomic DNA and uses high duplicate number short interspersed atomic components (SINEs). A large number of these pets and domestic animals usually shed hair, and hence these hairs could be gotten or abandoned at the scene of the crime by a culprit. The perpetrator may unconsciously have cat or dog hairs on their clothes or shoes, from a victim’s pet. They could have carried it away, and this might help in connecting the perpetrator to the victim and the crime scene. Animal DNA pieces of evidence can indicate to three different explanations: the animal could be a victim, a culprit, or a witness. DNA testing can help in cases of animal abuse or animal trafficking cases. The remaining evidence of a lost pet can be recognized through genetic or DNA examination. Normally genetic markers such as short tandem repeats (STRs) and mitochondrial DNA (mtDNA) are analyzed just like how human DNA is examined. In cases the point when creatures are associated with an assault on an individual, DNA composing might be utilized to recognize the creature culprit. In case that the victim has perished, at that point DNA proof might be the main evidence that the animal has done a particular crime. DNA testing of animals can “absolve” innocence of living beings (other than humans) so that they are not unnecessarily decimated. Animal DNA has been utilized effectively to interface suspects to wrongdoing scene. Research has been conducted on the exchange of animal hair during stimulating criminal conduct found that several feline hairs or canine hairs could be moved from the homes of casualties to a thief or an attacker. The number of hairs discovered was high to a great extent that it is practically impossible to find a house where a domestic pet lives, without being “polluted” by hairs of the pet (cats or dogs), when the proprietor depicts their animals as not a good source of the hair. The issue with

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the hairs which are shed regularly is that they don’t contain roots, so nuclear DNA may not be available in adequate amounts for STR typing. Mitochondrial DNA might be a more suitable option for a considerable lot of these sorts of shed hair to get transferred. Previously, forensic analysis of animals or animal hair was based upon morphological models’ species-level identification. Isoelectric centering of keratins were the principal endeavors utilized toward the molecular identification of animal samples. Genetic individualization was done by utilizing profoundly recurring minisatellite loci to create singular explicit “fingerprints” of human DNA. Jeffreys et al. were additionally the first to illustrate the capability of DNA fingerprinting of animals by utilizing human minisatellite DNA to produce multi-locus DNA fingerprints of canine and feline DNA. The Locard’s Exchange Principle permits forensic investigators to interface unique source and target surface and has a point of view on primary and secondary movement. The animal proof isn’t excluded from Locard’s standard and has become important for recognizable proof and individualization of follow or move biomaterial from crime scenes. The DNA examination of animal’s biomaterial, nonetheless, just gives crucial analytical leads and remaking of the scene of the crime; the examination of animal DNA can’t add to the individualization of any human suspect, and as a rule, this sort of proof should be enhanced by different types of actual proof. For the production of the forensic DNA typing model, 49 tri-and tetranucleotide STR loci were disengaged from felid STR-enhanced genomic libraries. Tetranucleotide STRs have been utilized for human profiling since they limit the generation of “stutter band” items created during PCR by amplification which can convolute the translation of genotypes from mixed DNA exhibits. The loci were included in the genetic maps of the domestic feline comparative with 579 coding qualities and 255 STR loci to choose unlinked markers. Thusly, the loci were screened in a little board of outbred felines and 28 feline varieties (3–10 creatures/breed, n ¼ 213), to distinguish a board of markers with the most elevated separating power. A bunch of 11 exceptionally polymorphic loci was at first chosen for the typing process. The loci were unlinked, exhibited high heterozygosity over various feline varieties, and, furthermore, demonstrated the absence of cross-species amplification. A multiplex amplification procedure was composed so the loci could be amplified with as meager as a nanogram of DNA. It also incorporated a gender recognizing STS on the Y chromosome, enhancing a part of the SRY quality. The PCR results of the 11 loci were planned in a size ranging from 100 to 415 bp, named with one of 4 uorescent labels, with no allele cover with adjoining loci, and the SRY item noticeable at 96 bp. Approval investigations of the multiplex exhibited that total item profiles could be created with as meager as 125 pg of genomic DNA, with a nonappearance of “allele dropout.” A database of the genetic information of the domestic cat varieties has been created from the multiplex with which to register composite match probabilities. In contrast to felines, which can be forceful yet infrequently dispense critical injury, canines can be culprits that produce critical injury; a canine scientific genetic testing unit was created. This kit is known as the Finnzymes Canine Genotypes™ 1.1 Multiplex STR Reagent pack which was created and approved utilizing a board of canine-explicit STRs utilizing

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species testing utilizing loci on the mitochondrial genome which has become a standard technique in preservation biology and phylogenetic examinations (Harbison and Fleming 2016). The danger of a bioterrorist assault has been the subject of worldwide concern. Various US public intelligence estimates and reports have inspected the issues. Because of current innovation and an enormous expansion in information gathered about atomic science of microbial specialists, researchers can all the more viably and proficiently distinguish microorganisms from an assortment of lattices to incorporate those from the climate. Progress around there has been important to the customary general well-being network for an extremely long time. It is likewise important to those networks that will be liable for attribution following a criminal demonstration or psychological oppressor assault utilizing a bioagent. To eventually distinguish the people or associations liable for such an assault will probably require thorough utilization of current microbiological logical apparatuses combined with conventional measurable orders. Proof gathered as a component of an organic attribution examination will yield exceptional kinds of microbiological proof that might be explicit to the idea of the assault. As instances of such microbiological proof, they referred to the accompanying: feasible examples of the microbial specialist, protein poisons, nucleic acids, clinical examples from casualties, research facility gear, scattering gadgets and their substance, natural examples, tainted apparel, or follow proof explicit to the cycle that created as well as weaponized the organic specialist. This gathering recognized that there was a requirement for research and, also, advancement endeavors to improve current capacities in microbial crime scene investigation and that this would require exertion among numerous components of the public government. The proposals found in this public procedure might be applicable to numerous nations endeavoring to all the more likely plan for the danger of bioterrorism. Regardless of whether for a general well-being or a bioterrorism occasion, the distinguishing proof, assortment, and investigation of suitable examples are a basic essential for effective discovery of organisms. Not exclusively can clinical material from tainted patients be used for the recognition; however, regularly creature or natural examples will yield basic data. For fruitful ID of dubious specialists, it is critical to utilize both exemplary microbiological methods and current atomic science procedures microbial forensics and, likewise, with other customary legal controls, requires execution and also use of conventions and practices that will eventually yield results that can be utilized either by leaders in law implementation or potentially public/global security. Recognizable proof of fitting examples, “chain of care” records (documentation that tracks actual control of tests), the utilization of legitimate logical conventions via prepared staff, adherence to quality confirmation gauges, and guaranteeing the protected capacity and safeguarding of tests – all are of basic significance for effective attribution examinations. Fairly extraordinary to this sort of proof is guaranteeing the well-being of anybody associated with the assortment and treatment of microbial scientific proof. When managing pathogenic microorganisms, there are extraordinary dealing with techniques that should be continued to satisfactorily forestall extra mischief to staff and the encompassing network. All staff engaged with reaction or treatment of these

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bioagents should go through specific preparation to guarantee the well-being and security of the people included and the general climate.

Next-Generation Sequencing NGS is primarily a high-throughput system for DNA sequencing, also considered as cutting edge sequencing. It has quickly advanced in the course of recent years, and new strategies are consistently being popularized. As the innovation grows, so do increments in the quantity of comparing applications for essential and applied science. This NGS innovation, with its high-throughput limit and minimal effort, has quickly become popular, and it has turned into a significant insightful instrument for some genomics scientists. In the field of forensic scientists, new opportunities have been emerged crediting to the NGS technology. NGS innovation can be applied various areas of scientific interest regarding genetic contexture, for example, mitochondrial, autosomes, and sex chromosomes. Besides, NGS innovation can likewise have expected applications in numerous different parts of exploration. These incorporate DNA information base development, phenotypic derivation, monozygotic twin examinations, body liquid and species recognizable proof, and criminological creature, family line/ancestry, and plant and microbiological investigations. Here we audit the use of NGS innovation in the field of legal science with the point of giving a reference to future criminology studies and practice. This technology alludes to nonSanger-based high-throughput DNA sequencing innovation. Millions or billions of DNA particles can be sequenced in equal, consequently expanding the throughput considerably and limiting the requirement for the fragment cloning technique that is frequently utilized in Sanger sequencing. It incorporates second-age sequencing innovation dependent on circle exhibit sequencing, which can examine countless examples at the same time, just as third-age sequencing innovation, which can decide the composition of single DNA particles. Roche presented the 454 Genome Sequencing System, the world’s first pyrosequencing-based high-throughput sequencing framework, in 2005. The initial 454 Genome Sequencer was equipped for creating around 200,000 peruses of 110 base sets (bp) long. To begin with, these advancements don’t need bacterial cloning of DNA parts; all things being equal, they depend on the readiness of NGS libraries in a sans cell framework. This technique, rather than many sequencing responses, can parallelize the thousands-to-many-a large number of sequencing response. The sequencing yield is generated thorough NGS straightforwardly identified with no requirement for electrophoresis. The tremendous number of peruses produced by NGS empowered the sequencing of whole genomes at an exceptional speed, and subsequently it came to be generally utilized in different fields of life sciences. Be that as it may, one disadvantage of second-age sequencing innovation is their moderately short understand lengths, which has brought about challenges in ensuing succession grafting, gathering, interpretation, and bioinformatics examination. Besides, standard PCR was used to arbitrarily intensify genomic sections during library planning. Due to the complex structure of genomes, factors, for

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Fig. 7 Next-gen sequencing steps

example, auxiliary structure and warm dependability, will in uence the productivity of PCR enhancement. The utilization of DNA techniques in forensic examinations has delivered DNA investigation a significant device in forensics. In the forensic DNA investigation, contrasted with different fields of life sciences, DNA analysis is based onto the low copy number, exceptionally debased and defiled examples, so there is the requirement for high precision and reproducibility, and time and cost examination. Today, most of scientific DNA tests utilize PCR- and narrow electrophoresis (CE)-based part investigation techniques to distinguish length variety in short tandem repeat (STR) markers. The CE-based Sanger sequencing has been utilized to examine explicit locales of mitochondrial DNA (mtDNA) (Fig. 7).

Conclusion This chapter analyzed the study of DNA typing methods and techniques used at present for the examination of DNA. It focused on the biology basics, technology, innovation, and genetic characteristics of short tandem repeats markers or (STR) markers. These markers incorporate the most well-known forensic DNA investigation techniques utilized today. The materials in this chapter are intended to provide information basically for forensic scientists and law professionals, and it describes the complex examination methods with simple language, for a clear understanding

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of DNA profiling. This content ought to likewise straightforwardly profit understudies studying criminological DNA investigation in a scholastic climate. These principles aims to improve the nature of work performed in scientific research centers by requiring specialized supervisors and DNA analysts to have preparing in organic chemistry, hereditary qualities and atomic science in request to pick up a fundamental comprehension of the establishment of criminological DNA examination. The research publications have developed significantly on the subject of STR composing and its utilization in criminological DNA testing. New points, for example, single-nucleotide polymorphisms (SNPs) and Y chromosome testing include picked up more noteworthy acknowledgment inside the legal network. A very thorough gander at mitochondrial DNA and its application to legal DNA examination is explained. There is refreshed data on new DNA extraction methods, ongoing PCR for DNA evaluation, and multi-narrow electrophoresis instruments that are currently utilized in numerous forensic DNA research facilities.

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Evaluation of the Autosomal STR Markers and Kits Vikash Kumar

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Autosomal STR Marker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nomenclature for DNA Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Types of Autosomal STR Marker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CODIS Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Autosomal STR Multiplex Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Earlier STR Multiplex Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . New Generation Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Forensic Relevance of Amelogenin Marker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Forensic Validation of Autosomal STR Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Usefulness of Autosomal STR Kits in Forensic Practice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

DNA profiling is one of the most important and ultimate techniques for identification of the individuals, biological dispute, family history, pedigree chart, etc. DNA profiling was developed by Dr. Alec Jeffrey and was first used to assist police in identifying a suspect in a rape case. Initially, it was based on RFLPbased VNTR markers. Later on, STR markers was widely accepted by scientific community to analyze the specific region of DNA for more discriminatory power and more precise results. Forensic Science Services, England started using STR markers to analyses specific region of DNA. Later on, a CODIS system was developed for creating national DNA databases of DNA profile by FBI under NDIS program. The CODIS and FSS markers were widely accepted by other countries and forensic science laboratories and became a standardized benchmark

V. Kumar (*) Centurion University of Technology and Management, Bhubaneswar, Odisha, India © Springer Nature Singapore Pte Ltd. 2022 H. R. Dash et al. (eds.), Handbook of DNA Profiling, https://doi.org/10.1007/978-981-16-4318-7_5

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for minimum analyzing criteria of the DNA profiling. On the basis of STR markers, many organizations developed different variant of autosomal kit which includes multiple markers in one mixture to analyze different region of DNA in one process. They have also added new markers in the kit to provide more discriminatory power and probability index to compare two DNA profile. Nowadays, many advanced autosomal STR kits are commercialized and used by forensic science laboratories, which are overcoming vast area of challenges and producing more precise and accurate results. Autosomal STR multiplex kit has become a concrete tool to analyze DNA profile and plays a vital role in criminal justice system. In this chapter, the different autosomal STR kits accepted widely among scientific and forensic community and used frequently in forensic science laboratory had been described. Keywords

DNA Profiling · STR markers · Autosomal STR markers · Autosomal STR Multiplex Kits · CODIS system · Nomenclature of Markers · Identifiler · Globalfiler · Verifiler · Powerplex 16HS · Powerplex Fusion 6C · Forensic Validation

Case Study: Narborough Murders: The Identification of Colin Pitchfork

The first forensic use of DNA profiling dates back to Narborough Murders also referred as Enderby Murders committed by Black Pad Killer – “Colin Pitchfork,” who was sentenced to life imprisonment for the murder of two young girls and also was sentenced 10 years for the two rapes along with 3 years for two indecent assaults. He was also given 3 years for the conspiracy to pervert the course of justice by avoiding giving a DNA samples at Leicester crown court on 22 January 1988. On 21 November 1983, a 15-year-old school girl Lynda Mann was raped and strangled in Narborough, Leicestershire. The body of Lynda Mann was found the next morning in the grounds of Carlton Hayes hospital next to Black Pad footpath. Semen sample recovered from Lynda Mann’s body was analyzed which indicated that culprit had blood group ‘A’ and enzyme profile shared by only 10% of the male population. With no further leads, the investigation never progressed further. In 31 July 1986, another 15-year-old school girl Dawn Ashworth was raped and murdered in the same area. The body of Dawn Ashworth was found 2 days later. The sample were recovered from body shows same characteristics as was found in Lynda Mann. These finding led the police to believe that both crimes were committed by the same person. On 8 August 1986, police arrested a 17-year-old local kitchen porter named Richard Buckland. He confessed the murder of Dawn Ashworth but he denied (continued)

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the murder of the first girl Lynda Mann. The police were convinced that they arrested the culprit of both murder but denying of first murder by Richard Buckland led the police to seek help from Dr. Alec Jeffrey of Leicester University as he had developed a unique technique that can individualize two people apart called DNA profiling. After analyzing all the questioned samples and reference sample of Richard Buckland, Dr. Alec Jeffrey’s findings indicate that both crimes were committed by same person but the profile did not match with Richard Buckland and the police released him on 21 November 1986. This emphasizes the need for caution even when a suspect confesses to a crime. With their chief suspect exonerated (another first for DNA profiling), the police undertook the world’s first DNA mass intelligence screening in which all the men in the area, 5000 in all, were asked to provide DNA either as a blood sample or a saliva swab. Of these samples, only those exhibiting the same blood group and enzyme pattern as the murderer were subjected to DNA profiling. This was a major operation, not least because the profiling techniques were much more time consuming than those in use today, and took 6 months to complete.After completing the entire DNA profiling, none of the profile matched with the questioned evidence sample. The operation meets a dead end without any lead and a year passed. In August 1987, a woman reported that she overheard a man named Ian Kelly in a pub saying that he had provided a DNA sample in place of his friend. When police questioned Ian Kelly he confessed that he took the blood test instead of his friend named Colin Pitchfork who was a local baker. On 19 September 1987, after the confession of Ian Kelly, the police arrested Colin Pitchfork who was then aged 27. The sample was taken from Colin Pitchfork and was sent to Dr. Alec Jeffrey for DNA profiling. The DNA profile of Colin Pitchfork was found to match of the semen samples recovered from the two murdered girls. Colin Pitchfork, who was a local baker, was therefore arrested for raping and murdering two girls. Colin Pitchfork was brought to trial on 22 January 1988 at Leicester Crown Court where he was found guilty and sentenced to life imprisonment.

Introduction As we all know that cell is the basic unit of life and a miniature factory having necessary capabilities to sustain life. Each cell of an individual contains same genetic programming. Within the nucleus of our cells the genetic material resides known as DNA and referred as nuclear DNA. Nuclear DNA is referred as genetic blueprint

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because it contains different types of genes having genetic traits necessary for genotypical character and phenotypical character. DNA is composed of nucleotides unit that are made up of three segments: a nitrogenous base, a sugar, and a phosphate group. The nitrogenous base plays a vital role to form different combination of nucleotides sequence in DNA. Humans have approximately 3 billion nucleotide position in their genomic DNA with zillions of combinations (Butler 2005). Within the nucleus, DNA is found in a specialized structure called chromosomes. Human cell contains 23 pairs of chromosomes among which 22 pairs are autosomal chromosome and 1 pair is sex chromosome. Autosomal chromosomes contain the information for the development of the body. Remaining sex chromosomes control the development of internal and external reproductive organ. Each chromosome contains a strand of tightly coiled DNA which is divided into small units called gene. Gene is the sequence of nucleotides having basic physical and functional unit of heredity. Each gene resides on a specific physical location referred as locus. A gene usually ranges from few thousands to tens of thousands base pairs in sizes. Genes consist of exons and introns and make up approximately 5% of human genomic DNA. Remaining noncoding region of DNA is called junk DNA. Identification of an individual has been an issue in the scientific community as well as in forensic community for several years. With the advancement of scientific knowledge of gene, genomic sequence and different types of markers plays a vital role for identification of the individuals, biological dispute, family history and pedigree chart, etc. In 1985, Dr. Alec Jeffrey of the University of Leicester, England, developed a unique technique known as DNA profiling. Dr. Alec Jeffrey first used DNA technology to assist police in identifying a suspect in a rape case. DNA profiling is basically involved RFLP/RAPD or AFLP. Although 99.9% of human DNA sequences are the same but approximately 0.1% DNA is different in every individual from each other. This 0.1% DNA is the basic concept of DNA fingerprinting that includes repetitive sequence of STR and VNTR. In court, an expert presenting DNA-based evidence talks about probabilities of a match between two samples. They do not state that that it is a direct match or not match. This fascinating technique is often explored and represented as a simple, exact, and infallible method of finding criminals and convicting the culprit to bring the justice. However, determining the match between a suspect and a crime scene is a complicated process that relies on probability of matching the profile.

Autosomal STR Marker DNA profiling for human identity testing is performed using markers on the autosomal chromosomes and gender determination is done with markers on the sex chromosomes. From a forensic aspect there is very little rationale in analyzing the 99.9% of human DNA that is common between individuals (Goodwin et al. 2012). Fortunately, the variable regions residing in DNA vary between individuals who

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become the center of attention for forensic community. Markers are exclusive small region of DNA which shows sequence and length polymorphism in different individuals referred as easily identifiable traits. A short tandem repeat (STR) is a unique tool that analyze and evaluate specific regions found in DNA when a pattern of two or more nucleotides are repeated and the repeated sequences are directly adjacent to each other. The repetitive pattern of nucleotide can range in length from 2–7 bp (Butler 2005). These repetitive patterns are highly polymorphic, which may be detected using PCR. Fluorescence detection by electrophoresis separation of the alleles of STR loci is distinguished from one another by the numbers of copies of the repeat sequence contained within the amplified region. With advancements in molecular biology and genetics techniques it is now possible to analyze any region within the 3.2 billion bases that make up the human genome. DNA loci that are to be used for forensic genetics should have some key properties; they should ideally: • • • • •

Be highly polymorphic (varying widely between individuals) Be easy and cheap to characterize Be easy to interpret and compare between laboratories Be not under any selective pressure Have a low mutation rate

The markers are capable of revealing the genetic variation at DNA sequence level and enable to distinguish two individuals from each other to discriminate between one individual’s DNA profile to another. The characteristics of the markers are following (Butler 2018): • • • • •

Inexpensive to develop and apply Unaffected by environmental and developmental variation Highly robust and repeatable across different tissue types and different labs Polymorphic, i.e., reveal high levels of allelic variability Codominant in its expression

The STR markers are widely used in forensic DNA typing for human identification and forensic DNA analysis. There are certain criteria for autosomal STR marker as follows (Butler 2011): • High discriminating power with observed heterozygosity 70% • Separate (or widely spaced) chromosomal locations to ensure that closely linked loci are not chosen • Robustness and reproducibility of results when multiplexed with other markers • Low stutter characteristics • Low mutation rate • Predicted length of alleles that fall in the range of 90–500 bp with smaller sizes better suited for analysis of degraded DNA samples

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Terminology • Locus or Loci: The specific location of a gene or DNA sequence or position on a chromosome. • Alleles: One of a number of alternative forms of the same gene or same genetic locus. • Polymorphism: The occurrence of more than one form or morph. • Heterozygous: If the two alleles at a genetic locus on homologous chromosomes are different they are termed as heterozygous. • Homozygous: If the alleles are identical at a particular locus, they are termed homozygous.

Nomenclature for DNA Markers The nomenclature for DNA markers is fairly straightforward. If a marker is part of a gene or falls within a gene, the gene name is used in the designation. For example, the short tandem repeat (STR) marker TH01 is from the human tyrosine hydroxylase gene located on chromosome 11. The “01” portion of TH01 comes from the fact that the repeat region in question is located within intron 1 of the tyrosine hydroxylase gene (Butler and Hill 2012). Sometimes the prefix HUM is included at the beginning of a locus name to indicate that it is from the human genome. Thus, the STR locus TH01 would be correctly listed as HUMTH01. Nomenclature of the DNA markers that fall outside of gene regions are designated on the basis of the chromosomal positions, chromosome number, their copy sequence, and physical location of the marker found on a particular chromosome (Butler 2012). On the basis of these criteria the nomenclature of the markers may be designated as follows: • “D” stands for DNA by their chromosomal position. • The next character refers to the chromosome number 5 for chromosome 5 and Y for the Y chromosome. • The “S” refers to the fact that the DNA marker is a single-copy sequence. • The final number indicates the order in which the marker was discovered and categorized for a particular chromosome. Ex: D16S539 – D – DNA – 16 – Chromosome 16 – S – Single-copy sequence – 539 – 539th locus described on chromosome 16

Types of Autosomal STR Marker On the basis of participation number of nucleotides, there are generally four types of autosomal STR marker such as:

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Table 1 Representation of tandem nucleotide repeats (AGAT or GATA motif is the most common motif for STR loci used by forensic scientist) S. No. 1. 2. 3. 4.

Types Mononucleotide repeats Dinucleotide repeats Trinucleotide repeats Tetranucleotide repeats

Representation A, C AC, AG, AT, CG AAC, AAG, AAT, ACG, ACT, ACC, AGC, AGG, ATC, CCG AAAC, AAAG, AAAT, AACG, AACC, AACT, AAGC, AAGG, AAGT, AATC, AATG, AATT, ACAG, ACAT, ACCC, ACCG, ACCT, ACGC, ACGG, ACGT, ACTC, ACTG, AGAT, AGCC, AGCG, AGCT, AGGC, AGGG, ATCC, ATCG, ATGC, CCCG, CCGG

1. Mononucleotide repeats: When one nucleotide is repeated sequences are directly adjacent to each other. 2. Dinucleotide repeats: When two nucleotides are repeated sequences are directly adjacent to each other. 3. Trinucleotide repeats: When three nucleotides are repeated sequences are directly adjacent to each other. 4. Tetranucleotide repeats: When four nucleotides are repeated sequences are directly adjacent to each other.

and repeated and repeated and repeated and repeated

The most popular STR repeats among different types of nucleotides repeats which drew attention of forensic community are tetranucleotide repeats. Tetranucleotide repeats are choice of STR types frequently used in different STR kits. Advance autosomal STR kits also contain trinucleotide and tetranucleotide repeats (Table 1). A narrow allele size range is more suitable to reduce allelic dropout from preferential amplification of smaller alleles and also permits multiplexing. The capability of generating small PCR product sizes benefits the recovery of information from degraded DNA specimens and reduced stutter product formation compared to dinucleotide repeats that benefit the interpretation of sample mixtures. For DNA typing markers to be effective across a wide number of jurisdictions, a common set of standardized autosomal markers must be used (Butler and Hill 2012). On the basis of repeating unit it is further classified into four types as follow (Peter et al. 2020): 1. Simple repeats: Units of identical length and sequence are repeated. 2. Compound repeats: Comprise two or more adjacent simple repeats. 3. Complex repeats: Several repeat blocks of variable unit length as well as variable intervening sequence. 4. Microvariant: When the tandem repeat ends with incomplete repeating sequence.

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CODIS Markers The commonly used STR loci today were initially characterized and developed either in the laboratory of Dr. Thomas Caskey at the Baylor College of Medicine or at the Forensic Science Service (FSS) in England. The Promega Corporation (Madison, WI) initially commercialized many of the Caskey markers, while Applied Biosystems (Foster City, CA) incorporated the FSS STR loci and also developed some new markers. The UK started using STR loci way before the USA with the help of Forensic Science Service. After utilization of STR by the UK, USA started a DNA project in early 1996 and in 1997 specific STRs were selected by FBI and established core STR loci with the help of NIST, and a national DNA database was created known as CODIS (Combined DNA Index System), a computer software program that helps law enforcement agency to compare DNA profile digitally with the help of 13 autosomal STR markers and one sex marker (Hares 2012). It is also referred as CODIS13. In 1994, US Congress passed an act called DNA identification act to create a national database of DNA profile. In 1998, FBI created the National DNA Index System referred as NDIS program (Christian et al. 2001), a part of CODIS to be implemented at national level to contain the DNA profiles submitted by local and state law enforcement agency and also by different forensic laboratories. As per a report provided by FBI, till 2013 CODIS contains over 10 million DNA profiles of different category which included to form a DNA database (CODIS Operating policies and procedures manual 2009). DNA profiles were recorded in different category of indexes which were included in CODIS system as database to generate investigative leads in crimes where biological evidence is recovered from the crime scene to find the victim, suspect, or culprit. CODIS uses computer software to search these indexes for matching the DNA profiles whenever required by law enforcement agency. The searching and comparing the DNA profile is automated by computer software, but can also be manually analyzed by users of respective authority to analyze the different probability required by law enforcement agency to get desirable perspective. The different category of CODIS index is represented as: • Forensic index: The forensic index contains all the evidence profile created from biological evidence collected from crime scene. • Convicted offender index: The convicted offender index contains DNA profile and data of the culprit who has been convicted of any crime. • Arrestee index: The arrestee index contains DNA profile and data of the suspect who has been arrested but not convicted. • Missing or unidentified index: The missing or unidentified index contains DNA profile and data of the individuals who went missing or unidentified. The DNA profile of different individuals and species are stored in CODIS on the basis of core STR markers and each DNA profile is given a tagline to identify an

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individual. Once any DNA profile is generated and analyzed with CODIS for a potential match, the respective authority is responsible to identify the individual with index category and obtain additional information about the individual such as name, number, address, case details, previous criminal records (if any), previous felony or conviction (if any), and any other information required. CODIS does not store any criminal history information, any case information, social information, or social numbers of any individual. The 13 autosomal STR markers used in CODIS are: 1. TPOX: It is a simple repeat of (AATG)n found on 10th intron of human thyroid peroxidase gene of 2nd chromosome. The mutation rate of TPOX STR marker is 0.01%. The physical location of TPOX is 1.436 Mb on chromosome 2. It was discovered and published by Anker et al. in 1991. 2. D3S1358: It is a compound repeat of tetrameric STR found on 3rd chromosome. The mutation rate of D3S1358 is 0.12%. It was discovered by Schmidt et al. in 1993. 3. FGA: It is a compound repeat STR marker of CTTT/TTCG found in the 3rd intron of human alpha fibrinogen gene of 4th chromosome. The mutation rate of FGA marker is 0.28–0.30%. The physical location of FGA STR marker is 156.086 Mb on chromosome 4 with microvariants for some alleles. It was discovered and published by Milis et al. in 1992. 4. D5S818: It is a simple repeat of ATCT tetrameric STR found on the 5th chromosome. The mutation rate of D5S818 is 0.12%. 5. CSF1PO: It is a simple repeat of ATCT tetrameric STR found in 6th intron of c-fma proto-oncogene of 5th chromosome. The mutation rate of CSF1PO is 0.16%. It was discovered by Hammond et al. in 1994. 6. D7S820: It is a simple repeat of TATC tetrameric STR found on 7th chromosome. The mutation rate of D7S820 is 0.10%. 7. D8S1179: It is a compound repeat of tetrameric STR found 8th chromosome. The mutation rate of D8S1179 is 0.13%. 8. TH01: TH01 STR marker is also referred as TC11 OR HUMTH01. TH01 is simple repeat of tetrameric STR found in 1st intron of tyrosine hydroxylase gene on chromosome 11 with physical location 2.156 Mb. It is a tetrameric STR of repeated unit of AATG with tri-allelic pattern. The 3rd alleles of TH01 in some cases may found microvariant (ATG) in nature. Mutation rate of TH01 is 0.01%. It was discovered by Polymeropoulos et al. in 1991. 9. VWA: It is a compound repeat of tetrameric STR found in 40th intron of 12th chromosome with physical location 5.963 Mb and responsible for Von Willerbrand factor. The mutation rate of VWA is 0.17%. It was discovered by Kimpton et al. in 1992. 10. D13S317: It is a simple repeat of TATC tetrameric STR marker found on 13th chromosome. The mutation rate of D13S317 is 0.15%. 11. D16S539: It is a simple repeat of GATA tetrameric STR marker found on 16th chromosome. The mutation rate of D16S539 is 0.11%.

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12. D18S51: It is a simple repeat of AGAA tetrameric STR marker found on 18th chromosome. The mutation rate of D18S51 is 0.25%. 13. D21S11: It is a complex repeat of tetrameric STR marker found on 21th chromosome. It also shows microvariant in some alleles. The mutation rate of D21S11 is 0.21%. Apart from 13 core STR markers, FBI announced additional 7 STR markers in 2015 to be introduced in CODIS program that become effective from 1 January 2017 and known as CODIS20. CODIS 20 comprises of seven additional STR loci along with 13 core STR loci. The additional STR loci are: 1. D1S1656: It is a compound repeat of tetrameric STR found on 1st chromosome. It has microvariant value of x.3 due to insertion of TGA. 2. D2S441: It is a simple repeat of TCTA tetrameric STR marker found on 2nd chromosome. Some microvariant alleles of x.3 have been also observed. 3. D2S1338: It is a compound repeat of tetrameric STR marker found on 2nd chromosome. 4. D10S1248: It is a simple repeat of GGAA tetrameric STR marker found on 10th chromosome. 5. D12S391: It is a compound repeat of tetrameric STR marker found on 12th chromosome. It is very highly polymorphic STR marker having microvariant of x.2 for some alleles. 6. D19S433: It is a complex repeat of tetrameric STR marker found on 19th chromosome. 7. D22S1045: It is a simple repeat of ATT trimeric STR marker found on 22nd chromosome.

Autosomal STR Multiplex Kits Designing primer, ladders, or optimization PCR multiplex is hard and problematic to most of the laboratories due to lack of time, lack of proper resources, as well as high costing equipment to synthesize these materials and monitor quality control of the product. To overcome this issue, many commercialized organization developed a ready-made kit based on different core autosomal STR loci with other required materials and exported to respective agencies. These kits are referred as “Autosomal STR multiplex kit.” It improved the accuracy of DNA profiling as well as probability of matching two DNA samples. Autosomal STR multiplex kits have been widely accepted by forensic community to generate DNA profiles and compare with the help of selected autosomal STR marker. These kits have been routinely used in all forensic laboratories and private detective agencies in human identity testing for various purposes to support the criminal justice system.

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Autosomal STR multiplex kits increase discrimination powers along with increasing the number of loci. All the loci are combined in one kit to produce analysis of multiple STR loci at one time. It also reduces the time of analysis and produces result faster than conventional method. In commercial autosomal STR multiplex kits there are usually five components included for DNA profiling such as: 1. Primer: It contains PCR primer mixture of oligonucleotides labeled with dye to amplify desired STR loci. 2. Buffer: It contains deoxynucleotide triphosphate, MgCl2, and other desired respective PCR buffer. 3. Polymerase: It contains DNA polymerase, the molecular precursor to synthesize the targeted DNA. 4. Ladder: It contains allelic ladder with respect to STR loci alleles. 5. Control sample: It contains positive DNA control sample to verify if kit is working properly and with standard protocol (Table 2).

Earlier STR Multiplex Kits STR multiplex kit contains number of different STR along with specific loci used to analyze genome. The first autosomal STR multiplex kit is developed by forensic science services comprised of quadraplex STR loci, also referred as “first generation multiplex.” The probability of matching two DNA profiles by first generation multiplex (FGM) is 1 in 104. The first commercial autosomal STR multiplex kit was developed by Promega Corporation and become available in 1994. This kit was referred by CTT triplex and had a matching probability of approx. 1 in 500. Promega and Applied Biosystem is the two frontier organization which started commercial STR multiplex kit with different variant of STR multiplex kit that contains specific STR markers. Both Promega and Applied Biosystem have commercialized many multiplex kits since 2000 that facilitate co-amplification of all 13 STR markers in one single reaction. Apart from that they commercialized different variant with extra STR markers added in kit. During starting period of adaptation of autosomal STR multiplex kit three cases were ruled out by the court of law where it was stated that DNA result cannot be admissible in court as evidence because primer sequence and validation of commercialized autosomal STR kit is not public information. After statement by the court, question mark arises on the credibility and reliability of the commercialized product of autosomal STR kit by both Promega and Applied Biosystem. After the statement made by court, the Promega Corporation decided to publish the sequence of their autosomal STR multiplex kit on 24 July to public and obtained several patients of autosomal STR multiplex kits. Meanwhile Applied Biosystem has refused to make the sequence publicly. But since 2002, Applied Biosystem provided the sequence of primer under protective court order.

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Table 2 Variant of autosomal STR marker multiplex kit commercialized by different organization Name of the kit AmpFlSTR Blue AmpFlSTR Green CTTv FFFL GammaSTR Powerplex 1.1 Powerplex 1.2 AmpFlSTR Profiler AmpFlSTR Profiler Plus AmpFlSTR COfiler AmpFlSTR SGM Plus Powerplex 2.1 (For Hitachi FMBIO users) Powerplex 16 Powerplex 16 BIO (For Hitachi FMBIO users) AmpFlSTR Identifiler AmpFlSTR profiler plus ID (Extra unlabeled D8-R primer) Powerplex ES AmpFlSTR SEfiler AmpFlSTR MiniFiler AmpFlSTR SEfiler Plus AmpFlSTR sinofiler Powerplex 16HS Powerplex ESX 16 & ESX 17 Powerplex ESI 16 & ESI 17 AmpFlSTR Identifiler Direct AmpFlSTR Identifiler Plus AmpFlSTR NGM Investigator ESSplex Investigator decaplex SE

Organization Applied Biosystem Applied Biosystem Promega Promega Promega Promega Promega Applied Biosystem Applied Biosystem Applied Biosystem Applied Biosystem Promega Promega Promega Applied Biosystem Applied Biosystem Promega Applied Biosystem Applied Biosystem Applied Biosystem Applied Biosystem Promega Promega Promega Applied Biosystem Applied Biosystem Applied Biosystem QIAGEN QIAGEN

Published/Released date Oct 1996 Jan 1997 Jan 1997 Jan 1997 Jan 1997 Jan 1997 Sept 1998 May 1997 Dec 1997 May 1998 Feb 1999 June 1999 May 2000 May 2001 July 2001 Sept 2001 Mar 2002 Sept 2002 Mar 2007 Nov 2007 Mar 2008 Mar 2009 Sept 2009 Sept 2009 Nov 2009 Jan 2010 Jan 2010 April 2010 April 2010 (continued)

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Table 2 (continued) Name of the kit Investigator triplex AFS QS Investigator triplex DSF Investigator ID plex Investigator HDplex Investigator hexaplex ESS Investigator Nanoplex SE Investigator ESSplex SE AmpFlSTR NGM Select Powerplex 18D

Organization QIAGEN QIAGEN QIAGEN QIAGEN QIAGEN QIAGEN QIAGEN Applied Biosystem Promega

Published/Released date April 2010 April 2010 Aug 2010 Sept 2010 Sept 2010 Sept 2010 Oct 2010 Dec 2010 Feb 2010

Identifiler Kit Identifiler Kit is Multiple PCR STR amplification Kit for the amplification of multiple autosomal STR markers. Identifiler kit is first developed and commercialized by Applied Biosystem as “AmpFLSTR Identifiler™” in July 2001. Identifiler kit was based on 13 CODIS STR markers and sex-typing marker. Apart from 13 CODIS STR markers, 2 additional autosomal STR markers were included in a single tube which was developed in collaboration with Forensic Science Service (FSS). The Identifiler Kit can amplify 15 autosomal STR loci and amelogenin gender determining marker in a single PCR amplification. Due to 16-Locus multiplex, Identifiler Kit gain advantages over previous and other kits that were accepted by forensic community. It reduces the sample preparation time, amplification time, laborious process, and analysis time in half as it completed the amplification of the entire locus in single multiplex PCR followed by one capillary electrophoresis. All 15 autosomal STR markers and one sex marker is included in one single tube. The autosomal STR markers are CSF1PO, FGA, TPOX, TH01, VWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, D2S1338, and D19S433. Identifiler Kit as stated by Thermofisher is the single most discriminating and widely used STR-based kit for human identification with two technologies. The first one is five-dye uorescent detection system. The spectral detection range was expanded with the help of five dyes set which enable high throughput analysis and maintain small amplicons. In five-dye detection system four different dyes are used to label the PCR product obtained by Identifiler kit STR markers. These four dyes are 6FAM™, VIC™, NED™, and PET™. The fifth dye is used to standardize the internal size to correlate the electrophoretic mobilities. The fifth dye is LIZ™ (User Guide, AmpFlSTR identifiler PCR amplification kit 2018). So, in Identifiler total five dyes are used unlike four traditional dyes that have been used previously in AmpFlSTR or Powerplex. In Powerplex kits four dyes were used and among those four dyes, three dyes were used to label PCR product, i.e.,

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Table 3 15 autosomal markers of Identifiler Kit with respect to labeling dye for each marker (Product Bulletin, AmpFlSTR Identifiler PCR amplification kit) Label dye 6FAM

STR marker CSF1PO D7S820 D8S1179 D21S11

Chromosome location 5q33.3-34 7q11.21-22 8 21q11.2-q21

5. 6. 7. 8. 9. 10. 11. 12. 13.

VIC

D2S1338 D3S1358 TH01 D13S317 D16S539 vWA TPOX D18S51 D19S433

2q35-37.1 3p 11p15.5 13q22-31 16q24-qter 12p12-pter 2p23-2per 18q21.3 19q12-13.1

14.

PET

FGA

4q28

D5S818

5q21-31

S. No. 1. 2. 3. 4.

15.

NED

Allelic variation 6–15 6–15 8–19 24, 24.2, 25–28, 28.2, 29, 29.2, 30, 30.2, 31, 31.2, 32, 32.2, 33, 33.2, 34, 34.2, 35, 35.2, 36–38 15–28 12–19 4–9, 9.3, 10, 11, 13.3 8–15 5, 8–15 11–24 6–13 7, 9, 10, 10.2, 11–13, 13.2, 14, 14.2, 15–27 9–12, 12.2, 13, 13.2, 14, 14.2, 15, 15.2, 16, 16.2, 17, 17.2 17–26, 26.2, 27–30, 30.2, 31.2, 32.2, 33.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 50.2, 51.2 7–16

5-FAM, JOE, and JOE, and one dye was used to standardize the internal size, i.e., ROX (Table 3). The data generated by Identifiler Kit was widely accepted and approved by forensic community such as FBI, Interpol, and European Network of Forensic Science Institutes (ENFSI) (Fig. 1). Identifiler expanded the spectral range to 660 nm. The expanded range allows maximum color separation with minimal spectral overlap. The second technology involves mobility modifying non-nucleotide linkers. The mobility modifying is composed of hexaethyleneoxide (HEO). The unique properties of mobility modifying non-nucleotide linker enables size shift to retain original primer sequence to avoid discovering primer binding mutation while STR allele size ranges may be altered during electrophoresis. These non-nucleotide linkers prevent overlapping of the size range of amplified alleles by providing a shift of approximately 2.5 nucleotides with each HEO unit. Non-nucleotide linkers are placed between the primers and the uorescent dye during oligonucleotide synthesis. The non-nucleotide linkers are used in primer synthesis for some of the loci in Identifiler such as: CSF1PO, D13S317, D16S539, D2S1338, and TPOX (Rabelo et al. 2015). Applied Biosystem released two more variants of the Identifiler kit with different enhancement in each version as follows:

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VIC

NED PET

LIZ

Normalized Emission

6-FAM

165

500

550

600

650

700

Wavelength (nm)

Fig. 1 Emission spectra of five dyes used by Identifiler kit

“AmpFLSTR Identifiler™ Direct” After 8 year of releasing of Identifiler Kit, Applied Biosystem released Identifiler Direct kit as AmpFLSTR Identifiler™ Direct in Nov 2009, also referred as next generation STR Kit. The main advantage of Identifiler direct kit over Identifiler kit is that Identifiler direct kit allows direct amplification of single source. Identifiler direct kit provides prep n-Go buffer for extraction of DNA. DNA can be extracted from prep n-Go buffer without the need for sample purification (User Guide, AmpFlSTR identifiler Direct PCR amplification kit 2018). “AmpFLSTR Identifiler™ Plus” After Identifiler direct, Applied Biosystem released a new variant of Identifiler kit called as AmpFLSTR Identifiler™ Plus in Jan 2010. Identifiler plus kit also referred as next generation STR kit which provides better sensitivity and better robustness than previous Identifiler kit. Identifiler kit uses modified PCR cycling condition for enhanced sensitivity. It also included a buffer mixture which gives better result with inhibited samples and provides cleaner electrophoretic background (User Guide, AmpFlSTR identifiler Plus PCR amplification kit 2015). The Identifiler kit is compatible with different genetic analyzer such as 310 genetic analyzer, 3100-avant genetic analyzer, 3130xl genetic analyzer, Gene Amp 9600, Gene Amp 9700, 3500 genetic analyzer, 3500xl genetic analyzer, and 3730 DNA analyzer, although optimization of the different process is required as per the standard protocol provided by Thermo fisher, Applied Biosystem.

Powerplex 16HS Kit Powerplex 16HS system is a multiple STR kit which is used for Forensic DNA typing developed and published by Promega Corporation in March 2009. Powerplex 16HS system is an updated version of the Powerplex 16 kit (Julio et al. 2013). The primer and dyes of Powerplex 16 HS kit is same as Powerplex 16 kit, but in

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Powerplex 16HS kit enhanced buffer system is provided that includes hot-start taq DNA polymerase that ensures reproducible result with low quantity of the DNA and robust performance. Powerplex 16HS kit contains 16 loci in a single tube and amplifies all 16 loci simultaneously in one processing. Among those 16 loci it includes all 13 CODIS STR markers along with amelogenin for gender determination and it also contains two new STR markers such as “Penta E and Penta D.” Penta E and Penta D is highly discriminating pentanucleotide STR markers having low stutter product (Jebor et al. 2015). • Penta D: It is a pentanucleotide repeat STR marker found on 21st chromosome. It is situated about 25Mbp away from D21S11. • Penta E: It is a pentanucleotide repeat STR marker found on long arm of 15th chromosome. It have very low stutter product with alleles ranging from 5–32 repeats. Powerplex 16HS kit contains three color detection label dye for 16 loci and one label dye for internal lane standard. The three labeling dyes for STR markers are uorescein (FL), carboxy-tetramethylrhodamine (TMR), and 6-carboxy-40 , 50 -dichloro-20 , 70 -dimethoxy- uorescein (JOE). One labeling dye for internal lane standard is carboxy-X-rhodamine (CXR). Powerplex 16HS kit can give reproducible results with 0.5–1 ng DNA but some experiment shows that it can also give good results with lesser than 0.5 ng DNA also. Penta D and Penta E allow high discriminating power for STR result with low quantity of DNA. Powerplex 16HS kit has been engineered to enable STR amplification in the presence of different PCR inhibitors such as heme, humic acid, tannic acid, and other inhibitors. This advantage allows biological samples to be directly amplified without purification (Technical Manual, Powerplex 16HS system 2016). The Powerplex 16HS kit is compatible with different genetic analyzer such as ABI PRISM 310-, 3100-, and 3100-avant genetic analyzer and applied biosystem 3130, 3130xl, 3500, and 3500xl genetic analyzer (Thatch et al. 2012), although optimization of the different process is required as per the standard protocol provided by Promega Corporation.

New Generation Kit Forensic DNA typing has been constantly evolving according to the different circumstances and challenges faced by forensic community and scientific community. Wide varieties of commercial STR kits have been designed to meet various needs of a forensic lab as STRs become the markers of choice for forensic DNA typing. Different challenges and requirement drive the scientific and research laboratories as well as commercial organization to overcome the other challenges in earlier generation autosomal STR multiplex kits and meet the

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Table 4 15 autosomal markers of Powerplex 16 HS autosomal STR multiplex kit with respect to labeling dye for each marker and allelic variation of each STR loci in kit

S. No. 1. 2. 3. 4. 5.

6. 7.

8. 9. 10. 11. 12. 13. 14. 15.

Label dye FL

STR marker D3S1358 TH01 Penta E D18S51 D21S11

Chromosome location 3p 11p15.5 15q 18q21.3 21q11-21q21

Size of allelic ladder bases 115–147 156–195 379–474 290–366 203–259

TMR

TPOX FGA

2p24-2pter 4q28

262–290 322–444

D8S1179 vWA CSF1PO D5S818 D7S820 D13S317 D16S539 Penta D

8q24.13 12p13.31 5q33.3-34 5q23.3-32 7q11.21-22 13q22-q31 16q24.1 21q

203–247 123–171 321–357 119–155 215–247 176–208 264–304 376–449

JOE

Allelic ladder variation 12–20 4–9, 9.3, 10–11, 13.3 5–24 8–10, 10.2, 11–13, 13.2, 14–27 24, 24.2, 25, 25.2, 26–28, 28.2, 29, 29.2, 30, 30.2, 31, 31.2, 32, 32.2, 33, 33.2, 34, 34.2, 35, 35.2, 36–38 6–13 16–18, 18.2, 19, 19.2, 20, 20.2, 21, 21.2, 22, 22.2, 23, 23.2, 24, 24.2, 25, 25.2, 26–30, 31.2, 43.2, 44.2, 45.2, 46.2 7–18 10–22 6–15 7–16 6–14 7–15 5, 8–15 2.2, 3.2, 7–17

criteria such as more accuracy, high robustness, simpler/automated processing, speedy process, sensitivity, and other parameters by different forensic science laboratories as well as others organization to produce new generation kits. These improvements enhanced the ability of forensic science laboratories to obtain a DNA profile from more challenging samples which were facing problem in accuracy and sensitivity in earlier generation kits. The new generation STR multiplex kits provide more accuracy with higher robustness and greater discriminatory power. These improvements and characteristics provide a higher reliability and credibility for differentiating trace DNA alleles from noise-related artifacts. The CODIS increased their core loci from 13 STR markers to 20 STR markers. The 20 CODIS system become effective from January 2017. All new generation STR kits contain 20 CODIS marker as base markers. Different commercial organizations incorporate these 20 CODIS STR markers as well as some other markers developed by them to provide better discriminatory power. With these improvements, many STR kits have been released by commercial organizations which provide high discriminatory power, robustness, and better performance in harsh conditions.

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The 13 autosomal STR markers of CODIS are (Gettings et al. 2015): 1. TPOX: It is a simple repeat of (AATG)n found on 10th intron of human thyroid peroxidase gene of 2nd chromosome. The mutation rate of TPOX STR marker is 0.01%. The physical location of TPOX is 1.436 Mb on chromosome 2. It was discovered and published by Anker et al. in 1991. 2. D3S1358: It is a compound repeat of tetrameric STR found on 3rd chromosome. The mutation rate of D3S1358 is 0.12%. It was discovered by Schmidt et al. in 1993. 3. FGA: It is a compound repeat STR marker OF CTTT/TTCG found in the 3rd intron of human alpha fibrinogen gene of 4th chromosome. The mutation rate of FGA marker is 0.28–0.30%. The physical location of FGA STR marker is 156.086 Mb on chromosome 4 with microvariants for some alleles. It was discovered and published by Milis et al. in 1992. 4. D5S818: It is a simple repeat of ATCT tetrameric STR found on 5th chromosome. The mutation rate of D5S818 is 0.12%. 5. CSF1PO: It is a simple repeat of ATCT tetrameric STR found in 6th intron of c-fma proto-oncogene of 5th chromosome. The mutation rate of CSF1PO is 0.16%. It was discovered by Hammond et al. in 1994. 6. D7S820: It is a a simple repeat of TATC tetrameric STR found on 7th chromosome. The mutation rate of D7S820 is 0.10%. 7. D8S1179: It is a compound repeat of tetrameric STR found 8th chromosome. The mutation rate of D8S1179 is 0.13%. 8. TH01: TH01 STR marker is also referred as TC11 OR HUMTH01. TH01 is simple repeat of tetrameric STR found in 1st intron of tyrosine hydroxylase gene on chromosome 11 with physical location 2.156 Mb. It is a tetrameric STR of repeated unit of AATG with tri-allelic pattern. The 3rd alleles of TH01 in some cases may found in microvariant (ATG) in nature. Mutation rate of TH01 is 0.01%. It was discovered by Polymeropoulos et al. in 1991. 9. VWA: It is a compound repeat of tetrameric STR found in 40th intron of 12th chromosome with physical location 5.963 Mb and responsible for Von Willerbrand factor. The mutation rate of VWA is 0.17%. It was discovered by Kimpton et al. in 1992. 10. D13S317: It is a simple repeat of TATC tetrameric STR marker found on 13th chromosome. The mutation rate of D13S317 is 0.15%. 11. D16S539: It is a simple repeat of GATA tetrameric STR marker found on 16th chromosome. The mutation rate of D16S539 is 0.11%. 12. D18S51: It is a simple repeat of AGAA tetrameric STR marker found on 18th chromosome. The mutation rate of D18S51 is 0.25%. 13. D21S11: It is a complex repeat of tetrameric STR marker found on 21th chromosome. It also shows microvariant in some alleles. The mutation rate of D21S11 is 0.21%. Apart from CODIS 13 STR marker, other seven STR markers which are part of both the CODIS 20 and ESS are as follows:

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1. D1S1656: It is a compound repeat of tetrameric STR found on 1st chromosome. It has microvariant value of x.3 due to insertion of TGA. 2. D2S441: It is a simple repeat of TCTA tetrameric STR marker found on 2nd chromosome. Some microvariant alleles of x.3 have been also observed. 3. D2S1338: It is a compound repeat of tetrameric STR marker found on 2nd chromosome. 4. D10S1248: It is a simple repeat of GGAA tetrameric STR marker found on 10th chromosome. 5. D12S391: It is a compound repeat of tetrameric STR marker found on 12th chromosome. It is very highly polymorphic STR marker having microvariant of x.2 for some alleles. 6. D19S433: It is a complex repeat of tetrameric STR marker found on 19th chromosome. 7. D22S1045: It is a simple repeat of ATT trimeric STR marker found on 22nd chromosome.

Global Filer Kit DNA profiling by targeting STR markers present in human genome proves an ultimate techniques to discriminate between two individuals for human identification in forensic cases. With the ever-growing needs and challenging samples for DNA profiling, different new generation STR kits were commercialized by different organization. Globalfiler kit is PCR amplification kit having comprised of 24 loci (Ludeman et al. 2018). Globalfiler kit was published in 2012 by Applied Biosystem and received approval soon after its release by FBI. The 24 loci comprised of 21 autosomal STR markers and 3 sex determination markers. Among those 21 autosomal STR marker, 13 CODIS STR marker were included and 7 markers were included from European standard set of loci (ESSL) and a highly discriminating locus was also added, i.e., SE33 locus. Sex determination markers comprised of Y-STR locus, Y-Indel, and amelogenin. Globalfiler PCR amplification kit allows for amplification of all 21 autosomal STR loci and 3 sex determination loci in one single reaction. Globalfiler kit is the 1st kit having six-dye technologies with 24 loci. Thermo Fisher Scientific (Applied Biosystem) is the only company to receive FBI approval for the six-dye technology. The 21 autosomal along with 3 sex determination marker provides maximum information recovery and amplification of challenging degraded samples (Bogas et al. 2015). Some of the key features introduced by Thermo Fisher Scientific company in Globalfiler kit are (Gouveia et al. 2015): • Reduced amplification time with the help of efficient protocol compared to previous generation kits and a specific enzyme included in optimized buffer system which also enables the expansion of DNA input volume which delivers maximum sensitivity for trace/low-level DNA samples • Easy interpretation of mixture samples with the help of improved intra-color balance • Maximize performance from degraded samples with the help of 10 mini-STR included in kit having less than 220 bp size.

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• Inclusion of CODIS core loci and FSS core loci increased global data compatibility and make this the only kit which includes all recommended markers by respective organizations. • The TPOX reverse primer has been redesigned to relocate the amplicon into the higher size range of the multiplex and optimizing marker spacing. • Addition of eight new SNP-specific primers for the D3S1358, VWA, D16S539, AMEL, D2S441, D22S1045, and D8S1179 loci. Globalfiler kit is a robust single-amplification STR multiplex kit that provides enhanced buffer system for enhanced sensitivity with inhibited and low concentration DNA samples (Brito et al. 2015). Non-nucleotide linkers are used in primer synthesis for the following loci: D19S433, VWA, CSF1PO, D2S441, TH01, FGA, and D12S391. Globalfiler kit satisfies the new recommendation based by FBI as well as ESS which gives them many advantages over others kits and it also included other STR markers to provide greater discrimination power to identify individuals. The seven markers from ESS were also accepted by CODIS system in 2017 in order to expand CODIS system from CODIS 13 to CODIS 20 (Hares 2012). So, in other words Globalfiler PCR amplification kit includes all 20 CODIS STR markers which were approved and become effective by 2017 and one additional loci of European STR kit. The details of all STR markers included in Globalfiler kit are given below: • CODIS 20 markers: CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, TH01, TPOX, VWA, D1S1656, D2S441, D2S1338, D10S1248, D12S391, D19S433 and D22S1045. Apart from these 20 STR markers, one more marker is included, i.e., SE33 which is the part of European standard set only. • SE33: It is a simple repeat of AAAG tetrameric STR marker found on 6th chromosome. It is highly polymorphic STR marker having microvariant for most of its alleles that provide greater discrimination power (Al-Snan et al. 2019). The mutation rate of this STR marker is 0.64% (Table 5). As mentioned above, Globalfiler kit is the first kit that used six-dye technology for labeling all the 24 loci. These dyes are 6-FAM, VIC, NED, TAZ, SID, and LIZ. Among these six dyes, five dyes are used for labeling the sample and allelic ladders, i.e., 6-FAM, VIC, NED, TAZ, and SID while one dye is used for labeling and standardization the internal lane standard, i.e., LIZ (Fig. 2). All of the24 loci of Globalfiler kit is having size lesser than 400 bp. The lesser size and allelic variation of all loci provide greater discrimination power to distinguish between two individuals. The matching probability between two individuals after analysis by Globalfiler kit is 1024 to 1028. The Globalfiler kit is compatible with different genetic analyzer such as SeqStudio genetic analyzer, 3500 genetic analyzer, 3500xL genetic analyzer, 3130 genetic analyzer, and 3130 xL genetic analyzer although optimization of

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Table 5 24 markers of Globalfiler STR multiplex kit with respect to labeling dye for each marker and allelic variation of each STR loci in kit S. No. 1. 2. 3. 4. 5. 6.

Label dye 6FAM

VIC

7. 8. 9. 10. 11.

12. 13.

NED

14. 15. 16. 17.

18. 19. 20. 21. 22. 23. 24.

TAZ

SID

Y Indel D8S1179 DYS391 D18S51 D21S11

Chromosome location 3p21.31 2p23-2per 5q33.3-34 12p12-pter 16q24.1 X: p22.1-22.3 y:p11.2 Yq11.221 8q24.13 Yq11.21 18q21.3 21q11.2-q21

D2S441 FGA

2p14 4q28

TH01 D19S433

11p15.5 19q12-13.1

D5S818 SE33

5q21-31 6q14

D7S820 D13S317 D22S1045 D1S1656

7q11.21-22 13q22-31 22q12.3 10q26.3

D2S1338 D10S1248 D12S391

2q35 10q26.3 12p13.2

STR marker D3S1358 TPOX CSF1PO vWA D16S539 Amelogenin

Allelic variation 9–20 6–15 6–15 11–24 5, 8–15 X, Y 1,2 5–19 7–13 7, 9, 10, 10.2, 11–13, 13.2, 14, 14.2, 15–27 24, 24.2, 25–28, 28.2, 29, 29.2, 30, 30.2, 31, 31.2, 32, 32.2, 33, 33.2, 34, 34.2, 35, 35.2, 36–38 8–11, 11.3, 12–17 13–26, 26.2, 27–30, 30.2, 31.2, 32.2, 33.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 50.2, 51.2 4–9, 9.3, 10, 11, 13.3 9–12, 12.2, 13, 13.2, 14, 14.2, 15, 15.2, 16, 16.2, 17, 17.2 7–18 4.2, 6.3, 8, 9, 11–20, 20.2, 21, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35, 35.2, 36, 37 6–15 5–16 8–19 9–14, 14.3, 15, 15.3, 16, 16.3, 17, 17.3, 18.3, 19.3, 20.3 11–28 8–19 14–19, 19.3, 20–27

the different process is required as per the standard protocol provided by respective organization. Value obtained for the same sample can differ between instruments platforms, because of different polymer matrices and electrophoretic conditions.

Verifiler Kit As we previously discussed the challenging samples in forensic work cases, the degrading samples in forensic area poses main threat and the circumstantial

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VIC NED TAZ

SID

LIZ

Normalized Emission

6-FAM

500

550

600

650

700

750

Wavelength (nm)

Fig. 2 Spectral graph of six dyes used by Globalfiler kit for labeling all 24 loci

evidence found from different types of environment and places with many inhibition factors in samples arise a lot of problem to get proper DNA profiling. To overcome this problem Applied Biosystem release a focused STR multiplex kit for these forensically challenged samples. Verifiler™ Plus PCR amplification kit was published and commercialized in the market in 2018. Verifiler kit is a 25-loci multiplex assay PCR kit for the amplification of single-source human genomic DNA. This kit is optimized for paternity testing and obtaining DNA profiles from single-source samples. It delivers highest discrimination power compared to previous ThermoFisher Scientific Human identification kit (User Guide, Verifiler Express PCR amplification kit 2017). Verifiler kit uses six-dye technology for the labeling of provided loci and standardization of internal lane standard. Verifiler kit provides amplification of all 25 loci in a single tube that can be amplified in a single reaction. The key features provided by Verifiler Plus PCR amplification kit includes (Pankaj and Dash 2019): • • • •

Very high discriminatory power across global populations All CODIS marker and Chinese national database loci required markers Concordance with previous thermoFisher scientific kit loci 11 mini STR having amplicon size less than 250 base pairs that increased the chances of alleles detection and improved the DNA profiling from degraded samples • Internal quality control (IQC) system that enables the distinction between inhibited and degraded samples • Next generation master mix formulation that speeds up the work ow process with 76-minute PCR cycle time for the amplification of the loci • Optimized buffer system that enhances the sensitivity to recover more alleles, hence recovery of more information for casework

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The Verifiler Plus PCR amplification kit loci comprised of: • CODIS 20 markers: CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, TH01, TPOX, VWA, D1S1656, D2S441, D2S1338, D10S1248, D12S391, D19S433, and D22S1045 • Three additional loci: D6S1043, Penta D, and Penta E • One Y chromosome marker: Y-Indel • One sex determining marker: Amelogenin • Two internal quality control marker: IQCS and IQCL (Table 6). The primers of Verifiler kit have been optimized to maintain concordance with pervious STR multiplex kit published by ThermoFisher Scientific. To simplify the interpretation of the results, primers are manufactured to maximize the assay signalto-noise ratio. Verifiler Plus PCR amplification kit provide next generation master mix formulation specifically designed for challenging samples such as touch samples, degraded samples, and samples contaminated with inhibition factors. It provides improved sensitivity and enhanced robustness against inhibition factors. The Verifiler kit use six-dye technology for the labeling of the loci. These dyes are 6-FAM™, VIC™, TED™, TAZ™, SID™, and LIZ™. Among these six dyes, five dyes are used for labeling the samples, allelic ladders, and control, i.e., 6-FAM™, VIC™, TED™, TAZ™, and SID™ while sixth dye is used for size standard, i.e., LIZ™ (Fig. 3). The combination of six-dye uorescent system and use of non-nucleotide linkers allow simultaneous amplification and efficient separation of all 25 loci. Non-nucleotide linkers are placed between the primers and uorescent dye during oligonucleotide synthesis which enable interlocus spacing which is used for primer synthesis for the following loci: D19S433, VWA, CSFAPO, D2S441, TH01 FGA, and D12S391. All the 25 loci of Verifiler kit have size lesser than 500 bp. The lesser size and allelic variation of all loci provide greater discrimination power to distinguish between two individuals. The matching probability between two individuals after analysis by Verifiler Plus PCR amplification kit of is 1028 to 1030. One of the unique advantages of Verifiler Plus PCR amplification kit is internal quality control (IQC) system. The IQC system has two synthetic targets, one low molecular weight (IQCS) and one high molecular weight (IQCL) (Al Janaahi et al. 2019). The IQC system identifies the presence of inhibitors and enables the distinction between degraded and inhibited samples and act as positive control for PCR amplification in Verifiler Plus PCR amplification kit. The Verifiler Plus PCR amplification kit gives the capability to obtain maximum possible information from the most challenging samples found in forensic casework. The Verifiler plus pcr amplification kit is compatible with different genetic analyzer such as 3500 genetic analyzer, 3500xL genetic analyzer, 3130 genetic analyzer, and 3130 xL genetic analyzer although optimization of the different process is required as per the standard protocol provided by respective organization. Value obtained for the same sample can differ between instruments platforms because of different polymer matrices and electrophoretic conditions (User Guide, Verifiler Plus PCR amplification kit 2020).

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Table 6 25 markers of Verifiler Plus STR multiplex kit with respect to labeling dye for each marker and allelic variation of each STR loci in kit S. No. 1.

Label dye 6FAM

Y Indel D8S1179 D5S818 D18S51 D21S11

Chromosome location Synthetic marker 3p21.31 5q33.3-34 6q15 12p12-pter 16q24.1 Synthetic marker X: p22.1-22.3 y:p11.2 Yq11.221 8q24.13 5q21-31 18q21.3 21q11.2-q21

D2S441 FGA

2p14 4q28

D10S1248 D19S433

10q26.3 19q12-13.1

TAZ

D1S1656

10q26.3

SID

D7S820 D13S317 D22S1045 Penta E TPOX D2S1338 TH01 D12S391 Penta D

7q11.21-22 13q22-31 22q12.3 15q26.2 2p23-2per 2q35 11p15.5 12p13.2 21q22.3

2. 3. 4. 5. 6. 7. 8.

D3S1358 CSF1PO D6S1043 vWA D16S539 IQCL VIC

9. 10. 11. 12. 13.

14. 15.

TED

16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

STR marker IQCS

Amelogenin

Allelic variation 1, 2 9–20 6–15 7–25 11–24 5, 8–15 1, 2 X, Y 1,2 5–19 7–18 7, 9, 10, 10.2, 11–13, 13.2, 14, 14.2, 15–27 24, 24.2, 25–28, 28.2, 29, 29.2, 30, 30.2, 31, 31.2, 32, 32.2, 33, 33.2, 34, 34.2, 35, 35.2, 36–38 8–11, 11.3, 12–17 13–26, 26.2, 27–30, 30.2, 31.2, 32.2, 33.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 50.2, 51.2 8–19 9–12, 12.2, 13, 13.2, 14, 14.2, 15, 15.2, 16, 16.2, 17, 17.2 9–14, 14.3, 15, 15.3, 16, 16.3, 17, 17.3, 18.3, 19.3, 20.3 6–15 5–16 8–19 5–26 5–15 11–28 4–9, 9.3, 10–12, 13.3 14–19, 19.3, 20–27 2.2, 3.2, 5–17

Fusion 6C Kit In forensic casework the evidence often contains a mixture of DNA from more than one individual. These mixtures can be very challenging to analyze and interpret. In most challenging cases where mixture of samples is found from crime scene, these

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VIC TED TAZ

SID

LIZ

Normalized Emission

6-FAM

175

500

550

600

650

700

750

Wavelength (nm) Fig. 3 Emission spectral graph of six dyes used by Verifiler Plus PCR amplification multiplex assay kit for labeling all the 25 loci

types of samples poses challenges to forensic community and scientific community to distinguish and identify the individuals. In January 2015, Promega Corporation released a powerful STR kit “Powerplex Fusion 6C system” to resolve these types of challenge and provide a solution to forensic community and scientific community. PowerPlex fusion 6C kit amplifies 27 locus multiplex which includes 23 autosomal STR loci, 3Y-STR, and one amelogenin marker for human identification, scientific testing, and research use. The Powerplex Fusion 6C system meets the requirement of CODIS and European standard enabling to achieve the international database compatibility (Ensenberger et al. 2016). The Powerplex Fusion 6C system is manufactured in accordance with ISO 18385:2016. The Fusion 6C kit uses six-dye technology to amplify all 27-locus multiplex. The key features provided by Powerplex Fusion 6C system include: • It reduces sample processing time by direct amplification protocols and rapid cycling capabilities • It minimizes the need of re-amplification enhanced inhibitors tolerance and sensitivity to minimize the need of re-amplify samples for casework • It meets the recommendation of CODIS marker and European standard set marker • It provides highest PI value for human identification. The 27 loci comprised of expanded CODIS core loci known as CODIS 20 along with amelogenin and DYS391 for gender discrimination. To increase more discriminatory power it also includes Penta-D, Penta-E, and SE33 loci (Shivani et al. 2019). Apart from these loci two rapidly mutation Y-STR loci are included, i.e., DYS570 and DYS576. The 27 loci are included in Fusion 6C kit as follows (Boavida et al. 2017):

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• CODIS 20: CSF1PO, D1S1656, D2S441, D2S1338, D3S1358, D5S818, D7S820, D8S1179, FGA, TH01, TPOX, VWA, D10S1248, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, and D22S1045 • Gender marker: Amelogenin and DYS391 • ESS markers: Penta-D, Penta-E, and SE33 • Rapidly mutating Y-STR marker: DYS570 and DYS576 This extended panel of STR marker in PowerPlex Fusion 6C system is intended to satisfy both CODIS and ESS recommendations (Table 7). The Powerplex Fusion 6C system is designed for amplification of extracted DNA and direct amplification samples. The primers included in the Powerplex Fusion 6C system have been designed to amplify human DNA. DNA samples encountered in forensic cases may contain trace or even excess nonhuman DNA along with human DNA. Hence, it is possible for some primers to recognize sites within nonhuman sources of DNA (Protocol, Use of the powerplex Fusion 6C system to amplify extracted DNA 2020). The possibility of mix source of human and nonhuman DNA may come from different circumstances such as: microbes in samples, environmental contamination, other animal biological uids, etc. Validation studies of Powerplex Fusion 6C system by different researchers show a threshold RFU peak to determine, identify, and distinguish the RFU peak of human DNA and nonhuman DNA. Powerplex Fusion 6C system uses six-dye technology for labeling all the loci. These dyes are FL, JOE, TMR, CXR, TOM, and WEN. These six dye hues are blue, green, yellow, red, purple, and orange, respectively. Among these six dyes, five dyes are used for labeling samples, allelic ladders, and control, i.e., FL, JOE, TMR, CXR, and TOM, while one dye is used for internal lane standard, i.e., WEN referred as WEN ILS 500 (Technical Manual, Powerplex Fusion 6C system for the use on the spectrum compact CE System 2020). All the 27 loci of Powerplex Fusion 6C system have size lesser than 500 bp. The lesser size and allelic variation of all loci and mini-STR provide greater discrimination power to distinguish between two individuals. The additional Y-marker provides greater discriminatory power for gender determination and human identification which increased the sensitivity for forensic cases such as paternity dispute, rape cases, etc. One of the main advantages this kit provides is distinguish between two individuals from the mixture of DNA. Different validation and mixture study shows that mixture of three individuals DNA can be analyzed and determined by Powerplex Fusion 6C system. Powerplex Fusion 6C system yields good DNA profile from touch DNA samples which enable the forensic community to face most challenging sample even in harsh environment and adverse circumstances. Powerplex Fusion 6C system provides highest probability of identity (PI), hence it decreases the likelihood of adventitious match of two individuals. The matching probability between two individuals after analysis by Powerplex Fusion 6C system is 1031. The Powerplex Fusion 6C system is compatible with different genetic analyzer such as ABI prism 3100 genetic analyzer, ABI prism 3100-avant genetic analyzer

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Table 7 27 markers of PowerPlex Fusion 6C STR multiplex kit with respect to labeling dye for each marker and allelic variation of each STR loci in kit (Technical Manual, Powerplex Fusion 6C system for the use on the Applied Biosystem Genetic Analyzers, 2018) STR marker D1S1656

Chromosome location 1q42

Repeat sequence TAGA complex

2. 3.

D2S441 D3S1358

2p14 3p21.31

4. 5. 6. 7.

D10S1248 D13S317 Penta-E Amelogenin D2S1338

10q26.3 13q31.1 15q26.2 Xp22.1–22.3 and Yp11.2 2q35

TCTA TCTA complex GGAA TATC AAAGA NA

CSF1PO D16S539 D18S51

5q33.1 16q24.1 18q21.33

Penta-D TPOX D5A818 D7S820 TH01

21q22.3 2p25.3 5q23.2 7q21.11 11p15.5

17.

vWA

12p13.31

18.

D21S11

21q21.1

SE33

6q14

AAAG complex

20.

D8S1179

8q24.13

21.

D12S391

12p12

22.

D19S433

19q12

TCTA complex AGAT/ AGAC complex AAGG complex

23. 24.

D22S1045 FGA

22q12.3 4q28

S. No. 1.

8.

Label dye FL6C

JOE6C

9. 10. 11. 12. 13. 14. 15. 16.

19.

TMR6C

CXR6C

TGCC/ TTCC AGAT GATA AGAA (20) AAAGA AATG AGAT GATA AATG (20) TCTA complex TCTA complex

ATT

Allelic variation 9–14, 14.3, 15, 15.3, 16, 16.3, 17, 17.3, 18, 18.3, 19, 19.3, 20.3 8–11, 11.3, 12–17 9–20 8–19 5–17 5–25 X, Y 10, 12, 14–28 5–16 4–16 7–10, 10.2, 11–13, 13.2, 14–27 2.2, 3.2, 5–17 4–16 6–18 5–16 3–9, 9.3, 10–11, 13.3 10–24 24, 24.2, 25, 25.2 26–28, 28.2, 29, 29.2, 30, 30.2, 31, 31.2, 32, 32.2, 33, 33.2, 34, 34.2, 35, 35.2, 36–38 4.2, 6.2, 8–12, 12.2, 13, 13.2, 14, 14.2, 15, 15.2, 16, 16.2, 17, 17.2, 18, 18.2 7–19 14–17, 17.3, 18, 18.3, 19–27

5.2, 6.2, 8–12, 12.2, 13, 13.2, 14, 14.2, 15, 15.2, 16, 16.2, 17, 17.2, 18, 18.2 7–20 (continued)

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Table 7 (continued) S. No.

25. 26. 27.

Label dye TOM6C

STR marker

Chromosome location

Repeat sequence TTTC complex

DYS391 DYS570 DYS576

Y Y Y

TCTA TTTC AAAG

Allelic variation 14–18, 18.2, 19, 19.2, 20, 20.2, 21, 21.2, 22, 22.2, 23, 23.2, 24, 24.2, 25, 25.2, 26–30, 31.2, 32.2, 33.2, 42.2, 43.2, 44.2, 45.2, 46.2, 48.2, 50.2 5–16 10–25 11–23

and Applied Biosystem 3500 genetic analyzer, 3500xL genetic analyzer, 3130 genetic analyzer, and 3130 xL genetic analyzer although optimization of the different process is required as per the standard protocol provided by respective organization. Value obtained for the same sample can differ between instrument platforms, because of different polymer matrices and electrophoretic conditions.

Forensic Relevance of Amelogenin Marker In forensic science DNA analysis plays a vital role in identification of individuals. In forensic community and research community, there is frequent need to determine the sex of individuals based on DNA for the identification of victims, accused, or culprit. The ability to determine the sex of an individual based on DNA evidence can be crucial in forensic investigation. In humans, a specific marker is used for gender determination known as amelogenin. The amelogenin locus has two homologous genes present on both the X and the Y chromosomes referred as Amel-X and Amel-Y. However, there are size differences in this gene between chromosomes enable to distinguish between male and female and have been used for sexing in forensic science. Forensic DNA typing of sex determination in human has been proven to be imperious tool to criminal justice system. The widespread use for amelogenin marker for gender determination or human identification helps forensic community in different type of cases such as: sexual assaults cases, parental dispute, gender identification, missing person investigation, and identification of victims of mass disaster. It also can differentiate the relative contributions and estimate the ratio of male and female DNA in mixed forensic samples. The amelogenin marker for gender determination is discovered by Sulivan et al. in 1993 and published in “A rapid and quantitative DNA sex test: uorescence-based PCR analysis of X-Y homologous gene amelogenin, BioTechniques, 15, 637-641.” The physical location of amelogenin gene AMELX on distal short arm of X-chromosome is Xp22.1–22.3 and AMELY on near the centromere of the Y-chromosome is Yp11.2. It encodes a matrix protein which is involved in forming of enamel known as amelogenesis.

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The British Forensic Science Service was the first to describe the particular primer sets that are used in forensic DNA laboratories today (Butler 2015). Most commonly used primer sets are designed by Sullivan et al. Presenting amplicon of AMELX/ AMELY are 106/112 and 212/218 bp, respectively. The PCR product of AMELX and AMELY can be distinguished from one each other by primer anking of a 6 bp deletion in the 3rd intron AMELX which is absent in AMELY. Both AMELX and AMELY can be amplified and analyzed in a single reaction along with other loci. After amplification of the amelogenin gene, if the shorter fragment is observed which gives a single peak this indicates that the DNA samples contain Xchromosomes and that will represent female phenotype but if both fragments are observed, i.e., longer and shorter fragments exhibit two peaks, then this indicates the presence of chromosome X and Y which represents male phenotype. This size variation of amelogenin gene in chromosome X and Y is used by forensic community to determine the gender of the individuals. However, there is several challenges that pose threat for a proper DNA profiling for forensic community and research community such as: degraded samples, PCR inhibitors, presence of mixed DNA, mutation, AMELX/AMELY deletion, primer site polymorphism, and aneuploidy. To overcome this problem, advanced next generation STR kit contain multiple marker for gender discrimination which decrease the anomalies that could make the sample falsely appear as a female in gender determination. Addition of multiple marker increases the gender discrimination and identification of an individual DNA samples.

Forensic Validation of Autosomal STR Kits Whenever a DNA profile is generated, questions always arise about the credibility and reliability of the DNA profile and the process of the DNA typing by jury, lawyers, and criminal justice system. Forensic validation of autosomal STR kits is a vital part of DNA typing, whenever a DNA profile is submitted to court. Validation is required to ensure that the method and process is accurate, efficient, without any error, relevant, and backed by scientific explanation (Jamie et al. 2019). Validation of autosomal STR kits is categorized into different category to check the efficacy of generated DNA profile such as: • Developmental validation: Extensive efficacy performance by manufacturer of the method and technology before commercializing or publishing (Melody et al. 2009). • Internal validation: Check the effectiveness of the introduced method, process, or technology by an individual’s laboratories. • Performance checks: Check the performance of instruments either new or repaired, calibration of the instruments, validation of the glassware and reagents, and assessment of the control samples or reference samples.

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Forensic validation of autosomal STR kits ensures proficiency test, standard operating procedure, sensitivities, reproducibility, precision, historical performance if any, limitation, probabilities, and chances of errors. A forum of technical working group known as scientific working group on DNA analysis methods (SWGDAM) comprised of forensic scientists as well as academic expert and other expert. SWGDAM assess the method, protocols, training, and research and evaluate the quality assurance to provide recommendation to the FBI (Julio et al. 2008). SWGDAM provides guidelines for validation of different autosomal STR kits known as validation guidelines. The following parameters are accessed for the forensic validation of autosomal STR kits: • Specificity: PCR primers target specific loci in DNA for amplification. These similar loci or sequence may be found in different animal such as chimpanzee, and other mammals. During PCR amplification of human STR marker crossreactivity may occur with nonhuman DNA which can generate allelic peak in electropherogram. To overcome these problem different parameters is used in autosomal STR kits. These STR kits are manufactured in a way that it will be specific to human DNA samples. Also, RFU is identified and determined to exclude the nonhuman allelic peak in an individual DNA profile. For any peak to be considered in analysis, it should be on or above-threshold RFU. Different STR kits provide the specific data and guideline with respect to RFU peak inclusion and exclusion and for different species after validation studies. • Sensitivities: Efficacy of the material, method, and instruments are very important during analysis. Different autosomal STR kits are PCR-based method which amplifies the target loci in the samples. For amplification of targeted segments, sample should be in enough quantity but it is not always possible to get enough quantity of the samples for analysis. The sensitivity of the STR kit determines the enough quantity is required for amplification of the targeted segments. Different STR kits shows different sensitiveness for the amount of samples and improving the STR kit increases the efficacy of sensitivity which can detect and processed the low amount of DNA samples (Hammond et al. 1994). The amount of required DNA for different STR kits may vary from ng to 0.5 pg. • Reproducibility: Whenever a method or materials is introduced to public, it need to be tested and documented for producing adequate information about the results. The results should be consistent when an experiment is repeated many times. The reproducibility of STR kits provide the information of allele size range, buffer, mixture, and other materials included in STR kits are consistent or not. The biological samples collected from crime scene are often contaminated or degraded which can be overcome by enhanced buffer system and material included in the kits for optimal amplification of the target segments of the DNA (Holt et al. 2002).

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• Inhibition study: The biological samples collected from the crime scene for DNA profiling is often contaminated and may contain inhibitor factors. The often-encountered inhibitor factors are hematin, humic acid, tannic acid, phenol-chloroform, and other inhibitors. These inhibitors may inhibit PCR amplification which may result in alter peak or lesser peaks of the alleles. • Stutter product: Allelic dropout and allelic dropout is often encountered during PCR amplification of the loci which resulted in loss of peak height. This phenomenon is known as stutter product. During DNA profiling stutter product is often encountered. This problem is generally handled with standard deviation method or threshold point inclusion and the guideline provided by respective manufacturer of STR kit. Although different validation studies show about the problem of stutters during PCR amplification of target sequence of DNA and often assessed by different calculation.

Usefulness of Autosomal STR Kits in Forensic Practice DNA fingerprinting was first used in forensic science by Sir Alec Jeffreys of University of Leicester to verify a suspect’s confession which he confessed for two rape and murders in 1986. With the help of DNA profiling test, it was founded that he had not committed the crimes. First paper publication on STR was published on 1991 while first STR kit was published by Applied Biosystem is AmpFISTR Blue in October 1996. There are many types of uses of STR kits in different scientific community such as: • • • • •

Environmental and health science Genetics Evolution Cell and molecular biology Forensic science

In forensic science, DNA profiling is an ultimate tool for forensic investigation based on DNA evidence. It helps criminal justice system to determine the victim, accused, or culprit. It helps in different types of forensic caseworks such as: • • • • • • •

Human identification Paternity dispute Mass disasters cases Historical investigation Missing person investigation Military DNA (dog tag) case work Preparation of DNA databases

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Conclusion The ultimate power of DNA profiling as an identification of individuals provides a tremendous technology which helps in forensic investigation and criminal justice system. Forensic community use the biological resource to obtain the DNA of an individual for the forensic DNA typing based on commonly used STR markers. STR sometimes also referred as microsatellite or simple sequence repeats. Human genome contains thousands of STR markers, only a small core set of loci have been selected for forensic DNA analysis and human identification. These STR loci is authenticated by different agency and organization such as Combined DNA Index System (CODIS) of the USA, core loci managed by FBI and NIST, European Standard Set (ESS), and many more organization to use these loci in forensic DNA profiling. Theses STR loci is amplified with different PCR amplification kit commercialized by different corporation. These organizations provide a variety of autosomal STR kit that meets the requirement of forensic community and used in forensic investigation for different types of casework. The development of new generation STR kit is required to meet the advance era scenario which can overcome limitation of old generation kit and provide result which can be interpreted easily. The harsh condition, environment, contamination, and many more issue are faced during DNA typing from the samples. The continuous advancement and improvement is the need of the hour to overcome all the problem faced by forensic community in DNA typing.

References Al Janaahi N et al (2019) Forensic evaluation of Verifiler plus 6-dye chemistry kit composed of 23 loci with casework samples. Forensic Sci Int Genet Suppl Ser 7(1):892–896 Al-Snan NR et al (2019) Population genetic data of the 21 autosomal STRs included in Globalfiler kit of a population sample from the kingdom of Bahrain. PLoS One. https://doi.org/10.1371/ journal.pone.0220620 Boavida A et. al. (2017), Powerplex Fusion 6C system: Internal Validation study, Forensic Genetics, Forensic Science Research 3(2). https://doi.org/10.1080/20961790.2018.1430471. Bogas V et al (2015) Testing the behaviour of Globalfiler PCR amplification kit with degraded and/or inhibited biological samples. Forensic Sci Int Genet Suppl Ser 5:e21–e23 Brito P et al (2015) Direct amplification of casework samples in fabrics using globalfiler PCR amplification kit. Forensic Sci Int Genet Suppl Ser 5:e487–e489 Butler JM (2005) Forensic DNA typing, 2nd edition biology, technology and genetics of STR markers. Academic, Elsevier Butler JM (2011) Short Tandem Repeat Loci and Kits, Science direct. https://doi.org/10.1016/ B978-0-12-374513-2.00005-1. Butler JM (2012) Advanced topics in forensic DNA typing: methodology. Academic, Elsevier Butler JM (2015) STR Profiles: Multiplex PCR, Tri-alleles, Amelogenin and partial profiles. https:// doi.org/10.1016/B978-0-12-405213-0.00005-1. Butler JM (2018) Short tandem repeat typing technologies used in human identity testing. Biotechniques 43(4):ii Butler JM, Hill CR (2012) Biology and genetics of new autosomal STR loci useful for forensic DNA analysis. Forensic Sci Rev 24:15 Christian RM et al (2001) STRBase: a short tandem repeat DNA database for the human identity testing community. Nucleic Acids Res NCBI 29(1):320–322

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Forensic Human Y-Chromosome Markers: Principles and Applications Arash Alipour Tabrizi

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Y-Chromosome Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Markers on Y Chromosome and Polymorphisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Y-STRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rapidly Mutating Y-STRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mini Y-STRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inheritance Pattern of Y-STR Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Y-SNPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Y-STR Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interpretation of Y-STR Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . No Interpretable Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inconclusive Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Exclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inclusion/Match . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mixture Interpretation Via Y-STR Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Forensic Use of Y-Chromosome Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sexual Assault Investigation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sex Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Paternity Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Disaster Victim Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Historical Investigations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Y-STR Haplotype Databases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Familial Searching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Haplogroup Prediction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

The human Y chromosome distinguishes males from females and passes through male lineages as a highly polymorphic data-field linked together which is called A. Alipour Tabrizi (*) Legal Medicine Research Center, Legal Medicine Organization, Tehran, Iran © Springer Nature Singapore Pte Ltd. 2022 H. R. Dash et al. (eds.), Handbook of DNA Profiling, https://doi.org/10.1007/978-981-16-4318-7_6

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Y-haplotype. Out of different types of polymorphisms mainly produced by the mutational process, Y short tandem repeats (Y-STRs) are the most popular markers implemented in commercial multiplex amplification kits and routinely used for forensic genetic casework analysis. The unique specifications of Y-STRs and mini Y-STR markers make them powerful alternative tools in the hands of forensic experts to analyze compromised crime scene evidence and solve complicated sexual crimes even in sperm-free samples or in the presence of minimal amounts of male DNA in a mixture. The exceptional features of rapidly mutating Y-STRs suggested them as the markers of the choice to differentiate among male paternal relatives. The application of national/international Y-haplotype databases and familial searching strategy empowers forensic investigators to narrow investigation domains. The characteristics of Y-chromosome single-nucleotide polymorphisms could be beneficial for haplogroup determination and historical investigations. In addition to the benefits of typing in sexual assault investigation, sex determination, and paternity dispute analysis, various technical limitations should be considered and addressed in standard interpretations and reporting guidelines. Keywords

Y chromosome · Forensic genetic · Y-STR · Y-SNP · Y-haplogroup · Y-haplotype database

Introduction In addition to visible phenotypic differences between males and females, which were remarkable for the humans of the distant past, several gender-specific feathers are assigned to modern human males. These gender-specific feathers include the endless use of the television remote buttons, the ability to remember the soccer scores while amnesiac for his wedding anniversary, driving hours without asking the right direction, and the inability to differ a lapis, azure, and blue bag. The Y chromosome as an indicator of maleness is crucial for phenotypic male traits, fertility maintenance, cerebral asymmetry, handedness, and tooth size.

Y-Chromosome Structure Since 1905, human sex determination has been based on the presence or lack of the Y chromosome. A normal karyotype of a human male has one Y chromosome; however, no Y chromosome is detected in human females. The full human Y-chromosome sequence was announced in 2003 for the first time. About 2% of the total human genome (i.e., approximately 60 million nucleotides) consists of the Y chromosome. This third-smallest human chromosome in a normal male karyotype contains the least number of genes in comparison with any other chromosomes.

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The determination of genetic gender in humans is based on the male determining genes (i.e., sex-determining region Y [SRY] genes). The SRY genes are located in the male-specific region of the Y chromosome (MSY) in the non-recombining region of the Y chromosome (NRY) nearby the distal end of the short arm (p) of the Y chromosome encoding the developmental factors of the testes. The MSY contains 78 functional protein-coding sequences encoding 27 different proteins. In addition to the coding regions, the MSY consists of repetitive sequences. Different from autosomes, only about 5% of the Y chromosome could have recombination with the X chromosome. These three portions are located at the distal tips of the Y chromosome called the pseudo autosomal regions (i.e., PAR1, PAR2, and PAR3). The PAR1 and PAR2 are located at the ends of the short arms and long arms of the X and Y chromosomes, respectively. PAR3 defined in only 2% of the general population has over 98% of sequence homology with Xq21.3 which was identified in 2013. Due to this lack of recombination, the mutational processes are the most responsible for diversity along the Y chromosomes of paternal lineages. These mutations will be passed to the next generations and not be repaired due to the lack of crossing over. Therefore, as the only known mechanism for variation between the males of a paternal lineage is a mutation, more mutations in a marker lead to further power of discrimination and greater use in forensic application and human identification. As a mosaic aneuploidy, the loss of chromosome Y (LOY) can be typically detected in the blood samples of males. There is a direct correlation between the incidence of LOY and chronological age in healthy males older than 16 years. Since buccal or white blood cells are sampled and routinely employed in forensic cases, the LOY may affect the results. It is necessary to carry out further studies on the effect of the LOY on the forensic analyses of these samples for better clarifications (Barros et al. 2020).

Markers on Y Chromosome and Polymorphisms Y-chromosome variations and polymorphisms were revealed in searching for the 1000 Genomes Project data set. Different types of polymorphisms, including microsatellites, minisatellites, and single-nucleotide polymorphisms (SNPs), are located on the Y chromosome along the non-recombining region and make it a suitable target for polymerase chain reaction (PCR)-based analysis for forensic purposes. In a simple classification, the mostly employed forensically relevant Y-chromosome markers are divided into two main categories, namely, biallelic markers with only two possible alleles and multi-allelic markers. Repeating microsatellites or Y short tandem repeats (Y-STRs) as the main multi-allelic forensic genetic markers and Y-chromosome single-nucleotide polymorphisms (Y-SNPs) in the subset of biallelic markers are widely used for human identification objectives. Another type of polymorphism defined as different internal sequence variations among alleles with a similar length is called SNPSTR. An example of this type of polymorphism on the Y chromosome revealed by base composition analysis is two “17” iso-alleles reported for

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DYS635. Both of them appear the same alleles in capillary electrophoresis (CE) analysis, indicating different sequence variations in the next-generation sequencing (NGS) (Butler 2014).

Y-STRs Genetic information based on the autosomal short tandem repeat (aSTR) markers are inherited half from the mother and half from the father; however, Y-STRs, as popular non-autosomal markers, are located on the human Y chromosome. A Y-STR haplotype consists of a combination of alleles noticed at all tested loci as a single entity. Therefore, Y-STRs are indicated as paternal lineage markers, and each of the loci has a single allele, rather than two alleles in aSTRs, and is considered a Y-haplotype and not a genotype. Since there is one Y chromosome, instead of homo
/heterozygosity observed in aSTRs, there is hemizygosity in Y-STRs. Approximately 4,500 Y-STRs were identified and utilized in different applications and other multidisciplinary studies (He et al. 2019). The Human Genome Organization Gene Nomenclature Committee assigned names for Y-STR markers based on the standards for human gene nomenclature. Accordingly, a marker name consists of two parts, including the DYS that is the abbreviation for “deoxyribonucleic acid (DNA) Y-chromosome single-copy sequence” followed by a unique identification number re ecting the order in which the Y-STR is discovered. The DYS- and DYF-numbers are referred to as single-copy and multi-copy markers, respectively. The first forensically useful Y-STR locus applied for human identification was Y-27H39, currently better known as DYS19, which was described by Lutz Roewer et al. in 1992. Besides, the potential application of the Y-STRs in forensic analysis has been determined for almost two decades. In a short time following the characterization and assessment of the first Y-chromosomal STR polymorphism, its practicality in crime casework was shown when a mixed stain from a vaginal swab of a raped and murdered female victim was resolved using Y-STR analysis, and a falsely convicted male was excluded in 1992 (Gill et al. 2001; Roewer et al. 1992). The first set of nine Y-STR loci includes DYS19, D YS389I/II, DYS390, DYS391, DYS385 a/b, DYS392, and DYS393selected as a standard for data transfer and population data management by the Scientific Working Group on DNA Analysis Methods (SWGDAM) and European Community and referred to as the minimal haplotype set in 1997. All minimal haplotype loci are included in commercially available Y-STR typing kits. Moreover, to date, tens of the new additional Y-STRs are included in the forensic grade Y-STR typing kits by different kit providers. Although most Y-STR loci included in Y-STR typing kits have tetranucleotide repeats (i.e., four-base pairs), the loci with five- or six-base pair repeats have been selected for forensic applications. In addition to a high degree of polymorphism, penta- and hexanucleotide repeat loci show a much lower degree of stutter product formation; as a result, they are considered valuable tools for mixture interpretations (Butler 2003).

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According to the comprehensive determination of the physical map of human Y-STRs, it was shown that most of the Y-STR loci were located within the Yq11.221, Yq11.222, and Yq11.223 segments and Yp11.2 segment on the long and short arms of the chromosome, respectively. No Y-STR loci were located in the telomeric regions of the Y chromosome, Yp11.32, and Yq12. Furthermore, only three loci (i.e., DYS631, DYS716, and DYS707) were located in a centromeric region on Yp11.1 (Hanson and Ballantyne 2006). Several markers in commercial kits always show more than one allele due to the identical copies of sequence on the Y chromosome (e.g., DYS385 and DYF387S1, each of which comprised of two loci) despite the fact that each Y-STR locus demonstrates a single allele. DYS464 is the most impressive polymorphic Y-STR discovered, with at least four copies in the Y chromosome as a duplicated locus (Butler 2003; Roewer et al. 2020). The DYS389 locus is complex consisting of two polymorphic regions. DYS389I is a polymorphic element within DYS389II as the whole locus. The forensic application of Y-STRs is the result of their high levels of polymorphism in human populations, small size in base pairs (range: 100–400 bp), and the ability for automation via a multiplex Y-STR PCR reaction (Hammer and Redd 2005).

Rapidly Mutating Y-STRs The evaluation of mutation rates for Y-STRs indicated that most of them mutate at a rate similar to that of aSTRs. Although it might be different in different loci in various populations, it is estimated that Y-STRs mutate about 1–4 per thousand per generation. As previously discussed, the only known mechanisms for variation among Y-STRs are the mutational events of slipped strand during the replication of DNA. Even though all Y-STRs are located and linked on the same chromosome, the mutation of various Y-STR loci occurs independently from each other. The structure of the locus and quantity of repeats (i.e., allele number) are for the most part associated with the mutation rate of Y-STRs, as more repeats lead to more DNA slippage during replication. Therefore, it is estimated that fathers passing greater Y-STR allele numbers have an increased chance of mutation to occur in comparison with fathers passing short Y-STR alleles at the same loci (Ralf et al. 2020). As a result, Y-STR mutation rates show inter-locus and intra-locus variations. The Y-STR loci, including compound repeats, tend more to mutate than simple repeat ones. They almost change to a single repeat gain, more common than multirepeat changes, and the mutation rates differ based on the locus-specific and allelespecific manners. In order to decrease the suspect pool via the separation of distant male relatives from close ones and further diversification of the population, forensic geneticists tend to apply an increasing number of Y-STRs, especially the application of rapidly mutating (RM) Y-STRs, to provide more chance of the detection of Y-STR mutations allowing reaching higher discrimination capacities. Out of the first 17 Y-STR loci in use, only the mutation rates of DYS458 and DYS439 are higher

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than 0.5%. Nevertheless, a higher rate of mutation is shown for several more recently added loci, such as DYS570 (2.23%), DYS576 (1.91%), and DYS481 (0.955%) (Butler 2014). The estimation of Y-STR locus mutation rates is based on the Y-haplotype comparisons from direct father-son pairs ((Ballantyne et al. 2010; Claerhout et al. 2019). The results of comprehensive Y-STR mutation rate assay is almost 2,000 confirmed father-son couples revealing that out of 200 investigated Y-STRs, 13 Y-STRs (including single- or multi-copy RM Y-STR markers of DYF403S1, DYF404S1, DYS449, DYS518, DYS526, DYF387S1, DYF399S1, DYS547, DYS570, DYS576, DYS612, DYS626, and DYS627) were identified with exceedingly high rates of mutation (>10–2 per locus per generation) nominated for RM Y-STRs. The evaluation of the RM Y-STR set in the context of male differentiation and power of detecting hidden paternal relationships demonstrated that it could help to discriminate between two male relatives even in populations with high levels of endogamy and ethnic fragmentation with lower proportions of unique haplotypes of RM Y-STRs (Ballantyne et al. 2014; Adnan et al. 2016; Della Rocca et al. 2019; Almohammed and Hadi 2019). Manfred Kayser et al. investigated the mutability of Y-STRs, mutation rates, features, molecular bases, and forensic implications. The results of the aforementioned study showed that Y-STR mutations occurred only after 11 or higher homogeneous adjacent repeats. In addition, two of the Y-STRs (i.e., DYS570 and DYS576) represented much higher rates of mutation in comparison with those included in the commercial kits (Kayser 2017). The goal of increasing the discrimination power of multiplex Y-STR haplotype leads to implement RM Y-STR markers in newly emerged Y-STR kits. In 2010, Manfred Kayser et al. indicated that differentiating a male from his close relatives may be possible by the application of a set of 13 RM Y-STRs. The development of RM Y-STRs provides a new perspective on the future of the forensic analysis of the Y chromosome. The Y-STR markers are classified into four groups according to estimated rates of mutation, namely, slowly mutating (SM Y-STRs), moderately mutating (MM Y-STRs), fast mutating (FM Y-STRs), and RM Y-STRs. The mutation rates of SM Y-STRs are reported as 100-fold excess of female DNA. The provided allelic ladder is designed

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to have an average of 35 bp separating spaces between two adjacent loci for the reduction of the potential for locus overlaps and consists of 102 alleles (Promega 2003). AmpFlSTR YfilerTM PCR Amplification Kit (released in 2004 by Life Technologies, Foster City, CA, USA) is a five-dye multiplex PCR kit with reagents for the multiplex amplification of 17 Y-STR loci (i.e., DYS19, DYS385 [counted as two loci], DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS456, DYS458, DYS635, DYS448, and Y GATA H4) enabling full DNA profiles based on 125 pg of male DNA in only 3 h (Yfiler ® Plus PCR Amplification Kit 2016). The PowerPlex ® Y23 System (PPY23, Promega Corporation, Madison, WI, USA), released in 2012, as a five-dye Y-STR multiplex was developed for Y-STR typing at 23 loci in only 72 min. For the construction of the kit, beneficial properties, such as the short length of the fragments and an uninterrupted repeat structure, were considered. Six new markers (i.e., DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643), two of which (i.e., DYS570 and DYS576) classified as RM Y-STRs, were attached to the Yfiler ® panel (PowerPlex Y23 System Technical Manual 2012; Thompson et al. 2013; Purps et al. 2014). However, since even close relatives might present one or more mismatches, especially at DYS570 and DYS676, the application of the PPY23 kit in kinship analysis or familial searching will render these practices increasingly complex. For the aforementioned applications, compulsory utilization of likelihoodbased techniques should be considered which will be discussed later. The PowerPlex ® Y23 System benefits from a high sensitivity in case there is a female DNA (1: 1000 and faster time to obtain results with a higher power of discrimination mainly due to the included

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RM Y-STRs. Due to the additional loci, the Yfiler ® Plus kit® is expected to have a higher discrimination power and better ability to determine the correct number of contributors to a mixed profile with an almost equal cost of analysis per sample than the Yfiler or PowerPlex Y-23 System (Ferreira-Silva et al. 2018; Dash et al. 2020b). The amplification of degraded DNA samples by the Yfiler ® Plus ® is facilitated due to the enhanced polymerase and buffer systems together with improvements in a panel design with 11 mini Y-STR loci (below 220 bp in size) due to the inclusion of 6 dyes (i.e., FAM™, VIC™, NED™, TAZ™, SID™, and LIZ™). Mobilitymodifying non-nucleotide linkers are implemented in some of the primers to facilitate inter-locus spacing (Alghafri et al. 2015). There was no cross-reactivity of the Yfiler™ Plus Kit ® with any of these nonhuman species except for the DNA samples of chimpanzees and gorillas producing partial profiles in the 100–330-base pair region (Yfiler ® Plus PCR Amplification Kit 2016; Gopinath et al. 2016; Ambers et al. 2018). As the more Y-STR implemented and enzymes, master mix, and primer design improved, the results of the 27 loci become much better and reliable. However, these strategies have drawbacks. As more and more loci were profiled, PCR products inevitably become large and could not effectively amplify, especially in longer alleles. Apart from seven RM-YSTs added to the panel for the improvement of discrimination power, the RM Y-STR databases should be established for the estimation of match probabilities (Ballantyne et al. 2012). As Saadi of Shiraz, a major Persian poet, said in his masterpiece The Rose Garden that “Either makes no friends with elephant-keepers or build a house suitable for elephants,” before the utilization of such a sensitive Y-STR typing kit, every laboratory should adopt contamination prevention strategies, along with updated guidelines for reliable interpretation (Tootkaboni 2017).

Interpretation of Y-STR Results The interpretation of the results and accurate wording of the final reports are the two most critical steps in the Y-STR DNA typing process. A forensic investigator should be aware, fully experienced, and prepared for different situations happening in each step of the whole process. The interpretation step, by itself, should be objectively and consistently performed by two genetic experts. The guidelines for interpreting and reporting the results of Y-STR DNA are provided by national authorities and international societies. The application of these international standard protocols can terminate the local setups and personal wordings which may lead to misunderstanding, confusion, and misinterpretation of the unique analytical results in national/ international casework. An updated guideline of the application of Y-STRs in forensic analysis was published by the DNA Commission of the International Society of Forensic Genetics (Gusmao et al. 2006). The pattern of Y-STR peaks is obtained after the electrophoresis of multiplex PCR products, and haplotypic data are analyzed by the application of GeneMapper ® ID-X (GMID-X) ® software (Life Technologies, USA). Prior to the

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interpretation of the Y-STR typing results, some preliminary assessments should be applied. The process is initiated by confirming properly functioned positive and negative controls, correct fragment sizing and allele callings, considering artifacts (especially stutters), designating the consistency of profile with being from a single male or a mixture, and determining which loci are interpretable. As a wellcharacterized PCR artifact, stutter refers to the appearance of a minor peak one repeat unit smaller than the target Y-STR allele product (i.e., minus stutter) or less frequently one repeat larger (i.e., plus stutter) (Butler 2014). The next following step is the evaluation of the quality of a Y-STR profile for the determination of the sufficiency of the Y-haplotype information for application in further comparisons. Therefore, a forensic laboratory should establish guidelines to determine the qualification of the Y-STR haplotype for comparisons (SWGDAM 2017; Roewer et al. 2020). For the interpretation of a 23 Y-STR haplotype, there should be a minimum of 6 loci with data above the analytical threshold. Single source and single major contributor haplotypic data with a minimum of ten loci should be subjected to statistical calculation (North Carolina 2016). All the questioned samples should be solitarily qualified, analyzed, and interpreted before being compared to known (i.e., suspect) profiles. After the standard checkup of the data and comparison of questioned Y-haplotypes to known reference haplotypes, there are four possible results:

No Interpretable Results No signals (i.e., peaks) above baseline or signals above baseline but below the analytical threshold at one or more loci were detected on the electropherogram.

Inconclusive Results One or more loci provide interpretable peaks; nevertheless, no conclusive result can be achieved based on these limited data due to insufficient quality and/or quantity not supporting rendering a conclusion to be included or excluded. Partial haplotype as a result of allele dropout, stochastic effects, locus dropout due to inhibition, degradation, or limited amounts of DNA may cause inconclusive results. In contrast to what was previously discussed (no interpretable results), in this section there are limited or partial data; however, the haplotype is of no comparative value (i.e., uninterpretable) and not suitable for further comparisons. According to the updated version of Federal Bureau of Investigation Y-STR Interpretation Guidelines, at least 14 Y-STR data in a given Y-STR haplotype should be included to report a cannot exclude statement. The Y-STR haplotypes, including lower sets of data, will be used only for exclusionary purposes. Comparisons, in which exclusion cannot be made, will be assumed as inconclusive (FBI 2020).

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Exclusion In addition to the abovementioned benefits of Y-STRs in the forensic analysis of DNA, it has some special limitations because out of all Y-STR data directly inherited from a male to his male lineages, only an exclusion result of Y-STR analysis can be helpful. Once the Y-STR typing results were evaluated and approved for further comparison and decision-making, the haplotypes of known (i.e., reference) and questioned samples should be compared. The side-to-side comparison of two known and unknown Y-STR haplotypes may demonstrate discrepancies between the allele(s) of one or more loci. Functionally, all mismatches can be categorized into two main groups. The first class consists of incompatible Y-STR haplotypes due to contributors belonging to different male lineages, called factual mismatches/exclusions. No analytical errors, technical limitations, or scientific justifications could be attributed to this class of mismatch cases. The second group is composed of differences that could be explained via scientific reasons (e.g., dropout, drop-in, mutations, and duplications), called factitious mismatches/exclusions. As more Y-STR loci are included in modern Y-STR typing kits, a single locus inconsistency between a male and his biological father is relatively regularly expected, and cases with double paternal genetic inconsistencies have been more frequently reported. In some forensic/kinship cases, there could be a difference in the multi-locus haplotypes of several brothers at a single locus as a result of the mutation of the fathers’ germ cells which may not be observed in all of his sons (Andersen and Balding 2017; Mertoglu et al. 2018). No effective binding of a primer might result from a point mutation in the 30 end of the primer binding leading to the absence of a detectable amount of PCR product bringing about a null (silent) allele (especially in DYF387S1 and DYS385). Y-chromosome microdeletions caused null alleles at one or more loci. Accordingly, it is worth mentioning that such structural/sequence alterations might be interpreted as factitious exclusions. Suppose that two Y-STR haplotypes are compared from a buccal swab of a suspect and an unknown blood spot, detected on probative crime scene evidence. If the Y-STR haplotypes of the evidence and suspect differ by at least one allele, the suspect will be ruled out as a contributor to the unknown Y-STR haplotype given the haplotype interpretation and interpretation assumptions, including factitious mismatches (exclusion). A verbal statement (e.g., someone other than the suspect is the source of the DNA) should be mentioned to support the results. Forensic geneticists may have to deal with the structural/sequence changes of the Y chromosome. In case of such an occurrence, it is of great importance to know how to interpret the nature of these results and how to disclose them in final reports. A simple strategy for exclusion interpretation in paternity or kinship analysis should be based on a minimum of three or more differences between two male samples to avoid factitious exclusions due to mutational events (Kayser and Sajantila 2001). A more conservative and smart approach for the exclusion of paternity or other kinship questions would be according to a exible dynamic model, as an alternative to the application of a fixed rule-based upon ad hoc cutoffs (i.e., the exclusion of a

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minimum of three or more Y-STRs). Accordingly, four fundamental WH questions need to be answered before the assessment of exclusion: 1. How many Y-STR loci have been analyzed? 2. What is the estimation of locus-specific mutation rate in the relevant population? 3. How many repeat number differences were observed between mismatching alleles? 4. What is the difference between the generations of two individuals with incompatible Y-STR haplotypes? Although the profiling of Y-STR is advantageous for exclusionary goals due to the unambiguity of the results without the necessity to provide a statistical weight, the aforementioned considerations should be taken into account for the prevention of false exclusions. According to the guidelines of the SWGDAM, it is recommended to apply Y-STR mixtures not presenting a clear major contributor only for the exclusion (Ballantyne et al. 2014; Hampikian et al. 2017).

Inclusion/Match In a comparison of a reference haplotype to an unknown haplotype obtained from questioned evidence, they may possess similar alleles at each locus (i.e., identical length alleles in case of the application of the CE method or identical sequence variants in case of the use of NGS), or few non-match alleles were observed which could be explained via scientific or technical resonating. Moreover, the reference cannot be ruled out as a possible contributor to the evidence haplotype. A full match between a known suspect and evidence of Y-STR haplotypes only indicates that he could be one (and not the only) of the suspects contributing to the crime scene evidence as his male lineages could. Due to this linked inheritance, the strength of the match could be evaluated by the calculation of the rarity of the Y-STR haplotype in the relevant population. The estimation for a random match with Y-STR haplotypes, as a single locus by magnitudes more variable than single aSTR loci, is carried out via the counting method instead of the common product rule applying for aSTR genotypes. As the SWGDAM stated, an appropriate and conservative statistical technique for the assessment of the probative value of a match is provided using the counting method incorporating the upper-bound estimate of the count proportion. In the counting method, the profile should be searched in different databases for a possible match to determine the rarity of a Y-STR haplotype. Basically, it means that how frequently a Y-STR haplotype could be observed in the relevant database, and it can be calculated by dividing the number of Y-haplotype observation/s (the numerator) by the total number of haplotypes included in the utilized database as the denominator. Therefore, as the size of the database increases, the random match more rarely occurs. A more useful Y-STR database indicates better statistics in this regard. A match probability (MP) between the two Y-STR haplotypes of 0.001

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shows that there is a 1 in 1,000 chance of random selection of a second individual with this haplotype provided that it has already been noticed once. Given the chance of 1 in a billion commonly provided in forensic reports for the profiling of autosomal markers, 1 in 1,000 for YSTRs may not be persuasive enough. Consequently, the MP results of Y-STR typing could not effectively differentiate between two male individuals as aSTRs could, and aSTR typing is always preferred to solve forensic casework. Alternatively, the assessment of the weight of a Y-STR match is feasible using a likelihood ratio approach. An additional advantage of the likelihood ratio method is the possible management of population subdivisions with an increased potential for co-ancestry. Accordingly, in the case of two Y-STR matches, it is recommended to carry out a quantitative assessment of the value of the match using relevant population/metapopulation data according to the national guidelines. The evaluation of the findings via the likelihood ratio (LR) approach needs the formulation of alternative hypotheses. The alternative hypotheses may state as follows: Hypothesis 1: The source of the Y-haplotype is regarded as the known suspect. Hypothesis 2: The source of the Y-haplotype is regarded as a random male Y from the reference population, and Y is another man, not the suspect. The Y-STR profile detected in the crime stain is LR times more likely to be observed under hypothesis 1 than those under hypothesis 2. The remaining issue is the possibility that a male individual, with no close relation to the person of interest, has an identical Y-STR profile. This possibility can be evaluated by the application of the discrete Laplace (DL) method, approximating the proportion of haplotypes in the population/metapopulation to which the suspect belongs, with the same Y-haplotype. The DL method is a parametric statistical model applied for the estimation of the frequencies of Y-STR haplotypes considering a reference database. A report based on quantitative assessment via the LR approach could be that suspect (known) could have contributed to the male source of the detected DNA. In addition, all male relatives on the paternal line and approximately 1 in 1,000 (i.e., likelihood ratio) unrelated males cannot be excluded (Taupin 2017). It is recommended to apply RM Y-STRs to further analyze evidence and known samples in case of a match for possible exclusion and reduce the number of individuals sharing the haplotype of the suspect. National reference Y-STR databases are often established according to a historical concept of ethnic affiliation in countries, such as the USA, Brazil, the UK, or China, with a strong population substructure. Haplotype diversity is considered a very applicable indicator for the evaluation of the effect of population substructure. The future expansion of national and global Y-STR haplotype databases of much more loci included in a typical haplotype could help to draw more informative conclusions regarding Y-STR analyses and interpretations. Needless to say, the guidelines should be developed by laboratories for the number of Y-STR loci applied in searching population databases. If there is limited or irrelevant population data for the assessment of a Y-STR match, a qualitative statement could be reported in case the issue of

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male relatedness is properly addressed in the final report. A conservative qualitative statement for a Y-STR match report between a sample recovered from a crime scene and a known suspect could be made in the following format: The Y-STR haplotype of the crime scene sample matches at all the examined loci to the Y-STR haplotype of the known individual (suspect). As a result, the suspect cannot be ruled out (excluded) as the donor of the sample recovered from the crime scene. Furthermore, all patrilineal-related male relatives in addition to an unknown number of unrelated males cannot be excluded as donors of the crime scene sample (Taupin 2017; Roewer et al. 2020).

Mixture Interpretation Via Y-STR Analysis In addition to all the troubles that should be addressed in Y-STR interpretations, particular complications occur during Y-STR mixture interpretation. Mixture interpretation is defined as strategies applied to determine and separate possible genotype combinations of the contributors. Peak height variation across the whole Y-STR haplotype, differential degradation of the contributors to a mixture, and locus
/allele-specific mutation rates should be considered in mixture haplotype assessment. Rarely, it may be two or more detected peaks on DYS19, DYS390, and DYS391. Awaiting the phenomenon of duplication or rare triplication of these Y-STR loci which may pass across generations, for the exact interpretation of mixture, a conservative strategy based on testing additional Y-STR loci and consideration of the entire Y-STR haplotype are required while determining the sample as a mixture (Butler 2014; SWGDAM 2017). Additionally, a combination of Y-STR results and even partial aSTR data may improve the rarity of a match. The combination of autosomal and Y-STR profiling results needs a consistent method for the addressed statistical question. Based on the different natures of underlying population structures, it is concluded that a simple combination of the genetic data acquired from the Y-STRs with data derived from the aSTRs as a single likelihood ratio is debatable and necessary to be applied in a conservative way (Amorim 2008). In contrast, separate reporting of the autosomal and Y-STR results seems to be a more appropriate method of avoiding misunderstanding at the court scene. A conservative way to estimate the joint probability, under the assumption of negligible dependencies, is achieved by multiplying the highest value of the group of autosomal match probabilities by the calculated matching probability for the Y-STRs. The MPS empowers forensic DNA laboratories to overcome the limitations related to the CE and current STR typing techniques. Several sequence variants with similar core sequence lengths of the Y-STR typing products were noticed using MPS which could not be determined via CE-based typing methods, thereby further empowering them for forensic identification. In addition, MPS also provides a powerful automation capability for integrating STR genotyping with the detection of some other associated forensic markers in the same reaction (Hammer and Redd 2005; Zhao et al. 2015).

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Once more, it is worth mentioning that the multiplicity of forensic casework and associated potential complexities result in the need for professional judgment and expertise in terms of the Y-STR interpretation. A fixed regulation cannot or should not comply with every situation. Universal guidelines only help to plan a standard scientific framework supported by the analytical data and prevent laboratory report audiences (i.e., judiciary systems and clients) from any misconceptions and conclusions (SWGDAM 2014).

Forensic Use of Y-Chromosome Testing Y-STR typing is used for the analysis of variations on the Y chromosome in nuclear DNA. The special feathers of the human Y chromosome and Y-STR specifications proposed them as master keys to handle several investigative problems and solve forensic genetic cold cases. The high degree of polymorphism declared in the Y-STRs analysis of human populations, their small-size PCR products (range: 100–400 bp), and their ability to multiplexing and automating are the most critical specifications turning them into being useful or sometimes markers of choice for forensic investigations.

Sexual Assault Investigation One of the notable responsibilities in the life of a forensic genetic analyst is the examination of different types of evidence from crime scenes, especially sexual assault intimate samples consisting of one or more male perpetrators. Sexual assault is a ubiquitous crime occurring in every culture, in all levels of society, and every country around the world disproportionally in uencing adolescents and young women. Instead of all other crime types with no statistically significant changes within 2017 to 2018, the frequency of rape or sexual assault in the USA doubled in this period (victimizations per 1,000 individuals with the age of 12 years or over). Based on the national US statistics, the general population consists of 49% males and 51% females. However, male offenders involve in 77% of violent incidents, and the frequency of violent incidents involving female offenders is only reported as 18%. This rate was 1.6 times higher than the frequency of males represented in the general population (49%) (Morgan et al. 2019). The conventional forensic serological testing of sexual assault samples consists of body uid identification by colorimetric or antigen-based protocols and/or microscopic examination for sperm. Most of the guidelines described labor-intensive and time-consuming methods yielding false-positive/false-negative results. Therefore, many laboratories looked for new robust and reliable methods to streamline the examination of sexual assault cases. These alternative methods could act as the gatekeepers for which the samples proceed to STR analysis. The SWGDAM proposed a direct-to-DNA Y-screening case approach as the next-generation body uid identification technique.

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Direct-to-DNA Y-screening is a direct-to-DNA work ow using quantitative PCR to quantify male DNA, applying the male DNA quantity rather than conventional serology to direct downstream DNA STR testing more sensitively and accurately in shorter turnaround times. The most critical aspect is to choose a kit that is sensitive enough to detect low amounts of male DNA in the presence of high levels of female DNA. The results of Y-screening may indicate that these types of samples might not be suitable for obtaining male aSTR profiles; however, they would be good candidates for Y-STR analysis (SWGDAM 2020). Quantitative real-time PCR kits provided the capabilities of the calculation of the quantities of both male and female DNA, along with degradation and inhibition indices generally accepted for quantification pre-PCR step. It assisted the proper selection of an amplification strategy and maximizing the potential for the determination of the male component(s) of a mixture. A comparison of detection limits between the Quantifiler Trio ® Human Male DNA Quantification Kit ® (Thermo Fisher Scientific, USA) and Yfiler Plus ® indicated the low likelihood of obtaining a Yfiler Plus profile appropriate for comparison objectives in case of no detectable male DNA with Quantifiler Trio. This may be a proper indicator for triaging samples to a further step using Yfiler Plus (SWGDAM 2014; Henry and Scandrett 2019). Biological evidence resulting from sexual assault demonstrates multiple challenges the most important of which is the presence of small quantities of male (suspect) DNA occurring with a relatively high quantity of the victim’s (mostly female) DNA. Due to the nature of the different samples, differential DNA extraction strategy may not be efficient in all cases. In addition, the general assessment of an aSTR mixture profile may unintentionally mask minor male alleles not immediately discernible from the alleles of the female complainant (i.e., the major contributor). This is more pronounced if the victim and suspect have been closely related. Therefore, the development of male-specific markers targeting the male fraction of a mixed DNA sample to prohibit female DNA competition for reagents during the preferential PCR amplification or masking minor male alleles may be useful for saving the trace levels of male DNA and deconvolving the mixture DNA profiles, including a minimum of two individuals and at least one male. This is also used in cases with high amounts of female DNA and limited yields of the male DNA, such as azoospermia/vasectomized perpetrators, or in case of alleged digital penetration in which the epithelial cells of the perpetrator should be analyzed instead of sperm cells (Aramayo et al. 2007; McDonald et al. 2015). In the vaginal cavity, there is a decrease in the number of sperm cells with time and mimic sperm cell negative samples in which no sperm cells can be detected by microscopic screening via Baecchi’s staining or immuno uorescence method of Sperm Hy-Liter. It is affected by factors, such as the personal hygiene of the victim, natural drainage, menstruation, damage to the sperm cell membranes, and extended interval postcoital sampling. According to the results of a study carried out by Albani et al. on the background yields of male DNA in the vaginal cavity, it was indicated that even up to 6 days following intercourse and up to 44 h after a semen negative offense, full Y-STR haplotype could be successfully obtained. Nevertheless, individuals differed in time frames which may also rely on the cycle stage of the uterine

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(Miller et al. 2011; Albani et al. 2018; Henry and Scandrett 2019). The analysis of recovered male DNA under scratching female fingernails following a violent attack showed that although the exogenous debris quickly decays over time, Y-STR typing could provide highly useful data to single out the male perpetrator(s) up to 5 h after scratching. The Y-STRs also provide male-specific genetic data in amylase positive (licking, biting, or kissing samples) determined on the victim’s body and screened via saliva presumptive tests (Taupin 2017; Iuvaro et al. 2018). The interpretation of mixture aSTR profiles is an overwhelming duty of the forensic genetic expert, and sometimes it is needed to work with mixture analysis software. Different from the conventional approaches, Y-STRs may be preferred to aSTRs to determine whether a familial relationship between the victim and perpetrator shares many autosomal alleles. The Y-STR analysis works by the removal of an overriding female profile in case of multiple perpetrator rapes and helps to simplify the interpretation of male-male cases. The application of Y-STR markers would be beneficial to the reliable exclusion of male suspects from involvement in a crime through non-matching Y-STR haplotypes. Furthermore, they are helpful for the identification of the paternal lineage to which a trace donor belongs in such a highly complicated forensic casework. Therefore, guidelines defining owcharts, under which forensic samples are subjected to Y-STR typing, should be established by every laboratory. While expecting a combination of male and female DNA (e.g., vaginal swabs) or a sperm-negative sample, it is suggested that these samples should be conserved for further Y-STR typing (Taupin 2017; Roewer et al. 2020). As a general owchart, for the generation of an eligible profile of the male DNA from either a single-source DNA or mixtures, aSTR typing should always be selected as the priority. In the case of the inconclusiveness of autosomal findings for the male component, it is necessary to analyze the sample via standard Y-STR typing. Whenever the Y-STR profile of the sample was informative for an individual matching the questioned haplotype, sufficient amounts of remaining extracts would be subjected to the supplementary panel of RM Y-STR marker set for the third step to exclude available close relatives. Finally, a quantitative assessment using either the DL method or count estimates should be provided to support matching results (Roewer et al. 2020).

Sex Determination Sex determination based on molecular techniques has been demonstrated to be preferable to any other present anthropological methods which are mostly based on the visual examination of skeletal morphology and required intact bone(s). There are molecular tools applied for sex determination among which sex determination tests on the basis of the amelogenin gene as a part of the majority of commercial PCR multiplex reaction kits have been extensively applied in forensic casework, DNA database, medical/archeological specimens, preimplantation, and prenatal diagnoses (Rezaei et al. 2017).

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The amelogenin locus contributing to the tooth enamel matrix formation is encoded by two single-copy genes (i.e., AMELX and AMELY) located on the short arm of the X (Xp22.1–22.3) and Y (Yp11.2) chromosomes, respectively. The most frequently utilized primer set of amelogenin delimits a 6-bp deletion in the intron 1 of AMELX, producing 106 bp amplicon for X and 112 bp amplicons for the Y chromosome. Although amelogenin is an effective technique for sex-typing of the biological samples in most cases, in rare cases of chromosomal microdeletions or variations in primer binding sites, relying only on the use of amelogenin as the sole sex marker will not provide foolproof results due to the dropout phenomenon (i.e., null alleles). There have been detected cases of amelogenin-negative males worldwide reported to be especially high in individuals of Indian origin. Among several wellknown genetic mechanisms underlying AMELY dropout, including the Y-chromosome microdeletions of various sizes covering AMELY locus, there are more mutations in the primer binding site of AMELY allele shown to a lesser extent. From the medical perspective, AMELY expression was detected at only 10% of the amount of AMELX, and the medical investigation of two cases of AMELY deletion showed normal teeth, suggesting minimal or no effect of deletion on enamel formation (Jobling et al. 2007). In sexual assault cases and missing individual investigations, it is also the first and not foremost finding that the questioned sample belongs to a male or a female source. The false-negative results of gender determination for contributors of a biological sample and mixed interpretation of undergarments of victims in rape may be misinterpreted as a negative case or mistakenly identified as a female (Butler 2014; Borovko et al. 2015). The closest Y-STR locus to the amelogenin-Y (i.e., DYS458 and rarely DYS456) may also be missed from the Y-STR haplotype due to the deletion events on the short arm of the Y chromosome at the pericentromeric bands. A total of 5 different deletion classes among 45 AMELYnull males from 12 populations were reported by Jobling et al. in 2007, among whom several other Y-STR loci presenting within or close to the amelogenin (i.e., DYS570, DYS576, DYS458, DYS481, DYS449, DYS627, and DYS391) may include in the deleted part (Chen et al. 2014; Cheng et al. 2019; Roewer et al. 2020; Dash et al. 2020a). Therefore, it can be helpful to determine the relative position of loci on the Y chromosome. There have been reports of the simultaneous AMELY and DYS458 null alleles due to the Yp11.2 deletion in various populations, with a much higher frequency in Sri Lankan population (8.3%) following Nepalese and Indian populations (6.5% and 0.23–3.2%, respectively). As experienced in the South Asian tsunami victim identification of 2004, higher rates of null amelogenin observed in the affected populations raised the need for additional sex determination techniques, such as the presence/absence of SRY (Jobling et al. 2007). One suggestion regarding Y-null amelogenin cases is the real-time PCR quantitation assay of Y chromosome-specific targets, as they are used to confirm the presence of male DNA in the questioned sample (Kumar et al. 2019). The other known solution for incorrect sexual identification is provided by testing additional markers, such as SRY- or Y-specific markers, for accurate gender

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determination. Therefore, they should be applied in multiplex PCR genotyping kits. Three Y-STRs (i.e., DYS391, DYS570, and DYS576) and one Y indel loci were chosen as alternative sex-determining markers by the new generations of autosomal multiplex kits, such as GlobalFiler™ (Thermo Fisher Scientific, USA), PowerPlex ® Fusion 6C (Promega Corporation, USA), and Investigator 24plex GO! Kit (QIAGEN, Germany). As a result, in addition to the amelogenin loci, one Y-STR (i.e., DYS391) and one Y-indel locus (i.e., rs2032678) were recently incorporated in Amp STR Globalfiler™ (Thermo Fisher Scientific, USA) to reduce the risk of misidentification of male samples. Y-indel marker (originally known as M175) is located on Yq11.221 with two different conditions. A repeat of five nucleotides (TTCTC) deleted or mostly inserted in this position (referred to one or two alleles, respectively) illustrates male samples. The application of these additional markers assists male gender confirmation in cases of null amelogenin (Dash et al. 2020a). The results of fetal gender determination during the third trimester of pregnancy using the Y-STR analysis of maternal plasma indicated the feasible application of Y-STR typing as an accurate gender determining test in order to identify the fetus gender (Aal-Hamdan et al. 2015).

Paternity Testing The Y-STR haplotyping is also relevant to the paternity dispute investigations of male offspring, several types of patrilineal kinship allegations, and familial searching. In paternity testing, Y-STR haplotyping is especially appropriate in deficiency cases in which the putative father of a male child is not at hand for aSTR typing. In different scenarios of ambiguous paternity, in the case of a female with multiple male partners, it is desired to carry out the noninvasively prenatal paternity testing (as recently discussed for gender determination) through analyzing the circulating cell-free fetal DNA, retrieved from maternal plasma. The Y-STR haplotype investigation of DNA extracted from maternal plasma can effectively be used as an alternative for exclusion purposes. It can only be applied to mothers carrying a male fetus. The main limitation of this application is that the meaning of MP for two similar Y-haplotypes is observed for the alleged father and fetus. It could only be concluded that they belong to the same paternal lineage, and this alternative strategy should not be used in a population reported for a high rate of endogamy (Barra et al. 2015).

Disaster Victim Identification The Y-STR markers can be beneficial in disaster victim identification. Routinely, the results of DNA typing from remained bodies should be compared to personal objects (e.g., hair brass, toothbrushes, and razor blades), biological father, and reference samples from the alleged male lineages. In these situations, Y-STR typing can increase the number of alternative reference samples, simplify the sorting of the

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tissue samples that remained in mass disasters, and help the forensic expert to select possible distant male relatives as informative subsidiary reference samples (Fig. 1). However, as previously discussed, Y-STR kits are not appropriate enough for the identification of recovered male victims due to the fact that all the male relatives possess the identical YSTR haplotype (Kayser 2017; Ambers et al. 2018).

Historical Investigations Although the main benefits of Y-STR typing in forensic casework are shown in case of extremely low amounts of male DNA in mixture (male(s)/female) samples recovered from crime scenes, Y-chromosome testing is also applicable to investigate various anthropological issues, such as human identification, familial relationships, historical events, and male migration and mating patterns in different geographical regions. For this reason, researchers should apply many different Y-chromosome markers, including Y-STRs, Y-SNPs, and Y-indels, to pursue the male lineages living in or migrating across their geographical region. Nevertheless, in order to enhance haplotype diversity and lineage resolution among populations, several Y-STR markers appear more appropriate than others. By far, a global analysis of Y-chromosomal haplotype diversity for 23 STR loci was published in 2014 as the most extensive collection of Y-chromosomal STR haplotypes worldwide for one of the biggest studies on the Y-STR data. A total of 19,630 unrelated male Y-STR haplotypes obtained from 129 populations in 51 countries worldwide were collected from 84 participating laboratories. According to the results, DYS481 and DYS570 markers demonstrated the largest numbers of different alleles, out of the six Y-STR markers distinguishing PPY23 from Yfiler. The DYS643 marker was observed to be more variable among Africans but less variable in Native Americans of Latin America than that of other continental groups. Moreover, the highest number of null alleles was shown for the DYS448 locus. A large deletion was detected at Yp11.2 covering the AMELY region with DYS570, DYS576, DYS458, and DYS481 in nine samples (with Asian ancestry, from Singapore, Tamils, and British Asians mostly originating from or living in India (Purps et al. 2014). Yuxiang Zhou et al. investigated the extent of genetic differentiation between populations (i.e., genetic distance) in over 20,000 haplotypes of Yfiler Plus set gathered from 41 global populations in 2020. They defined the mean of allele frequency difference (mAFD) as a reliable value for the estimation of the variance of genetic markers in populations and evaluation of population differentiation. The results of their study indicated that DYS392 and YGATAH4, among the studied Y-STRs, were reported with the largest (0.3802) and smallest (0.1845) mAFD values, respectively (Barra et al. 2015). In another study carried out by Grugni et al., 10 Y-STR loci and 88 Y-chromosome biallelic markers were investigated in 15 ethnic groups of the Iranian population. The results showed that 65 different Y-chromosome lineages belong to 15 main haplogroups in which J (31.4%) and R (29.1%) are frequently observed in the

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northwest, and G (11.8%) and E (9.2%) are most frequently observed due to different migratory events in the history. A clear African component (i.e., haplogroup E-M2) is observed in Hormozgan, Iran, indicating the presence of sub-Saharan Africans. Another Y-STR haplotype analysis of the Eastern Iranian population in comparison with haplotypes from the neighboring countries (Afghanistan and Pakistan) shows small genetic discrimination between neighbor populations living in this close region. Such historical-based population genetic studies applied reliable Y-chromosomal variation portrait to shed light on the genetic structures and expansion patterns to help reconstruct ancient migration routes (Grugni et al. 2012; Tabrizi et al. 2015).

Y-STR Haplotype Databases The global utilization of Y-STR typing in forensic genetic laboratories raised the need for national/international Y-STR databases. As previously discussed, the quantitative evaluation of the informativeness of a match between two haplotypes requires the assessment of the haplotype rarity based on the frequency estimates of the Y-STR haplotype in a reliable, robust, and relevant database. Standing on the first step in 1997, the SWGDAM and European Scientific Committee announced a recommended minimal haplotype as a cornerstone for the establishment of large global databases. After several years, Y Chromosome Haplotype Reference Database (YHRD; available at http://www.yhrd.org/) and US Y-STR Database (available at http://www.usystrdatabase.org) are founded as the two most important Y-STRbased databases. Lutz Roewer et al. created the largest and most extensively utilized Y-STR database in 1999, currently announcing the 63rd release. Over 321,000 minimal haplotypes of anonymous male individuals have been imported into YHRD 3.0 since 2000 up to November 2020. Complementary to the Y-STR haplotypes, YHRD has been enlarged with Y-SNP data and at present includes 25,000 Y-SNP data. The US Y-STR Database has been founded under the management of the US National Center for Forensic Science and financial supports of the National Institute of Justice since 2007. The US Y-STR Database consists of 35,658 Y-STR haplotypes generated by 30 different forensic, academic, and commercial laboratories since 2018 (release 4.2). It has been permanently transferred to YHRD for the continuation of usage and finally was decommissioned after June 30, 2019 (SWGDAM 2014). In addition to forensic Y-STR databases, several Y-STR-based genealogy databases were created to aid forensic investigators to link between a particular Y-STR haplotype and family surnames in most human societies, where the male surnames are paternally co-inherited. A surname is a part of a personal name demonstrating the family, tribe, or community the individual belongs to. Despite the observation of strong co-ancestry for several rare surnames, common surnames showed low or no Y-chromosome correlations restricting the predictive value of Y-STR haplotyping for common surnames. King et al. in their study examined unrelated males accounting for 150 different British surnames. In this study, the prediction of a correct surname was observed in only 19% of the cases; nevertheless, the sensitivity of

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prediction increased to 34% for less common surnames (Kayser 2017; Claerhout et al. 2020). Another population genetic analysis conducted on the Y-STR haplotypes of the five most common surnames in South Korea indicated limited genetic variations among five surnames due to the genetic heterogeneity of each surname. In addition, various kinds of non-surname-related haplotypes were distributed among five surnames. Therefore, the use of surname-based prediction of Y-STR haplotype data for narrowing down the suspect searching domain has not been adequately effective (Kim et al. 2009). For simplicity, to increase the advantages of the Y-STR database, forensic genetic society should follow the common motivating quote of the bigger the better. Despite the importance of increasing the size of reference databases that are used to estimate the Y-STR match probabilities, it is important to note some population highlights. As the diversity of Y-STR haplotypes in small, isolated, and migrant populations decreases, estimated discriminatory power reduces. Simultaneously, the presence of subpopulations in the database with distinct geographical distribution patterns, different from those of the full population profiles, may result in biased frequency estimations for the Y-STR haplotypes. It was more obvious while the assessment of partial Y-STR haplotypes as evidence in forensic casework (Amorim and Budowle 2016). One solution for the effective application of Y-STR typing and Y-based database construction in populations with the aforementioned specifications (e.g., the Finnish population) might be the inclusion of more Y-STR loci based on national standard protocols (Palo et al. 2008). Another issue may arise when a Y-STR database containing different Y-STR sets of loci applied for Y-haplotype frequency discrimination is a paradoxical phenomenon. For clarity, if highly discriminating Yfiler Plus data were searched against even in a large database with 9000 minimal haplotypes and 1000 records of 27 loci haplotypes, a full match means 10 times less discrimination estimated (1=1000) for a more discriminative 27 loci set than a minimal set (1=10000) in terms of the frequency of matching haplotypes per number of compared profiles. The simple explanation is fewer database records of highly discriminating Y-haplotypes. Therefore, in addition to great efforts by the forensic genetics community that are currently underway to extend sample sizes for populations through including extended Y-STR haplotype records, it is necessary to provide guidelines for the number of Y-STR loci used for searching population databases. The application of reduced locus set criteria is of great importance when a partial Y-STR haplotype resulted from the evidence of a crime scene (SWGDAM 2014).

Familial Searching The Y-STR data inherited in a patrilineal way result in the appropriateness of Y-STR haplotyping for familial searching in forensic cases when a newly submitted autosomal DNA profile does not have a full match. It helps to discriminate among male

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offender(s) via searching for his male relatives which is called forensic familial searching. When multiple Y-STR haplotypes are consistent with the Y-STR haplotype of a lineage, Y-STR profiling could help in narrowing the extent of searching and accelerating the investigation. One critical point in successful familial searching is the appropriate selection of different Y-STR panels in developing and filtering the search of Y-STR haplotype databases. A filtered panel of slow/moderate mutation rate Y-STRs is a selective searching strategy to boost the chance of finding distant relatives; however, RM Y-STRs are more appropriate to discriminate among close relatives (Claerhout et al. 2019). Apart from the great extra advantages of familial searching in sexual offense investigations to solve cold cases, some ethical, social, and legal issues are raised and needed to be legislated before familial searching and even before establishing a database plan. In this regard, the extra Y-STR testing of the forensic samples stored in the national DNA database and then expanded voluntary DNA mass screenings require much more budget. As the nature of Y-chromosome data sharing among paternal relatives, they belong not only to the suspect but also his innocent relatives (Fig. 1). On the other hand, accumulating much more genetic data essentially means further requirements for strict security protocols. In countries with the regular inclusion of Y-haplotypes in their national DNA databases (e.g., Austria), with serious crimes leading to long imprisonment, a minimum of 27 Y-STRs are incorporated in the commercially available Yfiler ® Plus kit ® which could be extended to 46 Y-STR markers used to provide a lower number of false-negative surname matches and prevent false-positive errors (Claerhout et al. 2020).

Haplogroup Prediction The Y-haplogroups are determined by the pattern of SNPs which can also be analyzed by direct testing of SNPs. The outcomes of haplogroup determination are beneficial for the assessment of genealogical relationships between two or more males. However, the process of haplogroup determination can sometimes be expensive and long. Therefore, there is considerable interest in the prediction of the Y-chromosome haplogroup, as a group or family of Y-chromosomes related by descent from a set of Y-STR markers. The Y-haplogroup predictors are considered tools extensively applied to have access to haplogroups based on Y-STR values. Needless to say, the prediction of haplogroups using predictors requires slower mutating markers and knowledge of the allele frequencies for each haplogroup (Athey 2005). Several approaches implemented in Y-haplogroup predictors differ in strengths and limitations. The prediction algorithm applied by Family Tree DNA (FTDNA) in collaboration with the University of Arizona (UAZ), USA, is on the basis of the genetic distance of the Y-haplotype in question to other Y-haplotypes with a previously determined haplogroup. Although the FTDNA/UAZ approach claimed to be successful for 80% of customers, the main limitation is indicated in case the absence of haplogroup-confirmed haplotype is observed in the database within a threshold distance; accordingly, there is no estimate of a haplogroup.

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Another approach adopted on a website since 2004 is based on the allele frequency estimations for each haplogroup and the score in which a test haplotype fits the pattern of alleles in each haplogroup. The investigator could enter any numbers of the FTDNA set (out of including 37 markers), and the program estimates goodness of fit scores for 10 haplogroups (i.e., E3a, E3b, G, I1a, I1b, I1c, J2, N3, R1a, and R1b). Whit Athey’s predictor was developed by Whit Athey as the first publicly available haplogroup prediction tool. Nevgen Y-DNA haplogroup predictor applies the Bayesian allele frequency approach to estimate the intended haplogroup of a Y-STR haplotype, utilized by Athey’s haplogroup predictor. Predictions based on this tool for the abovementioned markers are usually elaborated by two values, including probability (initial 100% probability of an individual belonging to specific haplogroup (as a numerator) divided into all possible Y-haplogroups) and fitness score which should be assumed different for various haplogroups (Szargut et al. 2019). Diepenbroek et al. investigated 23 markers of the human Y chromosome in remains exhumed from historical Nazi-occupied regions located in Białystok (i.e., Eastern Poland). The Y-DNA haplogroup prediction by Nevgen revealed that over 80% of the studied samples were suggested with European origin and typical haplogroup for the Polish population other than one ethnic minority (Diepenbroek et al. 2019). Bouakaze et al. developed PredYMaLe as a versatile machine learning program for the prediction of Y-haplogroups based on 32 Y-STR multiplex which is mutationally balanced in 2020 (Bouakaze et al. 2020). Emmerova et al. compared several Y-chromosomal haplogroup predictors. The final ranking results of software packages based on the number of non-concordant estimates (the lower the better in only 19 STRs) is presented as Whit Athey’s HAPEST, Nevgen, YPredictor (by Vadim Urasin), Jim Cullen’s World Haplogroup, and Haplo-I Subclade Predictor. In the aforementioned study, it was concluded that minimally a Y-haplotype should consist of more than 12 Y-STRs; otherwise, it should not be applied for the accurate prediction of the haplogroup. It is suggested that the integration of further predictors reduces the risk of false haplogroup assignment. For more details, Y-haplogroup predictors are available at the following sites: Whit Athey: Haplogroup predictor-HAPEST available (www.hprg.com/hapest5) Jim Cullen: World Haplogroup and Haplo-‘I’ Subclade Predictor (members.bex.net/ jtcullen515/haplotest. htm) Nevgen Y-DNA haplogroup predictor (www.nevgen.org) Vadim Urasin: YPredictor (predictor.yDNA.ru) (www.y-str.org/2013/06/yhaplogroup-predictor.hTMl) (Emmerova et al. 2017)

Conclusion Forensic genetic experts benefited from the Y-chromosome analysis in different forensic scenarios of sexual assault investigation and disaster victim identification to paternity dispute analysis and historical investigations. Apart from the advantages

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of Y-chromosome marker analysis, several technical limitations shall be weighed and addressed in standard interpretations, reporting, and application guidelines.

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Vouropoulou M, Kovacevic L, Marjanovic D, Lindner I, Mansour I, Al-Azem M, Andari AE, Marino M, Furfuro S, Locarno L, Martin P, Luque GM, Alonso A, Miranda LS, Moreira H, Mizuno N, Iwashima Y, Neto RS, Nogueira TL, Silva R, Nastainczyk-Wulf M, Edelmann J, Kohl M, Nie S, Wang X, Cheng B, Nunez C, Pancorbo MM, Olofsson JK, Morling N, Onofri V, Tagliabracci A, Pamjav H, Volgyi A, Barany G, Pawlowski R, Maciejewska A, Pelotti S, Pepinski W, Abreu-Glowacka M, Phillips C, Cardenas J, Rey-Gonzalez D, Salas A, Brisighelli F, Capelli C, Toscanini U, Piccinini A, Piglionica M, Baldassarra SL, Ploski R, Konarzewska M, Jastrzebska E, Robino C, Sajantila A, Palo JU, Guevara E, Salvador J, Ungria MC, Rodriguez JJ, Schmidt U, Schlauderer N, Saukko P, Schneider PM, Sirker M, Shin KJ, Oh YN, Skitsa I, Ampati A, Smith TG, Calvit LS, Stenzl V, Capal T, Tillmar A, Nilsson H, Turrina S, De Leo D, Verzeletti A, Cortellini V, Wetton JH, Gwynne GM, Jobling MA, Whittle MR, Sumita DR, Wolanska-Nowak P, Yong RY, Krawczak M, Nothnagel M, Roewer L (2014) A global analysis of Y-chromosomal haplotype diversity for 23 STR loci. Forensic Sci Int Genet 12:12–23. https://doi.org/10.1016/j.fsigen.2014.04.008 Qian X, Hou J, Wang Z, Ye Y, Lang M, Gao T, Liu J, Hou Y (2017) Next generation sequencing plus (NGS+) with Y-chromosomal markers for forensic pedigree searches. Sci Rep 7(1):11324. https://doi.org/10.1038/s41598-017-11955-x Ralf A, Lubach D, Kousouri N, Winkler C, Schulz I, Roewer L, Purps J, Lessig R, Krajewski P, Ploski R, Dobosz T, Henke L, Henke J, Larmuseau MHD, Kayser M (2020) Identification and characterization of novel rapidly mutating Y-chromosomal short tandem repeat markers. Hum Mutat 41(9):1680–1696. https://doi.org/10.1002/humu.24068 Rezaei E, Beiraghi-Toosi A, Ahmadabadi A, Tavousi SH, Alipour Tabrizi A, Fotuhi K, Jabbari Nooghabi M, Manafi A, Ahmadi Moghadam S (2017) Can skin allograft occasionally act as a permanent coverage in deep burns? A pilot study. World J Plast Surg 6(1):94–99 Roewer L, Amemann J, Spurr NK, Grzeschik KH, Epplen JT (1992) Simple repeat sequences on the human Y chromosome are equally polymorphic as their autosomal counterparts. Hum Genet 89(4):389–394. https://doi.org/10.1007/BF00194309 Roewer L, Andersen MM, Ballantyne J, Butler JM, Caliebe A, Corach D, D’Amato ME, Gusmao L, Hou Y, de Knijff P, Parson W, Prinz M, Schneider PM, Taylor D, Vennemann M, Willuweit S (2020) DNA commission of the International Society of Forensic Genetics (ISFG): recommendations on the interpretation of Y-STR results in forensic analysis. Forensic Sci Int Genet 48: 102308. https://doi.org/10.1016/j.fsigen.2020.102308 SWGDAM (2014) SWGDAM Interpretation Guidelines for Y-Chromosome STR Typing by Forensic DNA Laboratories. Available at https://www.swgdam.org/publications SWGDAM (2017) SWGDAM Interpretation Guidelines for Autosomal STR typing by forensic DNA testing laboratories. Available at https://www.swgdam.org/publications SWGDAM (2020) Scientific Working Group on DNA analysis methods next generation body uid identification working group report on Y-screening of Sexual Assault Evidence Kits (SAEKs). Available at https://www.swgdam.org/publications Szargut M, Diepenbroek M, Zielinska G, Cytacka S, Arciszewska J, Jalowinska K, Piatek J, Ossowski A (2019) Is MPS always the answer? Use of two PCR-based methods for Y-chromosomal haplotyping in highly and moderately degraded bone material. Forensic Sci Int Genet 42:181–189. https://doi.org/10.1016/j.fsigen.2019.07.016 Tabrizi AA, Hedjazi A, Kerachian MA, Honarvar Z, Dadgarmoghaddam M, Raoofian R (2015) Genetic profile of 17 Y-chromosome STR haplotypes in East of Iran. Forensic Sci Int Genet 14: e6–e7. https://doi.org/10.1016/j.fsigen.2014.10.010 Taupin JM (2017) Introduction to forensic DNA evidence for criminal justice professionals. Taylor & Francis. ISBN: 9781439899106 Thompson JM, Ewing MM, Frank WE, Pogemiller JJ, Nolde CA, Koehler DJ, Shaffer AM, Rabbach DR, Fulmer PM, Sprecher CJ, Storts DR (2013) Developmental validation of the PowerPlex ® Y23 system: a single multiplex Y-STR analysis system for casework and database samples. Forensic Sci Int Genet 7(2):240–250. https://doi.org/10.1016/j.fsigen.2012.10.013

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9

Haplodiploid Markers and Their Forensic Relevance Antonio Amorim and Nadia Pinto

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Theoretical and Statistical Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Single Locus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Haplotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Association at Gametogenesis Level: Linkage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Association at the Population Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Individual Profiling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic Relatedness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Con ict of Interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

The interpretation of a DNA profile requires the knowledge of the mode of transmission of the involved genetic markers. Living beings show a remarkable variety of transmission fashions, and restricting our analysis to eukaryotic sexual organisms, we can formalize the main ones into three categories: uniparental (normally haploid, as happens with plastids or mitochondria), A. Amorim (*) Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal Institute for Research and Innovation in Health (i3S), University of Porto, Porto, Portugal Faculty of Sciences of the University of Porto (FCUP), Porto, Portugal e-mail: [email protected] N. Pinto Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal Institute for Research and Innovation in Health (i3S), University of Porto, Porto, Portugal Centre of Mathematics of the University of Porto, Porto, Portugal e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 H. R. Dash et al. (eds.), Handbook of DNA Profiling, https://doi.org/10.1007/978-981-16-4318-7_7

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biparental homogametic (usually diploid), and heterogametic or hybrid (uni- or biparental according to parental sex, haplodiploid). The haplodiploid mode of transmission, albeit widespread, has been overlooked, and the theoretical developments required for its analysis lagged. Worse still, haplodiploid markers are many times misused and analyzed under classical diploidy assumption. Here, we provide a synthesis of the theoretical framework required for the analysis of this type of markers, enabling a fast guide for their use in DNA profiling at individual, familial, and population levels, with a special emphasis on forensic applications. Keywords

Haplodiploidy · Haplodiploid markers · Genetic transmission · X chromosome

Introduction The production of a DNA profile from a biological sample simply requires the knowledge of the technique(s) used and the appropriate protocols. The interpretation of the results is however more demanding as it implies the theory behind the assumed mode of transmission of the involved genetic markers. Realizing that current life on the planet relies upon nucleic acids (and sensu stricto on DNA) for storage of genetic information, the variety of transmission modes observed is remarkable. Even restricting our analysis to eukaryotic sexual organisms (Ashman et al. 2014), we can formalize the main ones (and widespread in animals) into three categories: uniparental (normally haploid, as for plastids or mitochondria), biparental homogametic (nuclear, usually diploid), and hybrid or heterogametic (uni- or biparental according to parental sex, from now on referred to as haplodiploid). It is noteworthy also that, in all eukaryotic free-living organisms, at least two of these modes of transmission coexist (Yahalomi et al. 2020), corresponding to distinct genomic sections. The haplodiploid mode of transmission occurs in many forms and depths. In most mammals, for instance, with very few exceptions (Matveevsky et al. 2017), sex is determined by a karyotypic determination system, in which males XY are haploid for one chromosome, while females are diploid, XX. More radically, haplodiploidy determines the sex in all members of some entire insect orders (Hymenoptera, Thysanoptera): males develop from unfertilized eggs and are haploid, while females are diploid (see the consequences for genome assembly in [Yahav and Privman 2019]). A serious consequence of this gender genetic asymmetry is that the haplodiploid sex is affected by deleterious alleles at the chromosome(s) involved, even if they are recessive in the diploid sex, as the case of hemophilia in humans (Franchini and Mannucci 2012). Albeit widespread, haplodiploidy has been overlooked, and the theoretical developments required for its analyses lagged. Worse still, haplodiploid markers are many times misused and analyzed under classical diploidy framework. This is unfortunately the case in forensic applications (Ferragut et al. 2019), where, instead of being a problem, haplodiploidy can be a solution to various common casework situations (Pinto et al. 2011).

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With this motivation we provide here a synthesis of the theoretical framework required for the analysis of this type of markers, enabling a fast guide to their use in DNA profiling at individual, familial, and population levels, with a special emphasis on forensic applications. For clarity sake, we will begin a single, per marker, approach and then proceed to the simultaneous analyses, enabling to a global comprehension of the complexities brought by asymmetrical recombination and gametic association (linkage disequilibrium, LD). The case of mammalian XX/XY type of haplodiploidy will be specially used, not only for anthropocentric reasons and applications, but also because of the existence in both X and Y chromosomes of genomic sections (two in humans, one in the rest of known mammals), with a particular transmission behavior, which is neither strictly biparental nor haplodiploid, the so-called pseudoautosomal regions (PARs) (Otto et al. 2011). As introduced above, the haplodiploid mode of transmission is associated with sexual reproduction and, therefore, with meiosis. So we will brie y and schematically present an overview of the process. Sexually reproducing eukaryotic organisms do not transmit their genetic blueprint in bulk to offspring. Instead, through a special cellular division (meiosis), they halve (most of) their (diploid) genome, producing transitory gametes, which, if successfully fused into an egg, create a new being, with reconstructed double amount of information. This is not true not only for cytoplasmic genomes but also, as in many animals with genetic determination of sex, for some information carried by nuclear chromosomes. Figure 1 illustrates this process using the mammalian type as example and makes visible the very distinct pattern of recombination in different parts of the nuclear genome: while in female meiosis all chromosomes behave homogeneously, in males crossing-over is absent from the sex chromosomes with the exception of PAR(s). This heterogeneous distribution of recombination has a major impact on the distinctive transmission properties of haplodiploid markers that will be explored below.

Theoretical and Statistical Analyses Single Locus At the familial level, the haplodiploid formal model for a single locus is indeed very simple. Using again Fig. 1, we can easily design a predictive matrix in the form of a Mendelian chessboard, in which the extension to population proportions assuming Hardy-Weinberg equilibrium (HWE) is shown, associating the frequency of the observed alleles in the gene pool (p is the frequency of A1, and 1-p the frequency of A2, which can be either an allele or the result of pooling all non-A1 alleles) – see Fig. 2. Note that, besides HWE assumptions for homogametic systems (infinite population size, random mating, and absence of selection, migration, and mutation), we must also assume equal allele frequencies in males and females. There is no need to

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♀ A

A

♂ X

A

X

A

X Y

PROGENITORS

} PAR A

X

A

} PAR A

X

X

A Y

Gametes

A

X

A

X

A

X

A

Y

OFFSPRING

♀

♂

Fig. 1 Chromosomal basis of haplodiploidy. Schematic representation of the auto- and heterosomal structure and mode of transmission and recombination, in the mammalian type of sex determination. Coloring of autosomes (A) and heterosomes (X and Y) identifies the parental origin of the chromosomes and allows to trace the occurrence of recombination. Note that recombination can occur between X and Y but only at the pseudoautosomal region (PAR) and that haplodiploid markers do not recombine in male meiosis

X

A1 p

X

A2 (1-p)

X

X

Y

A1 p

A2 (1-p)

-

♀

♀

A1A1 p2

♀

A1A2 p(1-p)

♂

-

A1A2 p(1-p)

A1 p

♀

♂

A2A2 (1-p)2

-

A2 (1-p)

Fig. 2 Predictive matrix of genotypic distribution, considering haplodiploid transmission and Hardy-Weinberg equilibrium. Allele frequencies: A1 – p; A2 – (1-p)

assume equal absolute numbers of males and females nor equal proportions of Xand Y-bearing gametes in male gametogenesis, and real populations do not observe these proportions.

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Conformity of observed genotype distribution to HWE expectations is not as simple as in homogametic systems. In fact, the females’ sample can indeed be tested in the usual way (caveat: using allele frequencies estimated in this sex!), but we have an extra verification to make: are the allele frequencies in the two sexes the same? To test this, various statistical solutions are possible, but the simplest is to treat male and female gene pools as two populations and compare the two observed allele distributions (a contingency test 2
n alleles table). The computation of a Kolmogorov-Smirnov test could be a standard approach for this purpose. At this point we can already extract some conclusions relevant to practical DNA profiling and forensic application of a haplodiploid marker: 1. No individual male can show more than one allele (no recessiveness), and so, in males, genotype and allele frequencies are the same; observed discrepancies to this rule must be investigated (they can be due to previously undetected duplications, which would lead also to triple allelic patterns in females) or to primer unspecificity (Gusmão et al. 2009; Diegoli et al. 2014). 2. Any significant deviation from HWE compromises the use of the marker at least on the population under scrutiny, as standard statistical approaches assume such condition for the calculation of expected frequencies. Possible causes deserve a dedicated research as their origins are multiple (and some can be inferred in silico): technical problems, silent alleles (heterozygote excess), selection (genotypes’ deficiency – particularly detectable in males if an allele is severely deleterious), migration (sex biased or not), nonrandom mating, drift (caused by small effective male and/or female population sizes), or even mutation. This remark holds against the careless and misplaced use of pseudo-conservative approaches, artificially lowering the p-values of significance thresholds (Ye et al. 2020).

Haplotype Due to space limitations, we will analyze the simplest case (a pair of biallelic markers) with greater detail, the generalization being straightforward. Haplotype is here therefore defined as the pairwise combination of two alleles from different loci. In Table 1 we show, in a similar way to the one presented for a single locus in Fig. 2, haplotype distributions and intergenerational evolution. Needless to say, this analysis can only proceed if both loci involved have shown to be in HWE. The simultaneous consideration of two makers introduces a new degree of complexity: the association between them. This association has also two components: lack of randomness at gamete formation (linkage) and at the population level. In any case, the haplotype frequency results being different from the product of the frequencies of the non-alleles involved.

Male gametic output

Y

A2B2 / h4

A2B1 h3

A1B2 h2

A1B1 h1

Gametic output

Female

A1B1 A2B1 2h1h3

A1B1 A2B2 2h1h4

A1B1 ½ ½ ½ ½ ½ A1B1 A1B2 A1B1 A2B1 (1r) A1B1 A1B1 A1B1 A1B1 A1B1 A1B1 A1B1 A1B1 A1B1 A1B2 A1B1 A2B1 A1B1 h13 h12h2 h12h2 h12h3 h12h3 (1r) h12h4 A1B1 A1B1 A1B2 A1B1 A1B2 A1B1 A1B2 A1B2 A1B2 A1B2 A2B1 A1B2 h12h2 h1h22 h1h22 h1h2h3 h1h2h3 (1r) h1h2h4 A1B1 A1B1 A1B2 A1B1 A2B1 A1B1 A2B1 A2B1 A2B1 A2B1 A2B1 A2B1 h12h3 h1h2 h1h2 h1h32 h1h32 (1r) h3 h3 h1h3h4 A1B1 A1B1 A1B2 A1B1 A2B1 A1B1 A2B2 A2B2 A2B2 A2B2 A2B2 A2B2 h12h4 h1h2 h1h2 h1h3h4 h1h3h4 (1r) h4 h4 h1h42 A1B1 A1B1 A1B2 A1B1 A2B1 A1B1 Y Y Y Y Y Y h12 h1h2 h1h2 h1h3 h1h3 (1r) h1h4

A1B1 A1B1 A1B1 A1B2 h12 2h1h2 ½ (1r) A2B2 A1B1 A2B2 (1r) h12h4 A1B2 A2B2 (1r) h1h2h4 A2B1 A2B2 (1r) h1h3h4 A2B2 A2B2 (1r) h1h42 A2B2 Y (1r) h1h4 ½ (r) A1B2 A1B1 A1B2 (r) h12h4 A1B2 A1B2 (r) h1h2h4 A1B2 A2B1 (r) h1h3h4 A1B2 A2B2 (r) h1h42 A1B2 Y (r) h1h4 ½ (r) A2B1 A1B1 A2B1 (r) h12h4 A1B2 A2B1 (r) h1h2h4 A2B1 A2B1 (r) h1h3h4 A2B1 A2B2 (r) h1h42 A2B1 Y (r) h1h4 A1B2 ½ (1r) A1B2 A1B1 A1B1 A1B2 A1B2 h1h22 (1-r) h1h2h3 A1B2 A1B2 A1B2 A1B2 h23 (1r) h22h3 A1B2 A1B2 A2B1 A2B1 h22h3 (1r) h2h32 A1B2 A1B2 A2B2 A2B2 h22h4 (1r) h2h3h4 A1B2 A1B2 Y Y h22 (1r) h2h3

A1B2 A1B2 A1B2 A2B1 h22 2h2h3 ½ (1r) A2B1 A1B1 A2B1 (1-r) h1h2h3 A2B1 A1B2 (1r) h22h3 A2B1 A2B1 (1r) h2h32 A1B2 A2B2 (1r) h2h3h4 A1B1 Y (1r) h1h3 ½ (r) A1B1 A1B1 A1B1 (r) h1h2h3 A1B1 A1B2 (r) h22h3 A1B1 A2B1 (r) h2h32 A1B1 A2B2 (r) h2h3h4 A1B1 Y (r) h1h3 ½ (r) A2B2 A1B1 A2B2 (r) h1h2h3 A1B2 A2B2 (r) h22h3 A2B1 A2B2 (r) h2h32 A2B2 A2B2 (r) h2h3h4 A1B1 Y (r) h1h3

A2B1 ½ A2B1

A2B1 A2B1 A2B1 A2B2 h32 2h3h4 ½ A2B2

A2B2

A2B2 A2B2 h42

A1B2 A2B2 Y Y h2h4 h2h4

A1B2 A2B2 A2B2 A2B2 h2h42 h2h42

A2B1 A2B1 Y Y h32 h3h4

A2B1 A2B1 A2B2 A2B2 h32h4 h3h42

A2B2 Y h3h4

A2B2 A2B2 h3h42

A2B1 A2B2 h32h4

A2B2 Y h42

A1B2 A2B2 h43

A2B1 A2B2 h3h42

A1B2 A1B2 A1B2 A1B2 A2B1 A2B1 A2B2 A2B2 h2h32 h2h3h4 h2h3h4 h2h43 A1B2 A2B1 A2B1 A2B1 A2B1 A2B2 A2B1 A2B1 h2h3h4 h2h3h4 h33 h32h4

A1B2 A1B2 A1B2 A2B2 h22h4 h22h4

A1B1 A1B1 A1B1 A1B1 A1B1 A1B1 A1B2 A2B2 A2B1 A2B1 A2B2 A2B2 h1h2h4 h1h2h4 h1h32 h1h3h4 h1h3h4 h1h42

½ ½ A1B2 A2B2

A1B2 A2B2 2h2h4

Table 1 Simultaneous analysis of two loci: haplotype distributions and intergenerational evolution. Each locus is assumed in HWE. Allele frequencies: locus A – p, q (¼1-p); locus B – u, v (¼1-p); haplotype frequencies: A1B1 (h1; in HWE ¼ p
u), A1B2 (h2; in HWE ¼ p
v), A2B1 (h3; in HWE ¼ q
u), and A2B2 (h4; in HWE ¼ q
v), considered to be equal in males and females; r – recombination rate

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Association at Gametogenesis Level: Linkage By definition, haplodiploid loci are always linked, as they sit at a chromosome that does not recombine in one sex, but even in females, recombination rate (r) varies (0  r  0.5) with the physical distance between loci. Several mapping functions were designed to convert genetic distances into recombination rates, such as in (Haldane 1919) or (Kosambi 1944) functions for humans. This means that the general prediction for the production of gametes in doubly heterozygous females A1B1/A2B2 is ½(1r) A1B1 + ½(1r) A2B2 + ½(r) A1B2 + ½(r) A2B1, instead of the expectation under independence, ¼ for each type. Note also that double heterozygotes can harbor another, genotypically indistinguishable, haplotype phase, A1B2/A2B1, and then gamete distribution will be, symmetrically: §ðrÞ A1B1 þ §ðrÞ A2B2 þ §ð1  rÞ A1B2 þ §ð1  rÞ A2B1: The consequences for general profiling, forensics, and other applications, such as genetic counseling, are: 1. Simultaneous analysis of various haplodiploid markers requires haplotypic phasing, which can be difficult as it requires either familial inference (sometimes unavailable) or special technical approaches (long read sequencing). 2. Robust estimates of recombination rates between haplodiploid markers are always needed whenever their analysis exceeds purely descriptive statistics.

Association at the Population Level Association between markers at the population level is defined as the situation of a population in which haplotype frequencies differ from the expected ones, so that, in the absence of association, the frequency of haplotype A1B1 should be the product of the frequencies of the non-alleles involved, a1
b1. Unfortunately (and misleadingly, as unlinked markers can be associated), the situation is frequently referred in the literature as linkage disequilibrium (or simply LD) despite many attempts of replacement with better wording for the concept (Hedrick 1987). This simple definition is however often misunderstood, and the verification if LD is present in a specific population faces difficulties, both of theoretical and of statistical nature. One of the common misunderstandings is rooted on the fact that the LD definition addresses the association between a pair of alleles at two loci, implying that (with the exception of strictly biallelic systems) at these two loci can coexist pairs of alleles which show association and others that do not. In the case of haplodiploid markers, we must add an extra complexity: LD can be different in the two sexes. On the practical application, first of all, we face a sampling problem, driven by the number of (independent) parameters required. Instead of the estimates required for allele frequencies at each locus in both sexes (in codominance by simple gene

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counting), we now need haplotype frequencies. This estimation requires large datasets, as the number of possible haplotypes is the product of allele numbers at each locus (Amorim and Pinto 2018). Moreover, in the homogametic sex, haplotypes are normally not accessible to direct observation (remember the problem of double heterozygote phasing; note also that simple haplotype counting can be performed in males, but it does not inform us on the frequencies at the other sex). Realizing that, it is clear that when performing a HWE test on the female genotype distribution in a population sample, we will be testing simultaneously (i) the random association of alleles from different loci to form gametic haplotypes (gametic association or linkage disequilibrium) and (ii) the random association of gametes into genotypes (assortative mating). It is possible to disentangle the two effects, separately evaluating the latter through a test, in which expected values are calculated using haplotype instead of allele frequencies. However, those are themselves not directly observed and countable, so they must be estimated in statistically much less efficient methods that anyway cannot inadvertently assume random association. Unfortunately, a consensual framework for the theoretical foundation of an approach to the association between haplodiploid markers has not yet been reached (Sved and Hill 2018; Thomson and Single 2014; Gorelick and Laubichler 2004; Sabatti and Risch 2002) nor are publicly available software or reviews usable by non-aficionados. A useful guide to forensic applications can be found in (Tillmar et al. 2017), and we can nonetheless formulate the following cautionary conclusions: 1. All haplodiploid markers in a species with XX/XY-like sex determination system are linked, as they are located in the same chromosome, which does not recombine in the heterogametic sex; however, some behave as unlinked like if they were in different chromosomes, provided their physical location allows the recombination rate of independent transmission (0.5). 2. Linked markers can show absence of association in a specific population, and, conversely, unlinked markers can exhibit high LDs, depending on the evolutionary history of the population. 3. Association between haplodiploid markers is difficult to prove or disprove, as large sample sizes are required, not only for test robustness but also for estimation of parameters involved in the calculation of expected values, which may be different in the two sexes.

Applications Individual Profiling Individual profiling of haplodiploid markers is technically similar to homogametic loci and indeed simpler in the case of heterogametic sex, since genotyping equates to haplotyping. The situation in the homogametic sex is exactly the same as for autosomal markers, with identical problem of phasing. Establishment of haplotypes in a homogametic individual is however of little interest if the profiling purpose is

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merely descriptive, intending to characterize an individual. The need for haplotypes is, in contrast, essential if the purpose of profiling is comparative, since two individuals with the same genotype may have distinct haplotype configurations. They may, therefore, produce significantly different gametogenesis (if recombination rate is < ½) and expected frequencies of occurrence in the population (if LD 6¼ 0). The use of haplodiploid markers in profile comparisons, namely, in the inference of kinship, will be analyzed in the next section.

Genetic Relatedness The definition of genetic relatedness between two individuals is grounded in the concept of identity by descent. Two alleles are said to be identical by descent (or IBD) if they have descended from the same ancestral allele. Mutation is considered to break IBD, and therefore, IBD alleles are identical by state. Generally, two individuals are defined to be related if they share IBD alleles due to familial transmission (Jacquard 1970; Weir et al. 2006). In the case of haploid or haplodiploid transmission, this definition must be revisited as (autosomally) related individuals may not share Y- or X-chromosomal IBD alleles. Indeed, for a specific relationship, different degrees of genetic relatedness may be expected for different modes of genetic transmission (Pinto et al. 2011, 2012). For example, a pair of male mammals sharing the same father but with unrelated mothers, i.e., paternal halfsiblings, are related as identical twins from Y-chromosomal point of view and as unrelated from X-chromosomal one – see Table 2. This has practical implications leading, for example, that the mating between a pair of paternal half brother-sister does not carry additional risk for X-chromosomal related conditions than the one associated with the mating of two unrelated individuals – see Fig. 3a and c and Table 2. Notwithstanding, this is not the case for the mating between a pair of maternal half brother-sister, as the probability of individuals sharing a pair of IBD X-chromosomal alleles equates ½, as for autosomes – see Fig. 3b and Table 2 (Pinto et al. 2011, 2012). Indeed, the degree of haploid and haplodiploid genetic relatedness is sex dependent, contrarily to what occurs for autosomes. Considering one marker and a specific mode of genetic transmission, a set of IBD partitions can be associated with any pair of individuals, depending solely on their relationship. These IBD partitions, also known as Jacquard’s coefficients in the case of autosomes (Jacquard 1970), represent all the possibilities of identity by descent between and within individuals’ alleles. Considering autosomal transmission, any relationship between a pair of individuals can be genetically characterized through the probabilities associated with nine IBD partitions. When excluding inbreeding, the number of partitions reduces to three, representing the possibility of the pair of individuals sharing two, one, or none pairs of IBD alleles (Jacquard 1970; Weir et al. 2006). This is also the case for X-chromosomal transmission between two females, reducing the number of IBD partitions to four for a female-male pair (two if excluding inbred females). Finally, when a pair of males is at stake, two trivial IBD patterns are possible: either the pair

Female-female

Male-female

Sex of the analyzed individuals Male-male

Pedigrees Father-son Full brothers Paternal half brothers Paternal grandfathergranddaughter Paternal uncle-nephew Unrelated Full brother-sister Paternal half brother-sister Maternal half brother-sister Unrelated Mother-daughter Full sisters Paternal grandmothergranddaughter Paternal half sisters Unrelated 0 ½ 1/2 0 1 ½ ½

0

0 ¼ 0 0 0 ¼ 0

0

1

1 0 ¼ ½

1 ¼ 1/2

0

0 ½ 0

NA

0

0 ½ 0 ½ 0 1 ½ 1

Diploid Haplodiploid Probability of sharing n pairs of IBD alleles n¼2 n¼1 n¼0 n¼2 n¼1 0 1 0 NA 0 ¼ ½ ¼ ½ 0 1/2 1/2 0

Modes of genetic transmission

1

1 ½ 1 ½ 1 0 0 0

n¼0 1 ½ 1

NA

0 NA

n¼1 1 1 1

Haploid Y-chr.

NA

1 NA

n¼0 0 0 0

1 0 1 0 1 0 0 1 1 1

0 0

n¼0 1 0 1

0 1 0 1 0 1 1 0

n¼1 0 1 0

mtDNA

Table 2 Probabilities of two individuals sharing n pairs of IBD alleles, depending on the mode of genetic transmission and considering the pedigrees mentioned in the text (NA ¼ not applicable)

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Fig. 3 Examples of genetic transmission, considering one haplodiploid locus and a pair of available individuals related as paternal (a) or maternal half brother-sister (b) and as unrelated (c)

of X-chromosomal alleles is IBD or not. For both diploid and haplodiploid modes of genetic transmission, simple counting rules to determine IBD coefficients for relationships linking two non-inbred individuals were already developed (Pinto et al. 2010b, 2012). For any mode of genetic transmission, IBD coefficients are crucial for the quantification of the evidence, which is grounded in the Bayes’ theorem and in the computation of a likelihood ratio, comparing the probabilities of the observations (genotypes) given the alternative hypotheses (relationships) (Weir et al. 2006; Pinto et al. 2011). These probabilities depend on both the IBD probabilities of the relationships at stake and on the population frequency of the alleles. At this point it is noteworthy to highlight that (i) different kinships may have associated the same set of IBD probabilities and that (ii) a specific kinship may have associated different IBD probabilities when considering different modes of genetic transmission. Proposition (i) implies that different kinships may be theoretically indistinguishable when analyzing independent markers. The most commonly cited example is the case of avuncular, half-siblings, and grandparent-grandchild when a pair of

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individuals is analyzed for autosomal transmission. Assuming haplodiploid independent transmission, the same occurs for diverse sets of pedigrees, as is the case of father-son and unrelated males, or for paternal half sisters and mother-daughter (Pinto et al. 2011). In Table 2 we present three pedigrees, relating two males: paternal half brothers, paternal grandfather-grandson, and paternal uncle-nephew, theoretically indistinguishable by the analysis of independent markers considering diploid, haploid, and haplodiploid genetic transmission. On the other hand, proposition (ii) highlights that different proportions of IBD alleles may be expected for the same pedigree when different modes of transmission are considered. For example, a pair of full siblings does not necessarily share IBD autosomal alleles, contrarily to what occurs when considering X-chromosomal ones – see Table 2. This leads that some modes of genetic transmission are expected to provide stronger results than others, depending on the hypotheses under analysis. The most commonly cited example in forensics to highlight that haplodiploid markers can be a major added value comparing to autosomes is related with complex kinship problems, where the alleged father of a daughter is not directly available for testing, contrarily to his undoubted mother or daughter. In these cases, the main hypothesis considers females related as full or paternal half sisters, or as paternal grandmother-granddaughter, and in both cases the sharing of IBD X-chromosomal alleles is mandatory, unless mutation or a silent allele occur – see Table 2. This leads that Mendelian incompatibilities can be identified, which represents a major improvement in the power of statistical analyses (Pinto et al. 2013).

Conclusions Before concluding, let us clarify that since the molecular biology and technical aspects of DNA profiling were addressed in various chapters of this book, here we have approached haplodiploidy in the formal, statistical, and theoretical perspective. Readers particularly interested in the forensic typing of X-chromosome markers can find useful information in other chapters as well as in (Diegoli 2015; Gomes et al. 2020). Only when technical issues were important to the correct modeling of the approach – as in the case of haplotype phasing – were these questions lightly discussed. Also, despite the enormous importance of haplodiploidy in various branches of life sciences – from genetics proper to physiology and evolution – and the diversity of applications, namely, of medical nature, we have put emphasis on the use of haplodiploid markers in a forensic context. In this area, not only for historical reasons but also due to practical, legal, and ethical questions, autosomal markers have been – and will continue to be – the first (and in many times the only ones) to use. However, in many kinship cases, the current standard autosomal profiling (which is satisfactorily powerful for first-degree relationships, as maternity or paternity) fails in producing convincing evidence. In many of these, such as paternal sisters or paternal grandmother-granddaughter, the use of haplodiploid markers can provide a powerful contribution. Moreover,

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(unlinked) autosomal markers are unable to distinguish between alternative genealogies (Pinto et al. 2010a), when they belong to the same kinship class (as between avuncular and half-siblings). Here again the use of haplodiploid markers enables the distinction between alternative pedigrees (as between paternal and maternal auntniece, or maternal half brothers and maternal uncle-nephew). Finally, haplodiploid markers can also be used in sexing a sample of unknown or disputed sex, not only in humans but also in any species with haplodiploidy (as in birds, for which sometimes sexual dimorphism is scant [Henderson et al. 2013]). At this point it may be interesting to recall that all commercial (autosomal) kits for generating STR (short tandem repeat) profiles to be deposited in forensic genetic databases (as CODIS) include a sexing marker, AMEL (amelogenin), a gene that occurs on the sex-specific region of both the X and Y chromosomes (Issan et al. 2019). This marker is prone to many amplification failures and therefore risks sex misidentification (Thangaraj et al. 2002; Alves et al. 2006). We believe some of these problems could have been solved if haplodiploid markers were included in these kits, as recently shown by (Zajac et al. 2020).

Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential con ict of interest. Acknowledgments IPATIMUP integrates the i3S research unit, which is partially supported by the Portuguese Foundation for Science and Technology. This work was partially financed by FEDER (Fundo Europeu de Desenvolvimento Regional) funds through the COMPETE 2020 Operational Program for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT (Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Inovação) in the framework of the project “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274).

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Single-Nucleotide Polymorphism A Forensic Perspective

10

Anubha Gang and Vivek Kumar Shrivastav

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SNPs and Their Attributes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Advantages of SNP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Categories of SNP Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Identity-Testing SNPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lineage SNPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ancestry Informative Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phenotype Informative SNPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Techniques of SNP Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular Beacons (MB) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microarrays/DNA Chips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SNaPshot Multiplexing Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Forensic Application of SNP Profiling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Human Identification (HID) from Skeletal Remains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Paternity Testing and Kinship Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phenotypic Information of a Missing Suspect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Constraints of SNP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SNP Information Databases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

236 236 237 239 240 240 240 241 241 241 245 246 246 247 247 248 249 250 250 251 253

Abstract

In forensic analysis, the conventional short tandem repeat (STR) markers are routinely used for examination of the biological samples. But, in the challenging casework studies such as mass disaster or natural calamities, in which DNA samples are either highly degraded or are present in very minute quantity, single-nucleotide polymorphism (SNPs) serve as a potential marker of choice over STRs. Various techniques of SNP analysis such as molecular beacons, A. Gang (*) · V. K. Shrivastav Regional Forensic Science Laboratory, Indore, MP, India © Springer Nature Singapore Pte Ltd. 2022 H. R. Dash et al. (eds.), Handbook of DNA Profiling, https://doi.org/10.1007/978-981-16-4318-7_8

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A. Gang and V. K. Shrivastav

SNaPshot, DNA microarray, ow cytometry, and mass spectrometry opened the channels of analyzing forensic samples. Though SNPs pose certain limitations like low discrimination power, less number of alleles per loci, still, SNPs play a fundamental role in human identification, kinship analysis of genetically related individuals, complex paternity disputes, identification of suspect ethnicity, establishing biogeographical ancestry, as well as phenotypic information of missing suspect. Various databases are available for collection, integration, and analysis of SNP for their application in forensic science. As per forensic perspective, the method of standardization and validation of SNP markers, in consensus with legislators and the scientific community, need to be established. Keywords

Single-nucleotide polymorphism (SNPs) · Short tandem repeat (STR) · SNaPshot · Kinship analysis · Molecular beacons · Paternity disputes · Mass disaster

Introduction In forensic DNA typing, the use of genetic markers for the characterization of biological samples serves as a hallmark in human identification. First approach made for SNP-based typing for forensic analysis was with HLA-DQA1 locus (formerly known as HLADQα) polymorphism. Another polymarker studied was Ampli ®TypePM which is an expansion of HLA-DQA1 analysis. It contained five different markers, viz., LDLR, GYPA, HBGG, D7S8, and GC with high power of discrimination. The method posed limitation for analysis of DNA samples with more than one contributor, which was overcome by development of conventional STR typing (Butler 2011). Although the use of short tandem repeat (STR) loci as a predominant genetic marker for human identification is well established, yet certain additional markers such as single-nucleotide polymorphism (SNPs) have been explored for their potential to be used by a genetic analyst (Budowle and Van Daal 2008; Canturk et al. 2014). The chapter provides insight on SNPs giving highlights about their advantages over other markers as per forensic perspective, categories of SNP markers, techniques involved in the analysis of SNP, and their applications in forensic science. The chapter also quotes the limitations of using SNP as a marker and certain information regarding various databases available for SNP.

SNPs and Their Attributes The human genome consists of about 3.2 billion base pairs with the number of repeated DNA sequences harboring an equal amount of difference in its genome. The variation in these repeat sequences arises largely due to size, number, and the length of core repeat units (Roewer 2013; Canturk et al. 2014). The variation in a

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Fig. 1 Single nucleotide polymorphism

sequence called polymorphism is an important characteristic of the human genome. Single-nucleotide polymorphism (SNP) refers to the sequence variants occurring in a genome with a single base pair change (Fig. 1). This change in sequence results due to base substitution, insertion, or deletion at a single site (Li et al. 2015; Brookes 1999). Every individual has millions of SNPs which add to provide a powerful tool in the forensic community for identification. There are nearly 10 million SNPs in the human genome, among these about 1.4 million SNPs have been identified. They comprise mainly the noncoding regions and sometimes coding regions of the genome (Li et al. 2015). Most of the SNPs are biallelic, but tri- and tetra-allelic SNPs have also been reported (Hüebner et al. 2007; Phillips et al. 2015).

Advantages of SNP SNPs are abundant within the human genome and can be used as markers for forensic applications. Although STR markers are currently been used in genome base analysis for human identification and DNA database preparation, the use of SNP as promising markers needs to be addressed. The most challenging capability of SNP markers is their potential to retrieve information from a highly degraded forensic sample. The devastating natural events or calamities often results in degraded DNA samples or the samples in very minute (i.e., 1 month), the extracts should be centrifuged, and the supernatant should be removed from the Chelex resin and stored in a new tube at 20  C

Whatman FTA® Paper Another extraction method that is easily employed in biological samples collected from fresh corpses is based on Whatman FTA ® paper. FTA paper is a cellulose-based absorbent substrate that contains additives which lyse cells, protect DNA molecules from degradation by nucleases, as well as prevent the growth of microorganisms. After the sample has dried, DNA on FTA paper is stable for several years under room temperature. A small punch of the paper is washed to remove unwanted substances while retaining the DNA. The clean punch is added directly to the PCR (Burger et al. 2005). If the above methods are not able to yield good enough results, another extraction method that provides improved purification, such as those described below, may be employed. Organic Method Organic, or phenol-chloroform, extraction receives its name due to the use of the organic solvents phenol and chloroform. It requires the addition of certain species of chemicals so it can achieve its purpose. A surfactant, such as sodium dodecylsulfate (SDS), is added to the aqueous buffer containing the sample to disrupt the phospholipidic bilayer that composes cellular membranes and denature proteins. The enzyme proteinase K is added to digest nucleases and proteins that surround the

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Table 2 Sample preparation for organic extraction from each type of biological matrix Biological matrix Blood or saliva Swab with blood or saliva Urine

Hair

Preparation Add 10 μL of whole blood or saliva to a microtube Cut a third of the swab and place it in a microtube Add 1 mL of urine to a microtube Centrifuge at 10,000 g for 3 min Discard supernatant except final 50 μL with pellet Cut 0.5–1 cm of the root end of a strand of hair with sheath Place the hair root in a microtube

DNA molecules. Next, a phenol-chloroform mixture is added to extract lipids and separate the denatured proteins from the aqueous solution containing the DNA. After centrifugation, two separate phases are observed: the organic lower phase and the aqueous upper phase. DNA is more soluble in the aqueous phase than in the organic phase. Collecting the clean aqueous phase, DNA molecules can be transferred for analysis (Butler 2009; Butler 2011; Cattaneo et al. 2006; Elkins 2012; Prinz and Lessig 2014). The organic method provides high-molecular-weight DNA with high purity, but currently there are other methods that use chemicals which are far less toxic than phenol. In addition, organic extraction is not yet amenable to automation, is time consuming, and requires multiple tube transfers, increasing the risks of error and contamination. For each biological substrate, sample preparation is illustrated on Table 2.

Organic Protocol 1. Add 400 μL of extraction buffer (SDS 2%, EDTA 0.01 M, pH 8) to the microtube containing the sample 2. Add Proteinase K enzyme (20 mg/mL) in a ratio of 10:1 (extraction buffer: Proteinase K) 3. Add DTT (0.4 M) (equal to the volume of proteinase K) 4. Vortex the sample for 5 s and then centrifuge brie y 5. Incubate the sample at 56  C overnight 6. Discard the swab, fabric, or substrate, if the case 7. Add an equal volume of phenol–chloroform–isoamyl alcohol (25:24:1) to the lysate. Vortex and centrifuge at 14,000 g for 5 min. Transfer the aqueous supernatant to a new microtube. Repeat this step 2–3 times until no impurities are seen in the interphase 8. Transfer the upper aqueous phase to a Microcon ® 100 Centrifugal Filter. Centrifuge at 4000 g for 20–30 min, or until the liquid has been reduced to the minimum retained volume 9. Wash the filtrated Microcon ® with 500 μL of DNA-free water and centrifuge at 4000 g for 20–30 min. Repeat this step 2– 3 times 10. Elute the DNA in 50–100 μL of DNA-free water by inverting the Microcon ® into a new 1.5 mL tube. Centrifuge at 4000 g for 3–5 min

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Solid-Phase A natural evolution from the organic method would be to use nontoxic chemicals and automate the DNA extraction process. Solid-phase extraction methods appeared with that proposal. In those methods, silica-based technology is involved, producing better DNA recovery and more efficient removal of inhibitors. The most common techniques are based on the same chemical principle: one method works with silica membranes in combination with a chaotropic salt-containing binding buffer. Another one uses silica-covered magnetic beads in combination with a chaotropic saltcontaining binding buffer. Other protocols rely on the use of a silica suspension. DNA is absorbed by the silica surface in the presence of a high concentration of salt and a pH 7.5. The method of choice should be decided by the quality of the samples. Depending on the biological substrate, the solid-phase employs a silica membrane having specific characteristics. Basically, all protocols differ in the use of decalcification and digestion buffer, incubation temperature, and time. Stringent washes purify the bound DNA molecules from proteins and other contaminants, and, in the end, a nonstringent wash elutes highly purified DNA (Greenspoon et al. 1998). The disadvantages are the higher costs, complexity, more hands-on time, and tube changes, which raises risks for error and contamination. Alternatively, there are several biotechnology companies that commercialize solid-phase DNA extraction kits using an automated platform, increasing costs but reducing time, complexity, error, and contamination (Prinz and Lessig 2014; Kitayama et al. 2020). QIAamp DNA Investigator Kit (QIAGEN, Hilden, Germany) Protocol Blood and Saliva

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Pipet 1–100 μL of whole blood or saliva into a 1.5 mL microtube Add Buffer ATL to a final volume of 100 μL Add 10 μL of proteinase K Add 100 μL of Buffer AL and close the lid Vortex for 15 s Incubate at 56  C for 10 min Brie y centrifuge the microtube to remove drops from inside the lid Add 50 μL of ethanol (96–100%), close the lid, and vortex for 15 s Incubate for 3 min at room temperature Brie y centrifuge the microtube to remove drops from inside the lid Follow the purification and elution (for all samples) step below

Buccal Swab

1. 2. 3. 4. 5. 6.

Cut off the cotton head of the buccal swab and place it in a 1.5 mL microtube Add 20 μL of proteinase K and 400 μL of Buffer ATL and close the lid Vortex for 10 s Incubate at 56  C for 1 h; Add 400 μL of Buffer AL and close the lid Vortex for 15 s

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Incubate at 70  C for 10 min with shaking at 900 rpm for 10 min Brie y centrifuge the microtube to remove drops from inside the lid Add 200 μL of ethanol (96–100%) and close the lid Vortex for 15 s Brie y centrifuge the microtube to remove drops from the inside of the lid Follow the Purification and elution (for all samples) step below

Urine

1. 2. 3. 4. 5. 6. 7. 8. 9.

10. 11. 12. 13. 14. 15. 16. 17.

Add 1 mL of urine to a 1.5 mL microtube Centrifuge at 10,000 g for 3 min to pellet cells Discard the supernatant except the final 50 μL containing the pellet Add 500 μL of Buffer AE Vortex for 5 s Centrifuge at 6000 g for 2 min Discard the supernatant Add 300 μL of Buffer ATL and 20 μL of proteinase K to the pellet Vortex for 10 s Note: Adding 20 μL of 1 M DTT may increase sensitivity, since urine can contain sperm cells which can only be lysed in the presence of reducing agents. Incubate at 56  C for 1 h Brie y centrifuge the microtube to remove drops from inside the lid Add 300 μL of Buffer AL, close the lid, and vortex for 10 s Incubate at 70  C for 10 min with shaking at 900 rpm Brie y centrifuge the microtube to remove drops from the inside of the lid Add 150 μL of ethanol (96–100%), close the lid, and vortex for 15 s Brie y centrifuge the microtube to remove drops from inside the lid Follow the Purification and elution (for all samples) step below

Hair

1. Cut off 0.5–1 cm from the root end of a strand of hair with sheath 2. Place the cut off root in a 1.5 mL microtube 3. Add 300 μL of Buffer ATL, 20 μL of proteinase K, and 20 μL of 1 M DTT, close the lid 4. Vortex for 10 s 5. Incubate at 56  C for 1 h with shaking at 900 rpm 6. Brie y centrifuge the tube to remove drops from the inside of the lid 7. Add 300 μL of Buffer AL, close the lid, and vortex for 10 s 8. Incubate at 70  C for 10 min with shaking at 900 rpm 9. Brie y centrifuge the microtube to remove drops from the inside of the lid 10. Add 150 μL of ethanol (96–100%), close the lid, and vortex for 15 s 11. Brie y centrifuge the microtube to remove drops from the inside of the lid 12. Follow the Purification and elution (for all samples) step below

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Purification and Elution (for All Samples)

1. Carefully transfer the entire lysate to the QIAamp MinElute column (in a 2 mL collection tube) without wetting the rim, close the lid, and centrifuge at 6000 g for 1 min Note: If the lysate has not completely passed through the membrane after centrifugation, centrifuge again at a higher speed until the QIAamp MinElute column is empty 2. Carefully open the QIAamp MinElute column and add 500 μL of Buffer AW1 without wetting the rim. Close the lid and centrifuge at 6000 g for 1 min 3. Place the QIAamp MinElute column in a clean 2 mL collection tube and discard the collection tube containing the ow-through 4. Carefully open the QIAamp MinElute column and add 700 μL of Buffer AW2 without wetting the rim. Close the lid and centrifuge at 6000 g for 1 min 5. Place the QIAamp MinElute column in a clean 2 mL collection tube and discard the collection tube containing the ow-through 6. Carefully open the QIAamp MinElute column and add 700 μL of ethanol (96– 100%) without wetting the rim. Close the cap and centrifuge at 6000 g for 1 min 7. Place the QIAamp MinElute column in a clean 2 mL collection tube and discard the collection tube containing the ow-through 8. Centrifuge at full speed (20,000 g) for 3 min to dry the membrane completely 9. Place the QIAamp MinElute column in a clean 1.5 mL microtube and discard the collection tube containing the ow-through. Carefully open the lid of the QIAamp MinElute column and incubate at room temperature (15–25  C) for 10 min or at 56  C for 3 min 10. Apply 20–100 μL of Buffer ATE or ultrapure water to the center of the membrane 11. Close the lid and incubate at room temperature for 5 min. Centrifuge at full speed for 1 min Notes: The extract is ready to be used in PCR-based amplification. For shortterm storage (up to 1 month), store extracts at 2–8  C. For long-term storage (>1 month), the extracts should be stored at 20  C

DNA Extraction from Decomposed/Skeletonized Bodies Overview Following death, the natural progression of the body’s decomposition or postmortem changes starts. At the cellular level, a complex series of biochemical and pathological processes initiates, resulting in considerable alteration of the structure and composition of the entire human body. These changes start immediately after death, occur sequentially, and continue for a prolonged time at different rates for different organs (Zhou and Byard 2011). The onset, the extent, and the rates of these changes are affected, accelerated, or decelerated, by multiple intrinsic and extrinsic factors, differing among geographical

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regions and also in the same geographic region from one season to another (Sutherland et al. 2013). Intrinsic factors primarily include body mass and the surface area of the body, while extrinsic factors primarily include the subject’s clothing and insulation, the environment of the death scene, and the storage of the body after death. Two mechanisms are involved in the postmortem process of decomposition: autolysis and putrefaction (Cockle and Bell 2017). Autolysis is a process that occurs due to leakage of hydrolytic cellular enzymes from cells after death, especially in those organs with high concentration of cellular enzymes like the pancreas. The leakage of cellular contents is a suitable environment for microbes normally composing the human microbiota which grow and degrade surrounding tissues, starting putrefaction (Cattaneo et al. 2005a). Unlike autolysis, putrefactive changes are evident on a macroscopic level: the skin shows discoloration and body parts such as the face, abdomen, breast, and scrotum start to bloat. It can appear in various forms, such as putrefactive uids and gases. There are five stages of decomposition, namely: fresh, bloated, active decay, advanced decay, and skeletal stage (Lee Goff 2009). These stages may occur at the same time in different parts of the same body, so for the forensic practitioner it could be challenging to label the decomposition of the corpse within a single stage. The speed of the onset of putrefaction and its rate of progression are affected mostly by ambient temperature. In addition, blow ies and esh ies are often the first insects to be attracted to a dead body, primarily during the bloated and decay stages of decomposition. In the bloated stage, body parts, including organs and soft tissues, swell due to the accumulation of putrefactive gases or other decomposition products from the putrefaction process (Gebhart et al. 2012). Active decay is the stage after bloating, where postmortem purging takes place and putrefactive body uids become forced out of body orifices. The detachment of hair or hair sloughing and black discoloration of ruptured skin are seen. Advanced or late decay is a stage where bones begin to get exposed, and the body assumes a “caved in” appearance. Degradation-resistant tissues such as hair (although already sloughed off) and cartilage are spared up to this stage. The skeletal stage, also called dry remains stage or skeletonization, begins when bone exposure is extensive (Swann et al. 2010). Remaining dry skin, cartilage, and tendons are minimal in this stage. Decomposition considerably decelerates at this stage, and it takes years or decades for the skeletal remains to disintegrate. Differential decomposition processes involving mummification or adipocere formation are also reported (Ubelaker and Zarenko 2011). The macroscopic changes discussed above re ect also on a cellular level. When an organism dies, the cells’ DNA start to degrade and become susceptible to damage and chromosomal fragmentation: the higher the degradation of the tissues, the lower the DNA yield. Primarily, two factors (time and condition of exposed environment) in uence the decomposition as well as degradation of DNA evidence (Dash et al. 2020). In particular, the process of degradation increases with time. The rate and the intensity of the decomposition process, and consequently the degradation of the samples, are dependent on several other variables: the environmental conditions such as climate, temperature, humidity, pH and ultraviolet radiation, the presence of

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scavengers, natural and artificial inhibitor substances, the rate of bacterial growth, and the body mass index of the deceased (Courts et al. 2015). In forensic biological samples, DNA damage occurs mostly due to hydrolysis, oxidation, and pyrimidine dimer formation. In these cases, humidity plays a crucial role in DNA degradation, as DNA is easily damaged by hydrolysis resulting in deamination, depurination, and depyrimidination. Also, prolonged exposure to heat, ultraviolet radiation, microorganisms, and microbial nucleases further increases the rate of DNA damage and degradation resulting in fragmentation of the DNA molecule into smaller pieces. More than 70% of soil microorganisms contain nucleases, which make them capable of destroying nucleic acids (Milos et al. 2007). On the contrary, desiccation and/or low temperature can slower these chemical processes (Davoren et al. 2007). The genetic material obtained from those samples can be both degraded and chemically damaged (Fattorini et al. 2009), implying that no or low copy number of entire templates are available for successful DNA typing.

Biological Samples Since tissues of a human cadaver decompose over time, forensic casework encounters a major chance of examining degraded biological samples such as soft tissues, organs, nails, teeth, and bones. Soft tissues like muscle and organs are the source of DNA of choice dealing with partially decomposed cadavers. In general, they contain large amounts of DNA, although different tissue types are affected differently by the above-mentioned degradation processes. Putrefaction tends to be more active in tissues such as the kidney and liver, resulting in early DNA degradation. The most advised soft tissues for DNA analysis include the brain, muscle, kidney, and heart. The required laboratory procedures are short, simple, and affordable (Schwark et al. 2011). With ongoing putrefaction in soft tissues and organs, DNA integrity will continuously decrease up to a point at which analysis and profiling using standard methods is no longer possible and the collection of other matrices are needed, such as nails and hard tissues (Caputo et al. 2011). Nails are made, like hair, of a tough protective protein called alpha-keratin with the aim of preserving the nail matrix, which contains nerves, lymph, and blood vessels. This structure ensures nail tissues high resistance to decay. They are a promising alternative as a source of DNA, even if nails appear to be less considered as a tissue of choice from decomposed bodies (Allouche et al. 2008). Although the DNA concentration in nail tissue is fairly low in comparison with hard tissues, it was shown that a certain constant concentration can be obtained regardless of the time elapsed after death (Kaneko et al. 2015). Skeletal elements are composed by hard tissues, as teeth and bones, which are often the only biological materials remaining after exposure to environmental conditions, intense heat, certain traumatic events, and in cases where a significant amount of time has passed since the death of the individual (Latham and Miller 2019).

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Teeth are one of the most useful sources of DNA for genetic analysis. They are a valuable source of DNA due to their unique composition and location protecting them from decay. The good state of preservation is granted by enamel, which is the hardest tissue of human body. Teeth roots, composed of cementum and pulp, are a rich source of DNA with high-quality and low-contamination. In case of extremely damaged or degraded human skeletal remains, bones are the most suitable samples for genetic analysis. Bone possesses a complex anatomical structure made of many organic as well as inorganic molecules such as hydroxyapatite, collagen, and noncollagen proteins. The combination of organic as well as inorganic substances renders bones their hardness and makes them less prone to degradation. Therefore, the collection of bones can offer higher probability of obtaining preserved DNA since desiccation and/or low temperature can slower decomposition processes (Hochmeister et al. 1991). The goal of skeletal DNA extraction techniques is to maximize DNA yield, minimize any additional DNA damage, and remove any inhibitors that may co-purify with the skeletal DNA and interfere with later genetic analyses. The relationship between the bone type and the generation of an informative DNA profile has been investigated: compact bone of the lower limbs tends to yield greater amounts of DNA than spongy bone. The main disadvantages of collecting samples from bones and teeth are the requirement of tedious and time-consuming extraction procedures due to the need of mechanical fragmentation and tissue decalcification (Madea et al. 2010). Also, the high number of steps required by these processes increases the risk of contamination, which has to be always considered (Vanek et al. 2017). Exogenous contamination plays a limiting role in the analysis and interpretation of DNA typing results. When the biological samples are contaminated by the abovementioned contaminating substances, these compounds are co-purified with the nucleic acids in spite of the accuracy of the protocol employed and act inhibiting the DNA polymerases (King et al. 2009). In these cases, it is mandatory to perform a new extraction by freshly prepared solutions and new consumables.

DNA Extraction DNA profiling of putrefied remains or skeletonized samples may be the only means to identify their human sources. With ongoing decomposition, the damage downgrades both the quality and quantity of DNA extracted from forensic samples and, consequently, minimizes the high discrimination power of routine forensic STR analyses by resulting in partial or no STR profiles (Alaeddini et al. 2010). In fact, although using STRs in DNA typing is currently the gold standard for human identification purposes, highly degraded samples often result in partial STR profiles because the larger loci (>250 bp) commonly fail to amplify due to fragmentation of the DNA structure (Sorensen et al. 2016). In cases of soft tissues, organs, and nails, DNA extraction protocols are the same for the fresh bodies as previously described. Differently, purifying DNA from bones

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and teeth requires modification of the usual DNA extraction techniques utilized for other biological samples (Latham and Miller 2019). Most of these methods includes a pre-extraction treatment aimed at removing contaminating DNA transferred to the surface of the bone or tooth. Decontamination can be achieved by physically removing the outer bone surface, by immersing the bone or tooth in a bleach solution, or by exposure to ultraviolet radiation (Latham and Madonna 2014). The hard tissues are often pulverized and subsequently incubated in extraction buffer and proteinase K, which work dissolving both the organic and inorganic portions of the bone tissue (Loreille et al. 2007). Grinding the sample into a powder (mechanically or using freezer mills) breaks the hydroxyapatite mineral matrix, increases the surface on which DNA extraction chemicals operate, and releases a greater amount of DNA. The amount of bone powder varies among protocols, requiring from 0.2 g to 2.5 g of starting material. The amount of powder needed for the extraction process has slowly been reduced thanks to the optimization of the DNA extraction process and the increased sensitivity of DNA kits which allow for minimal destruction of the skeletal samples (Hervella et al. 2015). Also, hard tissue extraction can require another pre-extraction treatment aimed at clearing mineralization within the bone, which represents physical barrier to the extraction reagents and therefore inhibit the release of DNA molecules (Loreille et al. 2007). Many protocols for bones and teeth are based on the incubation in an ethylene diamine tetra-acetic acid (EDTA)-containing extraction buffer, in order to reach total decalcification (Rothe and Nagy 2016). The EDTA both demineralizes the hard tissues and inactivates DNAses by chelating bivalent cations such as Mg++ or Ca++ (Loreille et al. 2007). As in the previous paragraphs, the following subheadings will illustrate the most common techniques employed in DNA extraction from advanced decomposed bodies and skeletonized remains. A brief introduction and a detailed protocol will be provided for each biological matrix.

Organic Muscle, organs, nails, bones, and teeth preparation are presented on Table 3, while the protocol is described below.

Organic Protocol 1. Add 300 μL of extraction buffer (10 mM Tris HCl, 100 mM NaCl, 39 mM DTT, 10 mM EDTA, SDS 2%) and 20 μL of proteinase K to the sample 2. Incubate at 56  C overnight 3. Add 300 μL of phenol:chloroform:isoamyl alcohol to each tube 4. Mix vigorously until a complete emulsion is formed 5. Centrifuge tubes for 3 min at 4000 g. There should be a clear delineation between the layers 6. Transfer the aqueous layer of each sample to clean tubes

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Table 3 Decomposed sample preparation for organic extraction from each type of biological matrix Biological matrix Muscle and organs Nail Bones and teeth

7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Preparation Add 10 mg of tissue to a tube Add nail clippings to a tube Cut a piece of bone or tooth roots (e.g., using a hammer or rotational device) Pulverize the bone or tooth piece (e.g., using a mill or blender) Add 0.5 to 1 g of powder to a tube Add 40 mL of EDTA 0.5 M, pH 7.5 to powdered sample for 24 h at 4  C Shake or invert the tubes gently to completely saturate the bone powder. Continue to gently shake until no dry spots are visible in the powder Centrifuge tubes for 15 min at 2000 rpm and discard the supernatant Wash the pellet with sterile water, vortexing for 15 s Centrifuge tubes for 15 min at 2000 rpm and discard the supernatant

Repeat steps 4–7 until the interface is clean (or a minimum of two times) Add 10 μL of NaAc 3 M and mix thoroughly Add 2 times the volume of cold absolute ethanol and incubate at 20  C for 2 h Centrifuge tubes for 15 min at 4000 g Discard the alcohol supernatant Add 200 μL of 70% ethanol Centrifuge tubes for 2 min at 4000 g Discard the alcohol supernatant Let the pellet dry thoroughly under hood Add 50 μL of sterile water and incubate at 56  C for 2 h

Solid-Phase Muscle, organs, and nails preparation is shown in Table 3, while the protocol for these samples is described in “Solid-Phase” For bone and teeth samples with prolonged time elapsed since death, DNA extraction with the silica-membrane method showed to have more DNA yield than with the beads method, since when using the extraction method with the beads, DNA loss could occur. Therefore, the silica-coated beads method should be chosen for more recent skeletal samples or samples with high amounts of inhibitors, while the silica-membrane method is preferred for samples with low amounts of DNA. Skeletal sample preparation for both solid-phase extraction methods is given in Table 4. QIAamp DNA Investigator Kit (QIAGEN, Hilden, Germany) Protocol 1. Add 360 μL of Buffer ATL, 20 μL of proteinase K, close the lid 2. Vortex for 10 s 3. Incubate at 56  C overnight with shaking at 900 rpm 4. Brie y centrifuge the tube to remove drops from the inside of the lid 5. Add 300 μL of Buffer AL, close the lid, and vortex for 10 s

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Table 4 Decomposed sample preparation for solid-phase extraction from each type of biological matrix Preparation

Biological matrix Bones

Teeth

6. 7. 8. 9. 10.

11. 12. 13. 14. 15. 16.

Silica-membrane Cut a piece of bone (e.g., using a hammer or rotational device) Pulverize the bone piece (e.g., using a mill or blender) Add up to 100 mg of bone powder to a tube Pulverize one tooth (e.g., using a mill or blender) Add up to 100 mg of tooth powder to a tube

Cut a piece of bone (e.g., using a hammer or rotational device) Add the bone piece in a solution of EDTA 0.5 M until it is demineralized

Add the tooth in a solution of EDTA 0.5 M until it is demineralized

Silica-coated beads ® PrepFiler BTA Forensic DNA Extraction Kit (Thermofisher, Waltham, MA, USA) Cut a piece of bone (e.g., using a hammer or rotational device) Pulverize the bone piece (e.g., using a mill or blender) Add up to 50 mg of bone powder to a microtube

Pulverize one tooth (e.g., using a mill or blender) Add up to 50 mg of tooth powder to a microtube

Incubate at 70  C for 10 min with shaking at 900 rpm Brie y centrifuge the microtube to remove drops from the inside of the lid Add 150 μL of ethanol (96–100%), close the lid, and vortex for 15 s Brie y centrifuge the microtube to remove drops from the inside of the lid Carefully transfer the entire lysate to the QIAamp MinElute column (in a 2 mL collection tube) without wetting the rim, close the lid, and centrifuge at 6000 g for 1 min Note: If the lysate has not completely passed through the membrane after centrifugation, centrifuge again at a higher speed until the QIAamp MinElute column is empty Carefully open the QIAamp MinElute column and add 600 μL of Buffer AW1 without wetting the rim. Close the lid and centrifuge at 6000 g for 1 min Place the QIAamp MinElute column in a clean 2 mL collection tube and discard the collection tube containing the ow-through Carefully open the QIAamp MinElute column and add 700 μL of Buffer AW2 without wetting the rim. Close the lid and centrifuge at 6000 g for 1 min Place the QIAamp MinElute column in a clean 2 mL collection tube and discard the collection tube containing the ow-through Carefully open the QIAamp MinElute column and add 700 μL of ethanol (96– 100%) without wetting the rim. Close the cap and centrifuge at 6,000 g for 1 min Place the QIAamp MinElute column in a clean 2 mL collection tube and discard the collection tube containing the ow-through

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17. Centrifuge at full speed (20,000 g) for 3 min to dry the membrane completely 18. Place the QIAamp MinElute column in a clean 1.5 mL microtube and discard the collection tube containing the ow-through. Carefully open the lid of the QIAamp MinElute column and incubate at room temperature (15–25  C) for 10 min or at 56  C for 3 min 19. Apply 20–50 μL of Buffer ATE or ultrapure water to the center of the membrane 20. Close the lid and incubate at room temperature for 5 min. Centrifuge at full speed for 1 min Notes: Extracts may be held at 20 or 80  C for extended storage. In the short term, 4  C is adequate. It is best to minimize freeze/thaw cycles as freezing may damage the DNA Silica-Coated Beads Protocol PrepFiler ® BTA Forensic DNA Extraction Kit (Thermofisher, Waltham, MA, USA) 1. Add 230 μL of the following lysis buffer solution to each sample: 220 μL PrepFiler ® BTA Lysis Buffer, 3 μL 1.0 M DTT and 7 μL Proteinase K 2. Cap the tubes, vortex for 5 s, centrifuge brie y, then gently ick to resuspend any powder or substrate 3. Make sure the tubes are well sealed, then place them in a thermal shaker and incubate at 1000 rpm and 56  C for 2 h 4. Allow the sample to equilibrate to room temperature 5. Centrifuge the sample tubes at 10000 g for 90 s, then carefully transfer the clear lysate to a new 1.5 mL tube 6. If needed, add PrepFiler ® BTA Lysis Buffer to bring the total sample lysate volume to 180 μL 7. Add 300 μL of PrepFiler ® Lysis Buffer to the sample lysate tube, vortex it brie y to mix, then centrifuge it brie y 8. Vortex the PrepFiler ® Magnetic Particles tube for 5 s, invert the tube to confirm that no visible pellet remains in the bottom of the tube, then centrifuge it 9. Pipet 15 μL of thoroughly resuspended magnetic particles into the sample lysate tube 10. After adding the particles, recap the PrepFiler ® Magnetic Particles tube to prevent evaporation 11. Cap the sample lysate tube, vortex it at low speed for 10 s, then centrifuge it brie y to collect any residual tube contents from the sides and cap of the tube 12. Add 300 μL isopropanol (99.5% molecular biology grade) to a sample lysate tube and mix one sample at a time to promote binding 13. Immediately after adding isopropanol, cap the sample lysate tube, vortex it at low speed for 5 s, then centrifuge it brie y to collect any residual tube contents from the sides, and cap of the tube 14. Place the sample lysate tube in a shaker, then mix at 1000 rpm at room temperature for 10 min 15. Vortex the sample DNA tube at maximum speed (approximately 10,000 rpm) for 10 s, then centrifuge brie y to collect any residual tube contents from the sides and cap of the tube

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16. Place the sample DNA tube in the magnetic stand and observe that the magnetic particles form a pellet against the back of the tube. Wait until the size of the pellet stops increasing (approximately 10 min) 17. With the sample DNA tube remaining in the magnetic stand, carefully aspirate and discard all visible liquid phase. Do not aspirate or disturb the magnetic particle pellet 18. Perform steps a through e three times: (a) Add prepared wash buffer to the sample DNA tubes: • First wash: 600 μL Wash Buffer A • Second wash: 300 μL Wash Buffer A • Third wash: 300 μL Wash Buffer B (b) Cap the sample DNA tubes and remove them from the magnetic stand (c) Vortex the sample DNA tubes for 15 s, then centrifuge brie y to collect any residual tube contents from the sides and cap (d) Place the sample DNA tubes in the magnetic stand for 1 min (e) With the sample DNA tubes remaining in the magnetic stand, carefully aspirate and discard all visible liquid phase. Do not disturb the magnetic particle pellet 19. Centrifuge the tubes brie y, place the tubes back on the magnetic stand for 30– 60 s, then collect any residual liquid 20. Add 50 μL of PrepFiler ® Elution Buffer to the sample DNA tube, then vortex at maximum speed until the pellet is resuspended 21. Place the sample DNA tube in a thermal shaker, then incubate at 70  C and 1000 rpm for 10 min 22. Vortex the sample DNA tube at maximum speed until there is no visible magnetic particle pellet on the side of the tube (approximately 2 s), then centrifuge brie y to collect any residual tube contents from the sides and cap 23. Place the sample DNA tube in the magnetic stand, then wait until the size of the pellet at the side of the tube stops increasing (approximately 5 min) 24. Carefully aspirate all visible liquid phase in the sample DNA tube 25. Transfer the eluate to a new, labeled 1.5 mL tube for storage Notes: The isolated DNA can be stored at 4  C for up to 1 week, or at 20  C for longer storage

Conclusions Successful individual-specific DNA profiles are routinely obtained from a variety of biological samples in the forensic context. The choice of matrices collected from cadavers for DNA extraction depends mostly on the preservation of the corpse. High-quality DNA profiles can be potentially obtained from any biological source. When dealing with fresh or partially decomposed cadavers, biological uids, soft tissues, and hair are the samples of choice as a source of DNA, requiring routinely laboratory procedures. Blood and saliva are better sources than urine, due to their

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high number of nucleated cells, while DNA extraction from hair is usually only possible when roots and adhering tissues are present. With ongoing putrefaction, samples may be degraded, which contain inhibitors or low DNA content. Soft tissues like muscle and organs are the source of DNA of choice dealing with partially decomposed cadavers. In cases of advanced decay, nails, which are resistant to decomposition, and hard tissues, such as bones and teeth, are alternative sources of DNA. Genetic identification can even be successfully carried out from aged, burned, and fragmented skeletal remains. The main disadvantages of collecting samples from bones and teeth are the requirement of tedious and time-consuming extraction procedures due to the need of mechanical fragmentation and tissue decalcification. Also, the high number of steps required by these processes increases the risk of contamination. The Chelex protocol for DNA extraction is among the most used due to its low cost and short execution time. It provides high DNA yield from samples that contain a large number of cells and no PCR inhibitory substances. Organic protocols have always been the preferred extraction method for high quantity and purity of DNA from any biological sample. However, since they require use of toxic chemicals, its use has shifted towards silica-based extractions. Silica-based protocols may provide somewhat less DNA quantity or purity than organic ones, but the difference is not believed to be significant. In addition, silica-based protocols are safer, easier, available in commercial kits and amenable to automation. Purifying DNA from bones and teeth often requires modification of the DNA extraction techniques utilized for other biological samples. The goal of skeletal DNA extraction techniques is to maximize DNA yield, minimize any additional DNA damage, and remove any inhibitors that may co-purify with the skeletal DNA and interfere with later genetic analyses. In challenging forensic scenarios, the collection of more than one sample from different tissues is advised. Also, if DNA is degraded or in low quantity, multiple extractions and amplifications may be necessary. Furthermore, since PCR is always required to produce DNA profiles in forensic genetics, the risk of contamination must always be considered. Contamination may be introduced at any stage, from recovery of the remains to the laboratory analysis. Precautions must be adopted when handling challenging/critical samples in order to optimize DNA profiling. Moreover, when the size of the sample is small, the risk of mistyping due to exogenous contamination increases exponentially. With the aid of the protocols provided herein, the practitioner should be able to extract high quality DNA from the most diverse range of samples for human identification and criminalistic purposes.

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DNA isolation from human remains

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María Saiz, Christian Haarko¨tter, X. Gálvez, L. J. Martinez-Gonzalez, M. I. Medina-Lozano, and Juan Carlos Alvarez

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sample Pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Decontamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pulverization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Manual DNA Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA Isolation with Commercial Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

The DNA analysis of human remains can be challenging despite the strong matrix in bones and teeth that helps preserve DNA. To isolate DNA, the correct procedures need to be applied. Factors such as temperature, pH, and humidity affect DNA degradation, while polymerase chain reaction inhibitors can affect or even prevent DNA amplification. If a sufficient quantity and quality of genetic material is obtained during DNA extraction, the key stage in DNA typing, a genetic profile can be obtained, thereby helping to identify the human remains. The aim of this chapter is to show various pretreatment strategies for bones and teeth (surface washing, chemical washing, enzymatic predigestion, milling and sanding, and M. Saiz · C. Haarkötter (*) · X. Gálvez · M. I. Medina-Lozano · J. C. Alvarez Laboratory of Genetic Identification, Department of Legal Medicine, Toxicology and Physical Anthropology, Faculty of Medicine, University of Granada, Granada, Spain e-mail: [email protected] L. J. Martinez-Gonzalez Department of Biochemistry and Molecular Biology III, Faculty of Medicine, University of Granada, Granada, Spain Pfizer-University of Granada-Junta de Andalucía Centre for Genomics and Oncological Research (GENYO), Granada, Spain © Springer Nature Singapore Pte Ltd. 2022 H. R. Dash et al. (eds.), Handbook of DNA Profiling, https://doi.org/10.1007/978-981-16-4318-7_38

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ultraviolet radiation), as well as pulverization methods (mortaring, freezer milling, or tissue lysis), manual DNA isolation protocols (total demineralization, organic use of Chelex resin, and manual purification), and available commercial kits for DNA extraction from human remains. Keywords

Ancient DNA · Automated DNA extraction · Human remains · Manual DNA extraction · Sample pretreatment

Introduction When discussing human remains, we usually refer to corpses or skeletons, and samples are most frequently found in bones and teeth. Both types of samples protect DNA from degradation and biological processes due to their physical and chemical robustness. However, accessing the DNA is not as easy as in other tissues due to these protective characteristics. Bone tissue consists primarily of proteins (mainly collagen and osteocalcin) and minerals. Approximately 70% of the mineral component of bone comprises hydroxyapatite, which includes calcium phosphate, calcium carbonate, calcium uoride, calcium hydroxide, and citrate. The DNA in bones is located in the osteoblasts, osteocytes, and osteoclasts. Osteons are the functional unit of bones and include osteocytes (located in spaces within the dense bone matrix called lacunae) and haversian canals, which contain blood vessels and nerves and are formed by concentric layers called lamellae. This structure favors the deposit and storage of mineral salts, which gives bone tissue its strength. Osteoblasts produce the organic components of the bone matrix and are situated at the surface of the bone matrix. Osteoclasts are responsible for bone remodeling and resorption during bone growth and are located on the surface of the bone matrix (Mescher 2018b) (Fig. 1). Teeth consist of dentin, a calcified material harder than bone that forms a large part of the structural axis of the tooth and surrounds the internal pulp cavity. Dentin consists of 70% hydroxyapatite, type I collagen, and proteoglycans. Dentin in the dental crown is covered by enamel, an extremely mineralized, hard, acellular, avascular tissue. Enamel is the hardest component of the human body and consists of 96% calcium hydroxyapatite, very few proteins, and no collagen. The dentin at the tooth root is covered by cementum, another type of calcified connective tissue that resembles bone. The soft tissue in the dental pulp is highly vascular and innervated and consists of odontoblasts, fibroblasts, endothelial cells, peripheral nerves, undifferentiated mesenchymal cells, and other nucleated blood components, making it rich in DNA (Muruganandhan and Sivakumar 2011; Mescher 2018a). DNA is recovered from the tooth pulp, where it is abundant and unlikely to be contaminated by nonhuman DNA (Girish et al. 2010) (Fig. 2). Bone and tooth tissues are compact and hard structures that preserve the DNA in their matrix. Isolating DNA from bones and teeth therefore requires several pretreatment steps before the DNA can be recovered from these cells.

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Fig. 1 Bone matrix. (M) Mesenchymal regions, (Ob) osteoblasts, (Oc) osteocytes, and (Ocl) osteoclasts (Mescher 2018b)

Fig. 2 Tooth formation. (A) Ameloblasts, (B) bone, (D) dentin, (DP) dental papila, (E) enamel, (PD) predentin, (O) odontoblasts, and (OEE) outer enamel epithelium (Mescher 2018a)

There are three main problems to solve at this stage: the introduction of modern DNA, the presence of too few DNA molecules to serve as templates for polymerase chain reaction (PCR) testing, and the co-purification of inhibitory substances that result in false negatives. The isolation of ancient DNA therefore has three requirements: 1) the samples have to be pretreated to reduce contamination, 2) extraction techniques that do not damage the DNA need to be employed, and 3) these techniques need to have a high purification power to reduce or eliminate the presence of inhibitors.

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Sample Pretreatment Decontamination Bone and tooth samples are typically obtained from excavations and are often improperly and carelessly handled. There is, however, a growing awareness among archaeologists and anthropologists regarding the importance of wearing protective clothes when working with ancient DNA samples. Conducting a pretreatment stage prior to DNA isolation is extremely important for eliminating any possible contamination by exogenous DNA and the remains of putrilage and impurities. There are several strategies for reducing or eliminating the possible superficial contamination of bones and teeth such as surface washing, acid washing, highly concentrated ethanol washing, bleach washing, hydrogen peroxide washing, milling and grinding, ultraviolet irradiation, and sampling of the inner part of the compact bone and combined techniques (Kemp and Smith 2005).

Surface Washing Vigorous surface washing can remove the external layer of exogenous material from the bone (Holland et al. 1993) and involves using sterile water prior to cutting (Merriwether et al. 1994) or repeated rinsing of the cut pieces in distilled water, with a final air dry step (Alonso et al. 2001). However, humidity has been widely reported as a factor in damaging DNA because it facilitates mineral dissolution and increases hydrolytic damage. Moreover, the interdependence between the organic and mineral components of bone supports the hypothesis of bone susceptibility to chemical and biological effects due to the increase in porosity (Emmons et al. 2020). Sodium Hypochlorite Washing For forensic and ancient DNA samples, one of the most common methods for eliminating exogenous DNA from bone and teeth is washing the surface or even the powdered bone or tooth with bleach. Although washing significantly reduces exogenous human DNA, it also results in a loss of endogenous DNA (Dabney and Meyer 2019). Sodium hypochlorite, an active component of bleach, rapidly attacks nucleic acids in a nonspecific manner, degrading purine and pyrimidine bases through oxidation reactions such as chlorination (Hayatsu et al. 1971). Given the destructive nature of bleach, the possibility of replacing it with other compounds such as phosphate buffer has been explored. The use of phosphate buffer is based on the competition between free phosphate ions and DNA phosphate groups attached to hydroxyapatite. Phosphate buffer has been shown to be less aggressive toward endogenous DNA than bleach washing and, although it can eliminate some microbial DNA, it is less effective with exogenous DNA (Dabney and Meyer 2019). Ethanol Washing An alternative to bleach washing is the cleaning of bone and teeth surfaces with sterile cotton previously moistened with a 95% (Fisher et al. 1993) or 70% ethanol solution (Stone and Stoneking 1998) in ultrapure water. Although ethanol exerts no degradative

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activity on DNA, its use for precipitating DNA is well known, and its usefulness for cleaning surfaces in forensic laboratories has long been accepted (Kampmann et al. 2017).

Acid Washing Similar to bleach and ethanol washing, the use of weak acids to remove exogenous DNA from samples is also common, as 30% acetic acid diluted in ultrapure water (Montiel et al. 2001). This technique is based on the power of denaturation of a low pH solution hydrolyzing the glycosidic and phosphodiester bonds of DNA. However, the effectiveness of this solution is lower if the DNA is rich in guanine and cytosine (Shapiro et al. 1978). Hydrogen Peroxide Washing Immersing human remains in a 3% (Ginther et al. 1992) or 3–30% hydrogen peroxide solution for 10–30 min (Merriwether et al. 1994) has also been shown to be an effective method for removing the exogenous component of samples. The decomposition of hydrogen peroxide into water and free oxygen radicals causes oxidative damage to DNA by radical-ionic mechanisms (Mouret et al. 1991). Enzymatic Predigestion One of the main problems with chemical decontamination methods is that they also attack the sample’s endogenous DNA. A less aggressive method is the predigestion of the samples with ethylenediaminetetraacetic acid (EDTA) lysis buffer and proteinase K, which significantly reduces the contaminating DNA without affecting the sample’s endogenous genetic material (Schroeder et al. 2019). Milling and Sanding The pretreatment technique of milling and sanding the surface of human remains consists of applying mechanical abrasion to their outermost part to eliminate the adhering exogeneous material. There are various approaches with these techniques such as air abrasion with 100 μm aluminium oxide particles (Richards and Sykes 1995), the use of sandpaper discs (Kalmár et al. 2000), scraping with a sterile scalpel (Lalueza-Fox et al. 2001), and the increasingly widespread use of precision rotary tools such as those manufactured by Dremel ® (Gaudio et al. 2019). The main problem with milling and sanding pretreatment is the formation of bone dust, which can contaminate the working surface, tools, other samples, and even the operator. The Laboratory of Genetic Identification of the University of Granada (Spain) developed a milling, sanding, and cutting methacrylate enclosure with a removable lid and two lateral holes to insert the operator’s hands and the milling/ sanding tool. The bottom of the enclosure is covered with filter paper, and the entire milling and sanding operation is conducted in a fume hood (Álvarez et al. 2001). The enclosure not only prevents the clogging of the filters of the hoods or cabinets in which the operation is performed but also facilitates the cleaning of the equipment. The external and internal surfaces of bones can be milled and grinded, and the bone can then be cut into fragments, being 0.5–1 cm2 fragments recommended (Fig. 3).

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Fig. 3 Methacrylate enclosure

Inner Part of the Compact Bone Sampling To minimize the risk of contamination with modern DNA, a 2
2 cm section of compact bone from long bones, such as the posterior femoral diaphysis, can be cut, followed by scraping of the inner and outer surfaces with a scalpel (Palmirotta et al. 1997), leaving only the innermost part of the compact bone, which might be free of or less exposed to exogenous DNA. Ultraviolet Irradiation Short-wave ultraviolet (UV) light (254 nm) induces the covalent bonding of thymine bases, preventing denaturalization of DNA double strands during PCR, making it inaccessible to polymerase during amplification. Therefore, exposing the surface of the samples to UV light for a few minutes is useful for eliminating exogenous DNA (Latham and Miller 2019). Variations in the UV exposure time, ranging from 10 min to up to 2 days (Carlyle et al. 2000; Kalmár et al. 2000; Matheson and Loy 2001), have been described and are sufficient to affect exogenous DNA but not endogenous DNA. The distance between the irradiation source and the irradiated surface is also important: the closer the source is to surface, the greater the irradiation power (Champlot et al. 2010; Hall et al. 2014). However, 10 min for both sides of the sample is the most frequently employed exposure time. Combination of Techniques Many laboratories combine two or more of the previous decontamination protocols to reduce potential exogenous contamination. The Laboratory of Genetic Identification of the University of Granada first mills, sands, and cuts the bone samples into pieces measuring approximately 0.5 cm2 and then irradiates them with UV for 10 min on both sides before pulverizing the samples.

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Pulverization After decontaminating the sample, each laboratory employs its own extraction method, processing the bone and tooth samples in small pieces or grinding them into powder through various processes.

Manual Mortar Manual mortars and pestles have typically been employed to grind bones and teeth. Numerous laboratories still use Te on pestles in ceramic mortars to generate the fine bone powder needed for extractions (Cafiero et al. 2019). Freezer Mill Freezer/Mill ® cryogenic grinders are widely used in laboratories to grind samples such as teeth, bones, and other animal and human tissues. Samples are placed in a sealed cryogenic grinding vial in the grinder and then immersed in liquid nitrogen. The samples are cooled to cryogenic temperatures and then pulverized by magnetically shuttling a steel impactor back and forth against two stationary end plugs (Fig. 4). TissueLyser To grind and disintegrate the tissues, a TissueLyser II system is recommended. The TissueLyser II grinds bone and teeth samples by shaking them with metallic balls without requiring liquid nitrogen. However, liquid nitrogen can be used with this technique to prevent the samples from heating, thereby facilitating the grinding (Fig. 5).

Fig. 4 Grinding vials and Freezer/Mill ®

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Fig. 5 Grinding vials and TissueLyser II sytem

DNA Isolation DNA isolation is the most important stage in the DNA analysis process, because it will determine the outcome of the entire process. If there is insufficient starting DNA, amplification will fail, yielding no results. Therefore, the key is to obtain as much DNA as possible. Molecules of genetic material have to be isolated from other cell components before the genetic material can be analyzed, because the cell proteins that package and protect the DNA can inhibit the analysis (Butler 2005). The other major problem is the presence of inhibitors that need be eliminated or minimized (Barrio-Caballero 2012), because they either inactivate DNA polymerase or compete with other components of the DNA synthesis reaction. The presence of extrinsic substances from bone such as humic and fulvic acids from the soil and intrinsic substances such as calcium have to been eliminated (Eilbert and Foran 2009).

Manual DNA Isolation Total Demineralization The total demineralization method was first developed in 1991 with well-preserved animal and human bones from archaeological sites. The method employs 0.5 M EDTA, proteinase K, and N-lauroylsarcosine at 37  C for 18–24 h, followed by an extraction with phenol-chloroform. Bone decalcification is made possible by incubating cut samples with EDTA for 72 h, despite this approach reducing the total DNA yield (Hagelberg and Clegg 1991). The basis for this method is the chelating activity of EDTA, which binds to iron and calcium ions. The problem of reduced DNA yield is solved by making EDTA part of the lysis buffer, so that DNA can be purified with phenol-chloroform-isoamyl alcohol in a 25:24:1 proportion after an overnight lysis. The resultant is then concentrated with centrifugal filter units (Edson et al. 2004). A hybrid protocol of these two methods can be used by incubating

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0.6–1.2 g of bone powder with 15 mL 0.5 M EDTA, 1% lauroyl-sarcosinate, and 20 mg/μL of proteinase K in a rotatory shaker overnight. The bone powder is thereby completely dissolved. Organic extraction with phenol-chloroform, filtration with centrifugal filter units, and two washes with ultrapure water are then performed (Loreille et al. 2007).

Organic Extraction The well-known phenol-chloroform-isoamyl alcohol method, also known as organic extraction, was developed in 1991. Using this approach, 5 g of bone are powdered and decalcified with 0.5 M EDTA for 3–5 days, washed three times with ultrapure water, lysed with proteinase K and extraction buffer for 2 h, extracted with phenolchloroform-isoamyl three times, and concentrated and purified in a Centricon centrifugal filter (Hochmeister et al. 1991). The mixture is typically stabilized with 10 mM of Tris-EDTA, the proteins are unfolded with phenol, empowered augmented by chloroform. The chloroform also denatures lipids, while the isoamyl alcohol stabilizes the interphase and increases DNA purity. DNA will be trapped in the upper aqueous phase. Modified protocols introduce sodium dodecyl sulphate (SDS) and dithiothreitol (DTT) into the lysis buffer (Ferreira et al. 2013). SDS is an anionic detergent that linearizes the proteins present in chromatin, while DTT is a reducing agent that reduces the disulphide bonds present in proteins. Although the phenol-chloroform method yields a large amount of DNA, the main issue is that it is a dangerous reagent, both for the analyst and the environment. The solution should therefore be used in a fume hood and its residues properly treated and disposed of. The method is also time-consuming and requires significant handson time. Chelex ® Resin In 1998, a simple, chelating, single-tube, resin-based procedure, using minimal steps was suggested. The Chelex ®-100 (Bio-Rad Laboratories, Hercules, CA) is a chelating resin composed of styrene divinylbenzene copolymers and iminodiacetate ions that bind to polyvalent metal ions. In the basic procedure, samples are boiled in a 5% Chelex ®-100 suspension (Willard et al. 1998). A prior 30 min incubation at 56  C is recommended for bone samples. An adapted protocol for ancient bone samples starts with a 3 h incubation at 56  C of 100 mg of bone powder in Chelex ®-100, followed by a 20 min boiling period (Coulson-Thomas et al. 2015). To yield more DNA, proteinase K can be added prior to incubation (Tsuchimochi et al. 2002). Despite being fast and environmentally friendly, Chelex ® cannot remove PCR inhibitors. Manual Purification of DNA Extracts To maximize the chances of success, DNA extraction protocols need to obtain the largest amount of target DNA possible while reducing or even eliminating the presence of PCR inhibitors. To this end, there are two classical methods for analyzing ancient DNA (Yang et al. 1998): centrifugal filter units and silica particle

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columns. The first method uses Centri-con™ filters (Hagelberg and Clegg 1991) that consist of an anisotropic membrane that retains macrosolutes, such as DNA, while letting low-molecular-weight compounds pass through, which can also occur with PCR inhibitors. Other protocols include further washing with approximately 1 mL of distilled water or 2 mL of TE buffer with 0.01 M Tris and 0.001 M EDTA at a pH of 7.5 (Hochmeister et al. 1991). The second method uses silica particles (Höss and Pääbo 1993) with a high binding capacity for DNA molecules and are therefore retained while the inhibitors are washed out; however, the silica particles are themselves potential PCR inhibitors. The silica pellets are therefore washed twice with a 10 M guanidine thiocyanate and 0.1 M Tris-HCl buffer at a pH of 6.4, washed twice with 70% ethanol, and washed once with acetone. Another approach is to precipitate out any material that is nonnucleic by adding saturated sodium acetate (a process known as salting-out [Cattaneo et al. 1995]), adding 1 mL of the solution to the tube, shaking it manually for 30 s and centrifuging it for 10 min at 4000 g. Several DNA purification commercial kits are available, such us DNA IQ™ System purification (Promega, MA, USA), or QIAquick™ PCR purification kit (QIAGEN, Hilde, Germany) (Ye et al. 2004).

DNA Isolation with Commercial Kits There are currently numerous commercial isolation kits for bone DNA analysis (see Table 1), although most require mechanical pretreatment. Most of these kits can be automated with the appropriate equipment, which offers several advantages such as maintained sample integrity, increased reproducibility, constant performance, greater throughput, work ow integration, electronic audits, compatibility with laboratory information management systems (LIMS), sample switching and data entry error minimization, reduced hands-on time, and lower repetitive stress injuries (Lee and Shewale 2017). In this section we will describe some of these advantages. Table 1 Commercial kits for bone DNA analysis Kit PrepFiler™ BTA (ThermoFisher) Bone DNA Extraction (Promega) QIAamp DNA Investigator (Qiagen) EZ1 Investigator (Qiagen) Cells and Tissue DNA Isolation (Norgen Biotek) CrimePrep (Ademtech)

Format 100 reactions 100 reactions 50 reactions 48 reactions 50 reactions 96/48 reactions

Lysis time ~2 h

Protocol time ~2–3 h ~2 h

Automatization AutoMate™ Express Maxwell ®

~24–48 h

~30-60 min ~20 min

QIAcube Connect EZ1

~2 h

~60 min

–

~2 h

~60 min

Automag

~2,5 h + ~2,5 h Overnight

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PrepFiler ™ BTA Forensic DNA Extraction Kit PrepFiler™ BTA Forensic DNA Extraction Kit (Thermo Fisher Scientific Inc., MA, USA) was developed for isolating DNA from bone, teeth, and other forensic samples with adhesives (cigarette butts, envelope aps, tape lifts, and chewing gum). The extraction kit uses a format that provides for 100 reactions, and the protocol can be performed in approximately 2–3 h, which reduces the processing time by requiring a shorter lysis time than standard methods. The use of phenol-chloroform with this kit is not necessary. To release DNA from calcified tissues, the kit employs a sequence of washes, with various buffers and filter columns. DNA isolation is performed with a magnetic bead. The protocol is divided into four parts: lysis (PrepFiler ® BTA lysis buffer and DTT), DNA binding (PrepFiler ® magnetic beads), purification (PrepFiler ® wash buffer), and DNA reconstitution (PrepFiler ® BTA lysis buffer). The PrepFiler ® lysis buffer is composed of a thiocyanic acid compound with guanidine (1:1), while the PrepFiler ® BTA lysis buffer is based on sodium hydroxide. There are several variations to the method according to the samples’ complexity, ranging from increasing the quantity of powdered sample (and thus the volume of lysis reagents) to extending the lysis time to overnight. The elution volume can also be customized to concentrate the DNA extract. There is an automated option for these kits: the AutoMate Express™ (Thermo Fisher Scientific Inc., MA, USA), which is based on the above protocol. First, lysis is performed in a thermoshaker and then the lysate is automatically purified. The AutoMate Express™ uses prefilled buffer cartridges that reduce the handling of samples, thereby reducing potential contamination by the operator (Applied Biosystems 2012). Bone DNA Extraction Kit The Bone DNA Extraction Kit (Promega Corporation, WI, USA) is actually the joining of two protocols: a preprocessing protocol and a subsequent purification protocol. The kit was developed as a combination of classical purification protocols, created by various genetic identification laboratories, and uses a demineralization buffer (0.5 M EDTA, pH 8.0, 1% lauroylsarcosine) and an organic extraction protocol (proteinase K and 1-thioglycerol) to effectively and efficiently extract DNA from the calcium matrix. The first step can be performed using manual or automated Promega methods (using the Maxwell ® extraction instrument), using DNA IQ™ for DNA purification. The kit’s format provides for 100 reactions, and the estimated time to completion is more than 7 h due to the demineralization and subsequent digestion requiring an incubation time of 2.5 h each. Performing an extraction within a single working day is therefore problematic (Promega 2019). QIAamp DNA Investigator Kit As with the Promega protocols, the Qiagen protocol (Qiagen N.V. Hilden, Germany) has two differentiated phases: a pretreatment protocol for bones and teeth (which describes the decalcification and lysis using EDTA and proteinase K) and a subsequent DNA purification using MinElute spin columns (QIAamp DNA Investigator Kit) to obtain purified genomic DNA. The success of this purification phase depends

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on the combination of the selective binding properties of a silica-based membrane. The DNA purification can be automated using QIAcube Connect, an instrument widely used in genetic identification laboratories to fully automate the purification of nucleic acids and proteins.

EZ1 DNA Investigator Kit Qiagen developed the EZ1 DNA Investigator kit, which uses a similar DNA extraction protocol for powdered bone and tooth to that of the PrepFilerTM BTA Forensic DNA Extraction Kit. The Qiagen protocol is based on a lysis phase performed manually and an automated purification phase in the EZ1 Advanced automated sample preparation system. The lysis requires a decalcification step with 0.5 M EDTA (not included in the kit) for 24–48 h and digestion with proteinase K for 3 h. The lysate is then divided into various aliquots, and buffer MTL is added to load the sample into the device. The protocol supports three different quantities of powered bone or tooth, which can vary the volume of reagents and the protocol on the device. The automated system uses prefilled buffer cartridges, which reduces the handling of samples and thus potential contamination by the operator (QIAGEN 2013). Cells and Tissue DNA Isolation Kit The Cells and Tissue DNA Isolation Kit (Norgen Biotek Corp., Ontario, Canada) employs a protocol that purifies DNA from various tissue types using a magnetic bead system. The manufacturer recommends a decalcification step prior to isolating genomic DNA to improve the efficiency of the DNA recovery. To perform decalcification, the bone or teeth are crushed, incubated with EDTA at 4  C for 24 h, and centrifuged several times. The supernatant is then removed, and 20 mg of the sample is used as the substrate to perform the kit’s protocol. The kit’s format provides for 50 reactions, and the estimated hands-on time is 1 h and more than 24 h for the incubations (Norgen Biotek Corp 2015). Crime Prep Adem-Kits for Casework Ademtech (Pessac, France) commercial kits are based on calibrated particles with high magnetic content and controlled surfaces, specially designed for molecular biology. The Crime Prep protocol starts with 100 mg of bone powder and a 2h lysis, followed by binding, washing, drying, and eluting. Alternative protocols employ an overnight lysis. Crime Prep comes in a 96-sample format and an estimated hands-on time of 1 h plus 2 h of lysis. The process can also be automated with an Automag device for 48 samples with a reduced time cost (Ademtech 2019). Incorporation of Manual Pretreatment Protocols to Commercial Kits Over the years, various DNA extraction protocols have been developed to extract DNA from skeletal remains by incorporating manual pretreatment to the commercial kits. This section discusses the protocols capable of extracting DNA without requiring that the samples be pulverized. In 2007, a new DNA extraction procedure that did not require pulverization of samples was described (Kitayama et al. 2010). In their article, the authors presented a new experimental kit that combined a

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conventional phenol-chloroform DNA extraction procedure with the QIAamp DNA Mini Kit for DNA isolation kit. In this protocol, mechanical grinding was replaced with gentle stirring during overnight incubation. The results were inconclusive due to the low number of samples and differences in the quality of the extracted DNA with respect to grinding protocols. However, there is certain value in exploring protocols that do not require the pulverization of samples. In 2020, De Donno et al. described a DNA isolation from a saponified sternum from a limbless human body recovered at sea. The authors extracted DNA using a Macherey-Nagel kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany), modifying the usual procedure described for NucleoSpin® DNA Trace Kit 2. The authors made numerous modifications to the original protocol, halving the quantity of bone material, volumes of reactant, proteinase K, and lysis buffer (B3 buffer, included in the NucleoSpin DNA Trace bones buffer set). The authors also changed the number and type of columns used for binding DNA to the silica membrane (Piglionica et al. 2012; De Donno et al. 2020). Cartozzo et al. described a similar DNA isolation method for waterlogged bones. The extraction was based on an organic isolation method followed by the use of a Thermo Fisher kit. The extraction began with digestion with proteinase K, purification with phenol-chloroform-isoamyl alcohol and subsequent drying of the aqueous phase using a Speed Vac Concentrator. The dried pellet was reconstituted with deionized water. This eluate was the substrate for starting the DNA extraction using the ChargeSwitch ® gDNA Plant Kit (Thermo Fisher Scientific Inc., MA, USA). The protocol was performed according to the manufacturer’s procedures (Pagan et al. 2012; Cartozzo et al. 2018). After the experiments, the authors concluded that the magnetic bead technology of the ChargeSwitch ® gDNA Plant Kit might be the most efficient method for recovering DNA from waterlogged bones, a surprising statement after using a kit recommended for fungi and plants. In any case, each bone presents its own set of challenges, requiring manual procedures and commercial kits to be adapted to ensure the success of the DNA extraction. Embalmed bones, for example, not only involve issues with extracting DNA from bones but also bring to the table the exposure to various compounds such as glutaraldehyde and formalin, which can induce molecular cross-linking. In these cases, modifying the existing grinding techniques and combining them with decalcification buffers, phenol-chloroform treatment, and commercial kits will produce efficient methods for extracting sufficient high-quality DNA (Gielda and Rigg 2017).

Conclusions In conclusion, this review shows how to overcome the drawbacks of isolating DNA from mineralized tissues, in order to identify them through forensic genetics. Bone and tooth tissue consist primarily of proteins and minerals, which are a major inconvenience in the laboratory; however, these tissues protect against degradation of the large DNA molecules. Several techniques aimed at preventing the introduction

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of exogenous DNA into the study samples have been reported. These techniques reduce or prevent the loss of the scarce DNA molecules and also reduce or eliminate the co-purification of inhibitory substances. The complications caused by the time elapsed between death and the laboratory procedures are increased by the characteristics of where and how the body was found. The success of DNA extraction and isolation is affected by variables such as relative humidity, temperature, UV light exposure, and microbiome (amount and type of microorganisms). These factors significantly affect the degradation of the cadavers and their skeletal remains. These variables that increase degradation and alter the mineral concentration make each bone an enigma. There is therefore no single solution for extracting DNA from bones. Although there are many valid solutions, several of which have been covered in this chapter, there is no ideal protocol for extracting DNA from bones and teeth, as this will depend on the circumstances surrounding each sample. The most advisable strategy is to use more than one extraction method. There are protocols that eliminate all contaminants and inhibitors from the sample. Due to purification, however, there is an excess of DNA loss, and the final concentration obtained is therefore low. Other methods that obtain more DNA can contain mineral remains or DNA from other organisms. As described in this chapter, the most effective approach is to use different protocols depending on the origin of each bone or, as numerous authors have done, combine stages from different procedures. Nevertheless, this approach should only be taken with a deep understanding of each step and the reactions in each stage. This is the only way to successfully obtain extracted DNA without an excessive number of attempts. To ensure success, certain tests should be performed before extracting DNA from bones belonging to the same mass grave, same burial type, or similar types of catastrophes. If the skeleton is highly valuable and its identification is imperative, it is highly recommended that the sample be fragmented into 1- to 2-g pieces, so that more than one test can be performed. Problems occurring during the first DNA extraction can thereby be solved in subsequent extractions. Lastly, the use of commercial kits for the last stages of DNA extraction or for the purification of the isolated DNA is recommended. This approach is very common in laboratories and has been described by numerous authors in the literature. These kits become highly recommended due to their capacity for preventing PCR inhibition and obtaining genetic profiles, which is the ultimate goal in identifying victims.

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Christian Haarko¨tter, M. J. Alvarez-Cubero, Juan Carlos Alvarez, and María Saiz

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA Quantification Importance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA Quantification Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Quantitative PCR (qPCR) Fundamentals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Detection Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Calibration Curve and DNA Quantity Calculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Quantitative PCR Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . qPCR Customization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Current Applications of qPCR in Forensic Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Autosomal DNA Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X and Y Chromosome Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondrial DNA Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ancient DNA Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Non-human DNA Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Single-Nucleotide Polymorphisms (SNPs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Issues Associated with qPCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future Perspectives for qPCR in Forensic Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Digital PCR (dPCR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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C. Haarkötter · J. C. Alvarez · M. Saiz Laboratory of Genetic Identification, Department of Legal Medicine, Toxicology and Physical Anthropology, Faculty of Medicine, University of Granada, Granada, Spain e-mail: [email protected]; [email protected]; [email protected] M. J. Alvarez-Cubero (*) Department of Biochemistry and Molecular Biology III, Faculty of Medicine, University of Granada, Granada, Spain Pfizer-University of Granada-Junta de Andalucía Centre for Genomics and Oncological Research (GENYO), Granada, Spain e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 H. R. Dash et al. (eds.), Handbook of DNA Profiling, https://doi.org/10.1007/978-981-16-4318-7_39

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Abstract

Although most DNA studies in forensic genetics are focused on the sample, including DNA extraction and amplification, sample quantification is an important step that is required to determine the optimal DNA input for amplification. In addition, the effective quantification of DNA can provide information regarding the degradation and inhibition of DNA to optimize the amplification strategy or the extraction method and can be used to inform the decision to analyze another sample of the same specimen. In this chapter, quantitative PCR (qPCR, also known as realtime PCR, RT-PCR) will be described after a brief history of quantification methods, PCR fundamentals, current applications in forensic genetics [including the sequencing of short-tandem repeats (STRs), single-nucleotide polymorphisms (SNPs), Y-chromosomes, mitochondrial DNA (mtDNA), and non-human DNA], and future perspectives for this technique. Keywords

DNA analysis · DNA quantification · Forensic genetics · Quantitative PCR · Real-time PCR (RT-PCR)

Introduction Due to the emergence of direct amplification techniques, for which DNA extraction represents an optional step, DNA quantification has become an alternative technique for some forensic cases. However, in some cases, DNA quantification continues to represent a necessary step for the optimization of DNA concentrations required for successful amplification processes. Quantification not only provides information regarding DNA concentration but by applying quantitative polymerase chain reaction (qPCR), during which DNA amplification is monitored during each cycle, DNA degradation, sex determination, the presence of multiple contributors from different sexes, and the presence of PCR inhibitors can be assessed. All of this information that can be obtained from DNA quantification can assist investigators during the following sample analysis steps. qPCR offers several advantages to the field of forensic genetics, including cost and time saving for laboratories, providing the information necessary to optimize the entire analytical process, including information regarding DNA quantity and degradation. In this chapter, the qPCR fundamentals will be discussed, including a review of the history of DNA quantification and an exploration of the current applications for qPCR in the field of forensic genetics, such as autosomal and Y-chromosome quantification, degradation assessment, mitochondrial DNA (mtDNA) analysis, non-human DNA analysis, ancient DNA (aDNA) analysis, and SNP analysis applications, ending with future perspectives for this technique.

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DNA Quantification DNA Quantification Importance DNA quantification continues to be a mandatory process in most international recommendations, including the Federal Bureau Investigation Quality Assurance Standards, in which Standard 9.4 states that every forensic sample must be quantified before amplification unless the laboratory features a validated system for direct amplification and typing, which is a characteristic of some direct amplification kits. This standard is also included in the Scientific Working Group on DNA Analysis Methods Interpretation Guidelines for Autosomal STR Typing by Forensic DNA Testing Laboratories. DNA quantification is necessary for two reasons (Butler 2011). First, the majority of available short-tandem repeat (STR) commercial kits requires a narrow range of DNA input, typically 0.5–1 ng of DNA, a range that can be even lower for the current generation of higher-resolution genetic analyzers. The use of insufficient DNA may result in partial profiles, allelic dropout, allelic drop-in, or null profiles, whereas the use of too much DNA may cause artefacts in electropherograms, such as pull-up spikes, although most modern genetic analyzers have bioinformatic algorithms designed to mitigate the effects of large DNA quantities. Second, quantification saves time and money, as the quantification results can be used to reorient the laboratory work ow to reconsider the planned extraction or purification methods or to attempt the purification and extraction of another sample if one is available. Finally, DNA quantification is crucial when working with compromised samples, such as aDNA, DNA from bones, or potentially mixtures from multiple sources (Carracedo 2005).

DNA Quantification Methods Forensic DNA quantification has passed through two different stages: initially, forensic analysts quantified the total amount of DNA content in an extracted sample; however, this approach was not specific to human DNA (Nicklas and Buel 2003a). Therefore, additional methods have been developed to improve specificity (Lee et al. 2015): 1. Total genomic methods: These methods measure the total amount of DNA, including both human and non-human DNA, which is found in the sample. a) UV spectrophotometry: Ultraviolet (UV) spectrophotometry is the most common method used to perform DNA quantification, based on the correlation between nucleotide concentrations and absorption at 260 nm, which allows the A260/A280 ratio to serve as a measure of DNA purity. Today, compact UV spectrophotometers, with the Nanodrop™ Spectrophotometer representing the most common device, can quantify a 1-μl sample without requiring a cuvette (Joseph 2010).

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b) Fluorescent assays: During this method, a uorochrome selectively binds to double-stranded DNA, and the uorescence can be measured and compared against a standard curve made from DNA samples with known concentrations. This is a very stable method that allows assay exibility, detecting approximately at least 250 pg of DNA (Ahn et al. 1996). c) Gel-based quantification: Samples are loaded onto a 0.8% agarose gel in TBE (Tris-Borate-EDTA buffer), and an electric field is applied, resulting in DNA migration toward the positive pole. Ethidium bromide or other uorochromes, such as GelRed ® (which is less toxic), can then be used to visualize the DNA under UV light (Alonso 2012). This method is semiquantitative and used to determine the presence of total genomic DNA. This method has an approximately 1-ng resolution and can be used to assess the degradation state based on the appearance of a DNA smear line. 2. Human- and higher primate-specific methods. a) Slot blot: DNA is immobilized on a nylon membrane, and a biotinylated oligonucleotide probe (D17Z1, which is specific for humans and primates) hybridizes to the DNA, and they both conjugate to a peroxidase. Peroxidase then reduces hydrogen peroxide in a chemiluminescent detection reagent, resulting in the oxidation of luminol and the emission of photons that can be detected using autoradiography film. The DNA concentration can be calculated by comparing the sizes and densities of the dots produced on the film against a dilution series containing standard DNA with known concentrations which makes this method capable of detecting as little as 150 pg of DNA (Walsh et al. 1992). b) AluQuant: The AluQuant™ system was designed by Promega, based on 300-bp DNA fragments that are found in humans and primates, referred to as Alu elements because Arthrobacter luteus was used in the initial characterization. When the probe hybridizes to the denatured DNA in a sample, a pyrophosphorylation reaction is initiated, which liberates a deoxynucleotide that becomes transformed into adenosine triphosphate through the activity of a kinase. The adenosine triphosphate then activates luciferin, which produces measurable light. This system is capable of detecting DNA levels as low as 100 pg (Mandrekar et al. 2001).

Quantitative PCR (qPCR) Fundamentals Detection Methods TaqMan Probes PCR represented a breakthrough development when it was first invented, and its utility increased further with the introduction of Thermophilus aquaticus usage, which allowed the process to be optimized for specificity. One property of Taq polymerase is its 50 ➝30 exonuclease activity, through which the polymerase degrades any oligonucleotide that has hybridized between the two forward and

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Fig. 1 TaqMan probe diagram (Holland et al. 1991)

reverse primers (Holland et al. 1991). This functionality of the TaqMan probe represents the cornerstone of quantitative PCR (Fig. 1). TaqMan probes are 18– 30 bp oligonucleotides designed to feature melting temperatures near the optimal extension temperature of the DNA molecule of interest. Given the short length of probes, minor groove binder molecules are often added to increase the melting temperature of the probe, allowing shorter probes to be used (Kutyavin et al. 2000). TaqMan probes are bound to two different dyes that transfer energy from one to the other when they are physically close: the reporter (R) dye is typically attached to the 50 -end of the probe, whereas the quencher (Q) dye is attached to the 30 -end of the TaqMan probe. When the DNA polymerase initiates synthesis between the two primers bound to the DNA template, the 50 ➝30 exonuclease activity hydrolyses the probe, liberating both the reporter and the quencher, which frees the reporter from the in uence of the quenching dye, allowing the reporter to emit uorescence that can be measured when it is excited by a certain wavelength of light. Table 1 shows the most common reporters and quenchers that are used today.

SYBR ® Green qPCR Assays SYBR Green I is a uorescent dye that binds only to double-stranded DNA (see Fig. 2) and emits uorescence proportionally to the amount of bound doublestranded DNA. In a typical real-time PCR reaction, the starting level of input DNA is small; therefore, the only double-stranded DNA that can be detected represents the PCR product. During the exponential phase of the PCR, SYBR ® Green uorescence doubles during each cycle until it reaches the plateau phase; the initial amount of DNA can be calculated according to the difference between the uorescence cycle threshold of the sample relative to the cycle threshold of a standard (Ponchel 2006). SYBR ® Green is an asymmetric cyanine that is commonly used during gel electrophoresis because it binds between the strands of double-stranded DNA. Therefore, qPCR assays require the inclusion of the DNA template, this dye, and a pair of amplification primers, which can result in the detection of as little as 1 pg of

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Table 1 Different reporters and their recommended quenchers (BioCat Real Time PCR Dyes (2009) Colour

Reporter Biosearch Blue™ FAM TET CAL Fluor ® Gold 540 JOE VIC HEX CAL Fluor Orange 560 Quasar ® 570 ABY ® Cy™ 3.5 ROX CAL Fluor red 610 JUN ® Pulsar ® 650 Cy 5 Quasar 670 Cy 5.5 Quasar 705

Alternative reporter –

DYE-50 -T EX EM 352 447

Recommended quencher BHQ-1

– – VIC/TET/JOE

495 521 522

520 536 544

BHQ-1 BHQ-1 BHQ-1

– – – VIC/HEX/JOE

529 538 535 538

555 554 556 559

BHQ-1 – BHQ-1 BHQ-1

CY3

548

566

BHQ-2

– – – TEXAS RED/ ROX/ ALEXA FLUOR ® 594 – –

– 581 586 590

580 596 610 610

– – BHQ-2 BHQ-2

– 460

617 650

– BHQ-2

CY5

646 647

669 670

CY5.5

675 690

694 705

– BHQ-2, BHQ-3 – BHQ-2, BHQ-3

Color

DNA (Nicklas and Buel 2003b). This alternative quantification method is advantageous due to the reduced need to handle the sample and represents a low-cost assay compared with the slot blot; however, some studies have suggested preferential binding to DNA with higher G + C% and larger-sized amplicons, which, combined with the non-specific binding to any double-stranded DNA, can represent problematic characteristics for forensic contexts (Giglio et al. 2003).

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Fig. 2 SYBR® Green binding model

However, this method is particularly advantageous because this quantification assay can be freely designed by any investigator, based on the choice of primer pairs, which can be used to quantify exact regions of interest. Several commercial kits are available, including SYBR™ Green PCR Master Mix (Thermo Fisher, Waltham, MA, USA), SYBR ® Green JumpStart™ (Sigma-Aldrich, St. Louis, Mo, USA), and QuantiTect ® SYBR ® Green PCR (Qiagen, Hilden, Deutschland). It is important to know if specific products have been obtained or whether dimers or unspecific products are present in qPCR when SYBR Green I is used. That is why it is crucial the presence of a melting curve. DNA melting allows the detection of small amounts of wrong qPCR products and can even detect single-nucleotide differences between amplified targets and copy number. Most of the commercial qPCR devices have a melting rate of 0.01–0.4  C/s. This range gives enough resolution to discriminate different qPCR products, taking around 5 min per plate the generation of a melting curve. However, the interpretation of this data is not automatized in any qPCR platform, and neither is available an application for automatically evaluating the DNA melting curve of different curves. Nevertheless, there are web applications available to predict melting curves of an amplicon and to identify differences between melting curves (Ruijter et al. 2019). Other methods, less specific and practical, imply agarose gel analysis which implies the visualization of the amplicons in an agarose gel.

Molecular Beacons Molecular beacons were designed to focus on the annealing specificity between two complementary DNA strands. This method uses specific single-stranded DNA probes that contain a loop with a complementary sequence and two complementary arms, forming a bulb-like shape (see Fig. 3). The stem is attached to both a uorescence molecule and a quencher, which prevents the emission of uorescence in its native form. When the molecular beacon finds its complementary sequence, it anneals to the sequence, forming a more stable complex than the complex formed by the annealing of the two complementary arms. The uorophore and the quencher become detached, allowing the uorophore to emit detectable uorescence (Tyagi and Kramer 1996).

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Fig. 3 Molecular beacon hybridization model

Although the primary disadvantage of molecular beacons is a requirement for large complementary sequences to bind (Hopkins and Woodson 2005), several forensic applications have been proposed for these molecules. In the same reaction, multiple targets can be detected using various molecular beacons attached to different uorophores for each target, limited only by the detection capabilities of the realtime PCR instrument. These characteristics can be useful for the high-throughput genotyping of SNPs (Sobrino et al. 2005) or body uid identifications using tissuespecific RNA species (Young et al. 2018).

Calibration Curve and DNA Quantity Calculation Every PCR assay consists of three different stages, based on how the reaction progresses: 1. Exponential growth: At the beginning, assuming a 100% PCR efficiency, the number of amplicons will be doubled during each cycle. 2. Linear growth: As the PCR assay consumes dNTPs, the primer reaction speed decreases, shifting to arithmetic growth rather than geometric growth. 3. Plateau phase: During this stage, the reagents become fully consumed, preventing any additional PCRs from being performed, and the number of amplicons remains stationary. These phases can be visualized if the detected uorescence is compared against the cycle number in a graphical representation, which results in a sigmoid function

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Fig. 4 Plot showing PCR uorescence vs cycles

(see Fig. 4). The best point to measure the uorescence is during the exponential growth phase because the number of PCR products in the next cycle, which should be double, or in the previous cycle, which should be half, can be predicted. If we define an arbitrary point in which the level of uorescence can be detected above baseline noise, larger DNA samples will become detectible after fewer cycles. The cycle number at which uorescence above background can be detected is referred to as the threshold cycle, CT (Heid et al. 1996). If we graphically represent the threshold cycle values for various samples with known concentrations according to the logarithm of the DNA quantity, a linear model can be obtained through linear regression, representing a calibration curve that can be used to calculate the DNA concentrations of the samples, as seen in Fig. 5. When performing a qPCR assay, determining the coefficient of determination, R squared, or R2 is very important because this value indicates the goodness of fit of a model by directly measuring how much of the variance observed in one of covariate can be attributed to another covariate. This value is calculated as the square of the correlation coefficient, which ranges from 0 to 1, with values that approach 1, indicating optimal goodness of fit for the calibration curve (Lucy 2005).

Quantitative PCR Instruments To detect uorescence during the performance of PCR assays, various real-time PCR systems have been developed, which are typically accompanied by specific software that can automatically calculate the linear regression, the coefficient, and the DNA quantity that is present in each sample.

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Fig. 5 Real-time PCR uorescence vs cycle plot; the values of at the CT threshold can then be plotted against the logarithmic value of the DNA concentration to generate a calibration curve

qPCR systems combine two pieces of equipment that are familiar to the forensic biologist: a thermocycler and a uorimeter. qPCR systems feature four major components (Shipley 2007): the light source, which determines the range of reporter dyes that can be used by the instrument; the detection system, which determines the spectral range and sensitivity of the; the thermocycling mechanism, which aims to rapidly reach and maintain specific temperatures; and the software, which controls the assay and performs the calculations. The thermocycler performs the PCR, the reporters become liberated, the light source applies light to the samples, the reporters emit light, and the light is detected by an active pixel sensor, typically a complementary metal-oxide-semiconductor (CMOS) sensor. As shown in Fig. 6, applying an excitation filter and an emission filter facilitates the performance of multiple qPCR assays utilizing different reporters simultaneously (see Fig. 6). In order to ensure an optimal performance of the PCR instruments, regular calibrations are required that vary according to the instrument. There are different types of calibrations: i) ROI calibration that allows the software used to locate the wells on the sample block so the increases in uorescent can be associated to a specific well on the plate. ii) Background calibration that measures the basal uorescent generated from background electrical signals, sample blocks, water, and consumables. This calibration is essential to eliminate the background signal, and it increases the instrument accuracy. iii) Dye spectral calibration that enables the instrument to distinguish the different uorescent dyes that will be used in the system. iv) Instrument verification run that verifies that the instrument can generate a standard curve and calculate the quantities of two known samples.

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Fig. 6 qPCR instrument diagram

qPCR Customization There are many commercial kits available to work in qPCR analyses, thanks to the versatility and specificity of the technique; multiple assays can be developed just with the design of the correct primers and probes. Some examples of customized assays developed in qPCR are the assessment of the degree of DNA degradation using primers that amplify fragments of different sizes (Heß et al. 2021); the determination of the degree of inhibitors of the PCR reaction present in the DNA extract; sex determination by the analysis of specific regions of Y and X chromosomes (Ginart et al. 2019). However, not only data from human DNA can be determined but also presence and quantity of fungicidal and bacterial DNA in a human DNA sample (Maggi et al. 2020), the determination of the different species in a crime scene by the analysis of the DNA of common wild animals present in the area, and the identification of the individuals by the determination of their microbiome on different samples (Baggesgaard Sterndorff et al. 2020).

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It is crucial an excellent primers and probes design to obtain the best results on the customized qPCR experiments, and there are some fundamental parameters to be considered: a) Primer Design • Primer melting temperatures: Both primers should have similar melting temperatures to assure that both primers bind simultaneously and amplify efficiently the DNA product. • Primer annealing temperature: It determines the capacity of primers to anneal to the DNA template. Optimal annealing temperatures will result in the highest product yield with the correct amplicon. • GC content: An ideal GC content for primers is 50% to allow complexity and maintain a unique sequence. b) qPCR Probe Design • Double-quenched probes are ideal to lower the background noise and increase the signal. • Location: Probes should be located close to any of both primers but not overlap the primer binding site. • Probe melting temperature: Probes should have a melting temperature 6–8  C higher than primers in order to not compromise the sensitivity of the assay. • Probe annealing temperature: Should be no more than 5  C below the lower primer melting temperature. • CG content: GC content should be 35–65% and avoid a G at the 50 end to not quench the 50 uorophore. c) Both Primer and Probe Design • Complementarity and secondary structure: Both primer and probe design should analyze the possible development of self-dimers, heterodimers, and hairpins. • Specificity: It is crucial to assure that selected primers are unique to the target sequence. d) Amplicons • PCR product size: PCR product size has to be 70–150 bp to guarantee the optimal design of primers and probes (Prediger 2013). There are several online tools to design and analyze custom design primers and probes: Primer 3 (Untergasser et al. 2012), Primer Blast (Ye et al. 2012), Primer Express Software v3.0.1 and Oligoperfect (ThermoFisher, Waltham, MA, USA), PrimerQuest ®, real-time PCR design tool, OligoAnalyzer™ Tool and UNAFold Tool (Integrated DNA Technologies, Coralville, IA, USA), and OligoArchitect and OligoEvaluator (Sigma Aldrich, Saint Louis, MO, USA).

Current Applications of qPCR in Forensic Genetics Currently, DNA quantification in forensic genetics serves two primary objectives: assessing the quantity, or the DNA amount, and assessing quality, or the degree of degradation, of the extracted DNA. Based on this information, the following steps

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associated with the DNA analysis can be modified: the amplification dilution factor can be modified, an initial purification step can be applied to the sample, the analysis protocol or sample handling procedures can be performed with improved efficiency, or other DNA extraction assays can be attempted using a different protocol or another sample if one is available. Therefore, the following five qPCR applications hold promise for the field of forensic genetics (Bunce et al. 2012): 1. Optimization of DNA extraction: Several DNA extraction protocols are currently available, including both automated and manual protocols. Understanding the amounts of extracted DNA that can be recovered by various protocols will determine the extraction protocol that should be applied to different types of sample. 2. Detection of PCR inhibition: qPCR assays introduce an internal PCR control (IPC), which represents an artificial oligonucleotide that cannot be found in nature; the absence of any IPC result might indicate the presence of a PCR inhibitor in the DNA extracts, which would require different strategies. 3. Assessment of DNA degradation: Two fragments are amplified during the qPCR reaction, one longer fragment of approximately 200–300 bp and a second shorter fragment of approximately 70–100 bp, to assess a degradation index – the ratio between the small and long target concentration. Amplification can be optimized according to this index. 4. Sex determination and the detection of DNA contaminants or mixtures: Some commercial kits include a human DNA target and a second sample of human male DNA, to allow for the determination of the sex associated with the DNA sample and to calculate the male to female ratio and indicate whether the DNA is mixed or contaminated. 5. NGS libraries: When using a next-generation sequencing (NGS) platform, a library for high-throughput sequencing must be built and accurately quantified. Forensic samples are not typically characterized by the availability of large quantities of DNA; therefore, qPCR can be used to quantify DNA samples both before and after enrichment. Several qPCR strategies have been developed depending on the target, including assays designed for autosomal DNA, sexual chromosomes, and mtDNA (Alonso and García 2007).

Autosomal DNA Quantification The first published use of qPCR in forensic genetics was the performance of a duplex assay to evaluate both nuclear and mtDNA, using the nuclear-encoded, single-copy gene RB1, which is located on chromosome 13, as an autosomal target of 78 bp, using hair, bloodstains, fingerprints, skin debris, and saliva stains as samples (Andréasson et al. 2002). Another study used the human TH01 STR marker as a target, generating a 62-bp amplicon, using buccal swabs as the sample source (Richard et al. 2003), claiming that a human DNA quantification probe should be sufficiently specific. The next step was the assessment of DNA degradation, through the use of two TH01

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targets: one that was 170–190 bp in length and another that was 67 bp in length, using different casework samples and prequantified control DNA, inducing artificial degradation through the application of DNase I (Swango et al. 2006). • Moderately degraded samples: All STR alleles were detected with 1 ng DNA input. • Highly degraded samples: Approximately, 80% of STR alleles were detected with 1 ng DNA input. • Most degraded samples: Very low genotyping success. Currently, commercial kits are available for the quantification of specific human DNA. Quantifiler™ HP and Trio DNA quantification kits (Thermo Fisher Scientific, Massachusetts, USA 2014) both contain an 80-bp, small, autosomal target; a 214-bp, large, autosomal target; and a 130-bp, internal PCR control. Quantifiler™ Trio also contains a 75-bp human male target. Both can detect 4

Quantity 3 pg/μl Full or partial profile expected Max volume input DNA recommended Partial or no profile expected Max volume input DNA recommended

>33.3 pg/μl Full profile expected Regular DNA input recommended Partial profile expected High DNA input recommended

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can be very helpful in these situations because they can assist in the detection of lowcopy-number DNA samples; an IPC is used to detect PCR inhibitors, and the use of X and Y chromosome targets allows for the calculation of possible mixture imbalances. The use of two different sizes of amplicons allows for the evaluation of both degradation and copy number (Alonso et al. 2003). Quantitative PCR assays have also been useful during other DNA analysis steps, such as DNA extraction, and can be used to address low-copy-number DNA issues, suggesting the use of a more efficient DNA extraction protocol (Barta et al. 2014).

Non-human DNA Analysis Obviously, the primary focus of forensic genetics investigations is human DNA; however, qPCR can be helpful for forensic investigations that involve non-human DNA analyses. Common samples in this field include animal species, plants, and microorganisms, primarily due to the wide variety of genomic markers and technologies that can be included in genomic analyses (Arenas et al. 2017). Through qPCR analyses, specific genetic regions, such as cytochrome b (CYTB), cytochrome c oxidase I (COI), and ribosomal RNA (rRNA) genes (Arenas et al. 2017), can be evaluated. Mitochondrial DNA has the same characteristics in both humans and animals: uniparental inheritance, non-recombinant, rapid-evolution, high copy numbers, and simple structures. With a standard pair of primers, certain species-specific coding regions can be analyzed, and these sequences can be aligned with a DNA database to identify the species, such as BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Cytochrome B represents the most commonly amplified region (Kocher et al. 1989). Quantitative PCR has been used with animal DNA testing, for example, to distinguish domestic animals from human DNA samples in a multiplex PCR assay, capable of detecting even 0.4 pg of human DNA within 4.0 pg of animal DNA (Kanthaswamy et al. 2012). Other studies use qPCR for food authentication by applying a multiplex qPCR to the amplification of different simultaneous animal targets (Köppel et al. 2009). The DNA testing of plants offers two interesting fields for forensics: one is the analysis of pollen to link a suspect or a victim to a specific area, and the other can be used to link drug cultivations to other plants or individuals (Butler 2011). Studies have been able to identify Cannabis sativa species through the use of a nuclear and chloroplast duplex qPCR assay (Johnson et al. 2013). Microbiome DNA studies have also been developed in different fields. For example, RT-PCR has been suggested for three different oral bacteria DNA, Streptococcus salivarius, Streptococcus sanguinis, and Neisseria sub ava, for the identification of saliva in forensic samples, which can indicate that human DNA analysis can also be performed (Jung et al. 2018). A similar principle can be applied to the characterization of soil, particularly as soil evidence is quite common in forensic contexts, and the characterization of microbial DNA, combined with an exhaustive knowledge of biogeography, can place an unknown soil sample within a 25-meter area (Habtom et al. 2019).

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Single-Nucleotide Polymorphisms (SNPs) STRs are a core feature used for human identification, and DNA databases focused on STR analyses continue to grow rapidly. However, SNPs are being increasingly investigated for forensic purposes. Furthermore, SNPs are not only useful for human identification but also for ancestry studies and phenotyping (Butler 2011); SNPs can facilitate the identification of sex, ancestry, eye color, or hair type associated with an unknown sample. Several SNP sequencing methods have been developed for forensic use, including the SNaPshot ® Multiplex System (Thermo Fisher Scientific, Massachusetts, USA), which is a primer extension-based method that can analyze up to 10 SNPs in a single reaction and is widely used for mtDNA analysis. Again, RT-PCR may be useful for SNP analyses because qPCR is well suited for the performance of quality checks in mtDNA examined by SNP analysis; a recent study of mitochondrial DNA analysis performed in hair reported no negative SNP results when the qPCR assay was positive, using a total amount of just 6 μl DNA extract, and simultaneously while reducing laboratory costs and working time (Köhnemann et al. 2010).

Issues Associated with qPCR No perfect techniques exist, and although qPCR is a useful tool for forensic scientists, it is also associated with a variety of issues and limitations. Understanding these limitations will allow scientists to provide more accurate results. During this chapter, several RT-PCR advantages have been described; however, each positive aspect is associated with a respective limitation (Table 3). Quantitative PCR allows a large range of quantification; however, because PCR products grow exponentially, a linear model must be constructed to assess DNA quantities. Table 3 Advantages and limitations of qPCR (Klein 2002)

Sensitivity

Advantages A large range of quantification High sensitivity

Precision

High precision

Post-PCR steps Contamination

No operations are needed after qPCR assay Minimized risk of crosscontamination High-throughput

PCR product

Efficiency Multiplex

Multiplex approach is available

Limitations PCR product growths exponentially The more the cycles, the more the variation introduced Linear values while constructing the calibration curve increase variation Emission spectra may overlap if dye chemistry is not designed accurately Sample handling Increased risk of false-negative and falsepositive results Only a certain number of simultaneous reactions are available

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Although qPCR has a high sensitivity, an increasing number of cycles is also associated with the increased introduction of variations. Although qPCR is associated with high precision, the linear values that are introduced during the construction of the calibration curve, experimental variations may increase variations in this transformation. Post-PCR steps are generally not necessary; however, the emission spectra of the various dyes may overlap if primers and probes are not designed accurately; similarly, although a multiplex approach is available, this approach is limited to a specific number of simultaneous reactions and depends on the instrument being used. The ability to perform a high throughput of samples is a strong advantage; however, increased risks of obtaining false-negative and false-positive results may result from the application of the same PCR conditions to all gene targets. Finally, the minimization of cross-contamination risk strongly depends on correct sample handling. Trouble-shooting-related issues must also be considered, based on the following primary topics: primers, probes, and calibration curves. A few recommendations may improve any RT-PCR assay and even eliminate interpretation problems: 1. Primers and probes (ThermoFisher Scientific 2007): The use of suboptimal melting temperatures, incorrect concentrations, designs against low-complexity sequences, wrong uorophores, incorrect target region, overly long amplicons, wrong species, non-target-specific, and uorophores that are not supported by the instrument are the most commonly encountered issues. • Melting temperatures should be maintained between 58  C and 60  C for primers and 10  C higher for probes. • The concentration of primers should be 10–100 μM and 2–10 μM in probes. When reconstituting, the proper concentrations can be assessed by spectrophotometric absorbance at 260 nm. • In regions with low-complexity, designed primers and probes may be problematic, and an alternative region should be selected, if possible; if not, choose longer primers and probes with higher melting temperatures. • Verify that the correct quencher and reporter are being used, as the use of the wrong probe can change the melting temperature and affect PCR efficiency. • Low-integrity sequences can result in failed assays, and the presence of SNP sites should be verified by using various databases, including the National Center for Biotechnology Information (NCBI). Increasing the annealing temperature and primer length may be necessary to avoid NABI primer-template mismatch. • Amplicon lengths should be 50–150 bp, as longer amplicons may cause poor PCR efficiency. • Primers and probes should be verified as being targeted against optimal gene regions by BLASTing (Basic Local Alignment Search Tool) the sequence against those found in public databases. • BLASTing is also necessary to verify the species. • Probes should be labeled with dyes that have been calibrated or are supported on by the qPCR instrument being used.

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2. Standard curve: A standard curve can be used to assess PCR efficiency based on a clean matrix and the absence of interference. This curve may be different from the standard curve that is used to estimate target quantities in unknown samples (Svec et al. 2015). Some important issues should be considered: • The amplification efficiency curve should cover routine applications plus at least 20%. Extensive standard curve measurements at extremely high or low concentrations can deviate from linearity, and very-high target concentrations can cause problems related to the baseline. • Serial dilutions, which are typically used to generate standard curves, may be discouraged due to the carryover of errors. A large transfer volume, at least 5 μl is recommended, across dilutions may be useful for the avoidance of sampling and pipetting errors. • Five dilution steps, at six different concentrations, represent the minimum number of concentrations necessary to identify the linear range and to obtain reliable estimations; each concentration should be performed in at least two replicates. • When standard concentrations on the log scale are uniformly distributed, an even number of concentrations is recommended. • The collected calibration curve should be determined for a standard procedure, including the planned extraction kit or RT-PCR kit and the use of a specific instrument, a plate, and a sealing strategy. Any changes made to the protocol should start with the generation of a new calibration curve. • If different sample types are analyzed together, separate standard curves should be constructed for each type. • The performance of a single and precise estimation of PCR efficiency, under representative assays conditions, is preferable to the construction of minimal standard curves based on few concentrations in each run. The availability of a precise nucleic acid quantification method is very important for molecular biologists in general and forensic scientists in particular. To achieve this aim, a forum should exist to promote the exchange of ideas, techniques, tools, and applications (Editorial 2015), which can improve forensic science and other fields.

Future Perspectives for qPCR in Forensic Genetics Digital PCR (dPCR) PCR technologies have evolved from end-point PCR, through RT-PCR, to the absolute quantitative digital PCR (dPCR). dPCR is capable of detecting a minimal trace amount of nucleic acids with reliable, quantitative results. Most dPCR instruments adopt the Poisson distribution which, when combined with the use of uorescent probes and DNA dilutions, statistically results in one or zero molecules in each reaction chamber prior to amplification, which may explain the success of this method. This method is widely spreading due to its high sensitivity, precision, and accuracy for target quantification, requiring only a small amount of sample.

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This recent technology provides new methods for the detection and quantification of nucleic acids and is viewed as an alternative method to qPCR, particularly for the detection of rare alleles. This methodology is used to divide DNA or cDNA samples into many individual PCR reactions, increasing the likelihood that a single molecule will be amplified a million-fold or more. Furthermore, the use of nano uidic chips can provide convenient and straightforward mechanisms for performing thousands of PCR reactions in parallel (Scientific). In brief, dPCR is a method for absolute nucleic acid quantification, which hinges on the detection of end-point uorescent signals and the enumeration of binomial events [the absence (0) or presence (1) of uorescence in a partition]. Using Poisson statistical approximations, dPCR identifies the parameters that constrain the performance metrics of this analytical method (Quan et al. 2018). Most dPCR applications are in biomedical fields, such as the search for rare mutations and nucleic acid quantification. Moreover, dPCR can also play relevant roles in the detection of pathogens, chromosome abnormalities, tumor DNA in liquid biopsies from tumor patients, and prenatal diagnoses (Cao et al. 2017). In the forensic field, any minimal quantity of biological evidence is extremely valuable during the investigation of a crime or other forensic investigation. A primary challenge is the reduction of inhibitors, such as humic acid and hematin, which can impair DNA polymerase activity, or immunoglobulin G, which binds singlestranded genomic DNA, reducing template availability (Sidstedt et al. 2019). However, few publications have examined the application of dPCR to forensic casework. dPCR has been shown to be very accurate in the determination of plasmid DNA levels and is particularly sensitive to DNA loss caused by degradation or adsorption. These determinations are relevant for obtaining better data analyses at forensic scenes (Wang et al. 2019). In cases of rare mtDNA deletions, dPCR tool could also be used as an accurate method for improving the detection range (Manoj 2016). dPCR appears to be less affected by inhibitors than qPCR, due to the application of end-point measurements; therefore, there is no subordination to amplification kinetics. However, for RT-PCR, quantification cycle values are related to a standard curve; therefore, any inhibitory effects will affect quantification. In contrast, dPCR inhibitor tolerance cannot be explained by the use of the end-point strategy itself because complete inhibition has been observed for lower amounts during qPCR compared with dPCR. Sample partitioning may be another factor to consider, resulting in the reduced interaction between molecules during the polymerization process, including inhibitor molecules (Sidstedt et al. 2020). dPCR represents a promising technology that may surpass qPCR in clinical applications due to its strength and technical reproducibility (Quan et al. 2018).

Conclusions DNA quantification represents a mandatory step in forensic DNA analysis, as stated by the Federal Bureau Investigation DNA Laboratories Quality Assurance guidelines and the Scientific Working Group on DNA Analysis Methods (SWGDAM)

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interpretation guidelines. The importance of DNA quantification is due to the narrow range of acceptable DNA inputs when using commercial STR typing kits and the ability to save both time and money if the quantitation results are negative. However, quantification can be used for many other purposes. Quantitative PCR assays have many applications within the field of forensic genetics, including the optimization of the DNA extraction, the detection of PCR inhibition, the assessment of DNA degradation, the detection of contamination, and the preparation of NGS libraries. Several commercial kits are available for autosomal DNA quantification, which often includes two target fragments, a long one and a short one, a Y-chromosome target, and an IPC that allows human autosomal DNA quantity, the degradation rate, the male human DNA quantity, the male-female proportions of the total human DNA detected, and the presence or absence of PCR inhibitors. However, several RT-PCR assays have been used in the literature for many purposes, including the assessment of Y-chromosome degradation and quantification, X-chromosome quantification, fast sex determination, mtDNA multiplex qPCR assays to assess mtDNA quantity and quality, and even the presence of PCR inhibitors in a single assay. qPCR can also be applied to SNP typing, aDNA analysis, and emerging non-human DNA analysis, relative to animals, plants, and the microbiome. DNA quantification assessments performed during daily laboratory work can be used to adjust the DNA inputs of STR typing, saving both work and money. Moreover, several commercial kits are available for assessing DNA quantity and quality, to allow the optimization of DNA analysis, by continuing with the planned process if the DNA quantity and degradation rate are sufficient and changing the amplification approach, choosing another extraction protocol, or using another sample if the quantity and quality are suboptimal. Subsequently, qPCR represents a valuable technique for forensic genetics, which is a challenging field in which any help is welcome.

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Logical Errors and Fallacies in DNA Evidence Interpretation

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Andrei Semikhodskii

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Initial Assessment of DNA Evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Probative Value of DNA Evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Logic of Evidential Proof . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Logical Fallacies and Reasoning Errors in DNA Evidence Interpretation in Reports and the Courtroom . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Prosecutor’s Fallacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Defendant’s Fallacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Uniqueness Fallacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Probability of another Match Error . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Numerical Conversion Error . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other Interpretation Difficulties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions: Why Misinterpretation of Evidence Happens and What Can Be Done About It? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Logical errors and fallacies in assessing forensic evidence are a major issue recognized by both forensic and legal community. They have a propensity to affect interpretation of DNA and other types of evidence by the fact finder and can be a potential cause of miscarriage of justice. Even though they have been a subject of many criminal appeals and publications in forensic and legal journals, they keep on cropping up again and again. This chapter discusses how DNA evidence is assessed and evaluated by the forensic expert and the fact finder in the courtroom, reviews commonly encountered reasoning errors and logical fallacies, examines their causes, and considers the ways which need to be taken to avoid them.

A. Semikhodskii (*) Medical Genomics Ltd., Tver, Russian Federation e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 H. R. Dash et al. (eds.), Handbook of DNA Profiling, https://doi.org/10.1007/978-981-16-4318-7_40

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Keywords

DNA evidence interpretation · Logical errors · Probative value of DNA evidence · Weigh of DNA evidence · Random Match Probability · Evidetial proof · Prosecutor’s fallacy · Defendant’s fallacy · Uniqueness error

Introduction The strength of DNA evidence is re ected by the value of the probability of a random match between the DNA profile from the crime sample and the defendant. Any probabilistic approach introduces uncertainly into interpretation of scientific evidence which, if not properly understood, can give rise to a misinterpretation of the evidence and inaccurate conclusions as to what the evidence really means. To be able to deal with errors of evidence misinterpretation, it is important to know their origin and how they can affect court proceedings. The errors of misinterpretation of DNA evidence refer to logical errors by forensic scientists or lawyers that happen in the courtroom. These errors have a propensity to affect interpretation of evidence by the fact finder and can be a potential cause of miscarriage of justice. Most of these errors are related in one way or another to a tendency to interpret DNA evidence as means of giving a comment on the truthfulness or otherwise of the prosecution’s proposition (Aitken and Taroni 2004) as to the source of the crime stain or the guilt of the accused. It is not in the domain of the forensic expert to assess the prosecution or defense propositions. It is a matter for the fact finder to determine whether DNA evidence, together with all the other evidence adduced, can prove that the crime stain was left by the defendant or is sufficient enough to warrant a finding of guilt. Not to fall into danger of committing a reasoning error the scientist has to understand the meaning of DNA evidence, the questions DNA evidence can and cannot answer, how evidence is used to prove a fact at issue, and the role of a forensic expert during a criminal trial. Nearly all of the errors or interpretation stem from misunderstanding one of these points. In this chapter the logic of forensic DNA inference and the errors and fallacies commonly encounter in the court room will be examined. Interpretation of forensic evidence is typically performed within a framework appropriate for both scientific analysis and presentation in the courtroom. We start with how the propositions for evidence evaluation are formed and how, by comparing prosecution and defense theories of events, the weight of DNA evidence is obtained. Then, we will look at the random match probability and what it does and does not mean. We will examine the reasoning process employed by the fact finder to reach conclusion on the source of biological material and ultimately the issue of guilt and were the role of the forensic scientist and DNA evidence lays in this process. Thereafter, various logical fallacies and reasoning errors will be examined and elucidated. Last but not least we discuss why these errors happen and what can be done to minimize this.

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Initial Assessment of DNA Evidence Interpretation of DNA evidence happens within a framework of contrasting propositions. In order to evaluate the evidence correctly, the scientist must first formulate different propositions representing both prosecution and defense hypotheses. Here I follow Aitken and Taroni (2004) and Buckleton (2005) in describing different propositions available to the scientist for setting DNA evidence into the framework acceptable for scientific evaluation and presentation in court. The framing of the propositions is based on three key principles (Evett and Weir 1998): 1. Evaluation of DNA evidence is only meaningful when at least one of the two (or in some cases more) competing propositions is addressed. Usually these are the prosecution hypothesis (Hp) and the defense hypothesis (Hd). 2. Evaluation of DNA evidence considers the probability of evidence under the two competing propositions. 3. Evaluation of evidence is carried out within a framework of circumstances and is conditioned not only by the competing propositions but also by the structure and content of the framework. The propositions or hypotheses must be mutually exclusive (Robertson and Vignaux 1995) and are formulated so that they represent the prosecution and defense views (even though because of the presumption of innocence, the defense is not required to put forward any proposition whatsoever), for example: Hp: The DNA comes from the suspect Hd: The DNA comes from a male other than the suspect Commonly, there are only two propositions which are analyzed in pairs, but because the hypotheses do not need to be exhaustive, there may be three or more propositions although, in most cases, they can be reduced to just two alternatives. For example, the following propositions: Hp: The DNA comes from the suspect Hd1: The DNA comes from the father of the suspect Hd2: The DNA comes from the brother of the suspect can be reduced to: Hp: The DNA comes from the suspect Hd: The DNA comes from a male related to the suspect The choice of the propositions to be addressed depends on the circumstances of the case, the observations that have been made, and the available background information (Aitken and Taroni 2004). Setting appropriate alternative propositions is crucial for

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correct evaluation of DNA evidence as the inappropriate choice of proposition can make the evidence irrelevant in the circumstances of a particular case, leading to its dismissal. There will be various propositions considered by various parties involved in the case and ultimately the trial. Aitken and Taroni (2004) classify the hierarchy of the propositions into four levels: (i) (ii) (iii) (iv)

Offence (crime) Level Proposition Activity Level Proposition Source Level Proposition Sublevel (sub-source level) Proposition

The Offence Level Proposition is at the top of the hierarchy and deals with the ultimate issue such as “the defendant is guilty of raping the complainant.” This proposition is solely the domain of the fact finder and is above the level of forensic scientist. All evidence adduced at the trial (and DNA evidence usually is only one of many types of evidence) must be taken into account when assessing the guilt or innocence of the defendant. At this level, the fact finder evaluates whether all the necessary elements of crime (e.g., mens rea and actus reus for common-law jurisdictions) in relation to the defendant have been proved by the prosecution to the required standard. The second level of hierarchy, the Activity Level, deals with the act in question, for example, “the defendant had a sexual intercourse with the complainant.” This level of proposition can be addressed by the scientist, but, because many other types of evidence are not accessible to her, the Activity Level should also be left for the fact finder to consider. As an example, semen of the defendant can be found on a vaginal swab from the complainant, but it does not necessarily mean that the defendant had a sexual intercourse with her. Semen obtained from the defendant in a way other than vaginal intercourse (e.g., oral sex or masturbation) could have been planted inside the vagina by the complainant who had a grudge against him and then accused him of raping her. The evidence related to the possibility of oral sex or masturbation may not be available to the scientist, making her conclusion on the possibility of the intercourse between the defendant and the complainant not only groundless but a potential cause for miscarriage of justice. The Source Level is the penultimate level of the hierarchy. This level is an exclusive prerogative of the forensic scientist who considers questions such as “did the semen from the vaginal swab originate from the suspect?” using available background information for the case. At the very bottom of the hierarchy is the Sublevel (sub-source level) Proposition. Because of the sensitivity of DNA technology, there may be uncertainty regarding which body uid or cell type the DNA could have come from and at this level is required to answer such a question. Sublevel propositions consider just the DNA in isolation, without trying to attribute it to a particular body uid or cell type. The typical proposition considered at this level is “does the DNA come from the suspect?” Framing the propositions is the first stage of the analysis. Next, the scientist has to calculate the probative value of evidence and provide it to the court.

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The Probative Value of DNA Evidence When the fact finder evaluates the most probable origin of DNA evidence (evidentiary fact), it is done by weighing the prosecution and the defense hypotheses as to the fact at issue and determining which of the alternative explanations is most probable. This is done by considering the DNA evidence and all the other evidence adduced. The scientist employs a similar process. Although she cannot express her opinion directly as to the source of DNA evidence, she can provide the court with her estimation of under which of the alternative propositions observation of DNA evidence is more likely. This is done by comparing the likelihood of the evidence if the prosecution theory is true with the likelihood of the evidence if the defense theory is true. The relative values of these two likelihoods provide a measure of the meaning and the probative value of the evidence. This estimation is usually given to court as a ratio known as the likelihood ratio. Central to this approach (called logical or Bayesian approach) is Bayes’ theorem, formulated by Reverend Thomas Bayes and published posthumously in 1763 in An Essay towards solving a Problem in the Doctrine of Chances. Applied to DNA evidence, Bayes’ theorem deals with the likelihood of the fact at issue under two alternative propositions (the prosecution and defense hypotheses) before and after DNA evidence is adduced. Let: Hp be the hypothesis advanced by the prosecution Hd be the hypothesis advanced by the defense E be the event that defendant’s DNA profile matches the DNA profile from the crime stain I be all background (other) evidence in the case Then we can formulate probability Pr of the hypotheses advanced by the prosecution Hp and the defense Hd, given DNA evidence E and other evidence I as Pr(Hp | E,I) and Pr(Hd | E,I), respectively (the sign “|’ reads as “given’). Using laws of probability, it is possible to compare the two alternative probabilities as:


Pr Hp j E, I Pr E jHp , I Pr H p j I ¼  PrðH d j E, I Þ PrðE jHd , I Þ PrðH d j I Þ The expression:
Pr H p j I PrðH d j I Þ termed “prior odds” represents the view of the alternative hypotheses which is formed by the fact finder before DNA evidence was adduced. This is something which is not expressed numerically and does not need to be, as the opinion on the fact at issue is

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formed in the minds of the fact finder based on the evidence adduced prior to DNA evidence. Being the exclusive realm of the fact finder, “prior odds’ are beyond the scope of the forensic scientist who must not express her opinion on this matter. The expression:
Pr H p j E, I PrðH d j E, I Þ called “posterior odds” represents the view of the fact finder on the alternative hypotheses of events after DNA evidence was adduced. The expression:
Pr E jH p , I PrðE jH d , I Þ called “likelihood ratio” (LR) describes whether the DNA evidence is more likely under the prosecution (numerator) or defense (denominator) hypothesis and is the numerical measure of the weight of DNA evidence. When a forensic scientist assesses the evidence, she typically reports only the LR, which is then used by the fact finder to weigh the prosecution and defense hypothesis against each other. If the LR is greater than 1, the evidence supports the prosecution hypothesis on the origin of DNA in the crime sample. The higher is the LR, the more weight is put on the prosecution hypothesis. Alternatively, the smaller the LR, the more weight is put on Hd. When the likelihood ratio is close to 1, both prosecution and defense hypotheses are likely, and the probative value of DNA evidence is very small. To illustrate how the likelihood ratio is calculated, let us look at the following simple example. Assume that a crime was committed. Let c be the stain recovered at the scene. Suppose DNA analysis revealed that the profile of the stain is Gc. Later a suspect s was arrested whose DNA profile Gs is identical to Gc. The prosecution alleges that DNA in the crime stain comes from the suspect s who is the perpetrator of the crime. The defense’s case is that it was not the suspect s but the real criminal x who is the donor of the DNA found in the crime sample and that the suspect s and the real criminal x have identical DNA profiles due to chance. In this case, the prosecution and defense hypotheses will be: Hp: The DNA in the crime stain c came from the suspect s. Hd: The DNA in the crime stain c came from the real criminal x. In order to estimate the weight of DNA evidence, let us calculate the probability Pr of DNA evidence under Hp and Hd. This probability for Hp will be Pr(Gc | Gs,Hp) which is read as “the probability of finding the DNA profile Gc in the crime stain c, given that the suspect s has the DNA profile Gs.” As under Hp it was the suspect who had committed the crime and nobody else, Pr(Gc | Gs,Hp) ¼ 1. According to Hd the suspect s and the real criminal x have identical DNA profiles. Let us write the probability of finding this profile as Pr(Gc | Gs,Hp). Then, we can write the likelihood ratio as follows:

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Pr Gc j Gs , H p 1 LR ¼ ¼ PrðGc j Gs , H d Þ PrðGc j Gs , H d Þ The likelihood ratio is an objective estimation of the weight of DNA evidence because it is based on parameters and statistics that are obtained experimentally (e.g., allelic frequencies or the coefficient of co-ancestry). The expression Pr(Gc | Gs, Hd) is the random match probability which is the probability that a randomly taken individual has the same DNA profile, given that the defendant has identical DNA profile. The smaller is random match probability, the more the LR would favor Hp; on the opposite, the higher it is, the more weight is given to Hd. As the concept of the match involves two people, the match probability is not the same as the probability (frequency) of a DNA profile, and it is important to distinguish between the two. The probability of a DNA profile is a chance that a randomly taken individual will have a particular profile (e.g., a DNA profile of the crime stain), while the random match probability is the chance that a randomly taken individual will have a particular profile when it is already known that another individual (defendant) has this profile. Therefore, the random match probability explicitly requires statements about two profiles – the profile of the defendant and the profile of a randomly taken individual. It should also be noted that the random match probability is not the measure of (a) the probability that the accused has committed the crime in question, nor of (b) the probability that someone other than the accused has committed the crime, nor of (c) the probability that someone other than the accused is the source of DNA in the crime stain (Koehler 1993). Approaches to calculations of the random match probability for various types of DNA evidence can be found in Balding (2005) and Buckleton et al. (2016).

The Logic of Evidential Proof When DNA evidence is examined in court, there is one issue that is crucial for determining how much weight should be given to it in the proceedings. The issue, though simple, is not trivial: is it possible to relate the DNA match between the defendant and the crime stain with the probability of him being the perpetrator of the crime (the ultimate probandum)? What needs to be understood and remembered is that only the fact finder can assess this relationship and evaluate the probability of guilt. It is in no way the job of the forensic scientist to comment on this issue. The same is true for the link between the match and the probability that the defendant is the source of DNA in the crime statin. When evaluating the probative value of DNA evidence in the context of a particular case, the fact finder proceeds by building the following logical chain (given here in a very simplified manner) in reaching the decision on the ultimate probandum: 1. Evidence matches the accused; following from this: 2. The accused is the source of evidence; following from this: 3. The accused is the perpetrator of the crime.

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Every following statement is independent from the previous ones, and it is exclusively the domain of the fact finder to decide whether Statement 2 could be inferred from Statement 1 or Statement 3 from Statement 2. DNA evidence provides information only about the first statement. Using information about the strength of DNA evidence in conjunction with other relevant evidence in the case (to which the DNA scientist is not privy), the fact finder makes a decision whether Statement 2 can be inferred from Statement 1 and Statement 3 from Statement 2. The confusion and misunderstanding of DNA evidence occurs when, by giving information about Statement 1, the scientist expresses an opinion on Statement 2 or even Statement 3. In other words, instead of commenting on the evidence, she comments on the probability of the prosecution (or the defense) hypothesis. DNA is circumstantial evidence. Circumstantial evidence allows a fact at issue to be proven inferentially rather than directly by providing factual information from which an inference may be drawn on the probability of the fact at issue (Emson 2004). It is for the forensic scientist to provide the facts but for the fact finder to make this inference from them. The instances of misunderstanding of DNA evidence in the courtroom are far too common and are committed not only by lawyers who in most cases do not have special statistical training but also by forensic scientists who should have been trained in probabilistic reasoning. For example, when using DNA evidence in arriving at the decision as to the guilt of the defendant, the probability that the defendant’s DNA profile would match that of the criminal, given that he is innocent, may be confused with the probability that the defendant is innocent, given that his DNA profile matches that of the real criminal. This particular error of evidence misinterpretation is called “the prosecutor’s fallacy.” It can be detrimental to the case of the defendant and has been the subject of many appeal cases in various jurisdictions. Other logical errors of interpretation include the defendant’s fallacy, the uniqueness fallacy, and several other ones. All of them will be discussed below.

Logical Fallacies and Reasoning Errors in DNA Evidence Interpretation in Reports and the Courtroom The Prosecutor’s Fallacy The prosecutor’s fallacy is a specific case of a logical error of the transposed conditional (Thompson and Schumann 1987) which arises when the probability of DNA evidence under a particular hypothesis is confused with the probability of the hypothesis given DNA evidence. This fallacy tends to favor the prosecution and was a subject of numerous publications in legal literature. In spite of this, it still often happens, especially in cases when DNA evidence is involved. The error of the transposed conditional is an elementary logical error in which the evidence interpreter assumes that if A implies B, then B implies A. David Balding (2005) gives a simple explanation of this error. Let A denote “a cow” and B denote

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“has four legs,” then a statement “a cow has four legs” is not the same as “if an animal has four legs it is a cow.” A cow, of cause, is one of the possibilities but the animal can also be a sheep, a goat, or a dog. The reasoning behind the prosecutor’s fallacy is similar to this logical error, only in terms of probabilities. This can be illustrated by the following example. If there are 100 four-legged animals living on a farm, of which 10 are cows and 90 are sheep, then the probability of randomly picking a four-legged animal which will be a cow is 0.1 (i.e., every tenth animal is a cow). Assume that the probability of having four legs if you are a cow on the farm is 1 (i.e., every cow on the farm has four legs, disregarding here rare genetic anomalies and accidents leading to amputation of limbs). In such a case, the probability of the statement “a cow has four legs” is 1, while the probability of the statement “a four-legged animal on the farm is a cow” is only 0.1. The first statement is the probability of evidence (“four legs”) under a particular hypothesis (“is a cow”); the second statement is a probability of a particular hypothesis (“is a cow”) given evidence (“has four legs”). In relation to DNA evidence, the prosecutor’s fallacy is to confuse the probability of a match between the accused and the crime stain (Statement 1 in the previous section) with the probability that the accused is the source of the crime stain (Statement 2) or with the probability of accused being guilty of the offence (Statement 3). These two types of prosecutor’s fallacy are called the source probability error (the former) and the ultimate issue error (the latter).

The Source Probability Error The source probability error is the most common type of the prosecutor’s fallacy. This error happens when the probability of DNA evidence (e.g., the random match probability) is equated with the probability of the defendant being the source of the crime stain sample. Equating these two probabilities tends to exaggerate the strength of the prosecution hypothesis and is detrimental to the defendant. The information on the source is inferred from the factual information provided by the expert, and it is exclusively the prerogative of the fact finder to make such an inference from adduced evidence. Because of this, it is not for the expert to express her opinion on the source of evidence; this lies in the domain of the fact finder. Examples of the source probability error are numerous. When the probability of a random match is, say, 1 in 1 billion, the fallacious statement may look like this: “It is 1 billion times more likely that Mr X is the source of the biological material found at the crime scene, than an unknown unrelated to him individual”

A correct version of such a statement should always involve a conditional statement of the form “if the defendant were not the source, then the probability . . .” (Balding 2005), for example: “If Mr X were not the source of the biological material found at the crime scene then the profiles must match by chance. I estimate that the chance of obtaining matching DNA profiles if the biological material comes from an unknown unrelated to him individual is 1 in 1 billion”

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Forensic scientists are specially trained not to commit the errors of transposed conditional in their statements, but the above examples indicate these types of errors are likely to be committed when the scientist is asked about the meaning of evidence in court. A legitimate question arises about the extent to which experts are responsible for the mischaracterization of their scientific testimony by others. Under the pressure and stress of direct and cross-examinations, it may not be reasonable to expect an expert to catch and correct all subtle distortions and misunderstandings expressed within the courtroom (Koehler 1993). To avoid this type of error, one must always remember that the scientist provides the court with the weight of DNA evidence, expressed as the random match probability or the LR, and it is then for the fact finder to evaluate the hypotheses that the defendant is the source of DNA in the crime stain.

The Ultimate Issue Error Sometimes the source probability error is extended to comment on the probability of the guilt of the defendant. In this case the ultimate issue error is committed. When scientists report the probability of a random match between the accused and the crime samples as, say, 1 in one million, this may erroneously be interpreted by the fact finder or prosecutor as meaning that the probability of the defendant being innocent is also 1 in one million. This logic is fallacious, but it can still be encountered during criminal trials. An example of this type of erroneous reasoning is the following: “If the defendant has a DNA profile matching that obtained from the crime stain and the observed random match probability is 1 in 1 million then, considering the population of the United Kingdom being equal to 65 million people, he will be one of perhaps 33 men in the United Kingdom who share that characteristic. If no fact is known about the defendant, other than that he was in the United Kingdom at the time of the crime, the DNA evidence tells us that there is a statistical probability of 1 in 33 that defendant is the real perpetrator of the crime.”

The ultimate issue error often arises when the prosecutor or judge asks the scientist who presents the evidence a question in the form of a transposed conditional. Koehler (1993) gives an example of a dialogue between the prosecutor and the DNA expert witness after he reported a random match probability between the DNA profile of the accused and the crime stain samples as 1 in 5 billion: Q.: So that in the event that the accused sitting in this chair would happen to be White, you’re telling the members of this jury that there would [be] a one in 5 billion chance that anybody else could have committed the crime; is that correct? A.: One in 5 billion, correct.

As illustrated by this dialogue, it is very easy for an expert witness to get caught by such a question and give an erroneous testimony. As well as experts, it is thought that ultimate source errors are committed by the judges and jurors, even when the experts don’t commit them (Koehler 1993). As this is often the case, the expert in her testimony has to emphasize to the fact finder that DNA evidence does not provide

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any information as to the guilt or otherwise of the accused, but only indicates the likelihood of DNA originatig from another person with the DNA profile identical to that of the accused, should the accused be not the donor of the biological material in the crime stain. The ultimate issue error also can be found in DNA parentage testing reports. There it equates the Combined Parentage Index (CPI) with the likelihood that the alleged parent is the true biological parent of the child in question (the probability of guilt). The fallacious statement often reads like this: “The Combined Parentage Index value of 107,463 indicates that Joe Bloggs is 107,463 times more likely to be the biological father of Jane Bloggs as compared to an untested, unrelated male of the same ethnicity.”

The correct statement in this situation should be something like this: “The Combined Parentage Index value of 107,463 indicates that the genetic data obtained are 107,463 times more likely if Joe Bloggs and not an untested, unrelated male of the same ethnicity is the biological father of Jane Bloggs.”

As with random match probability, CPI is not a measure of guilt but a parameter indicating the likelihood of obtaining the observed results under two contrasting hypotheses: Hp, the alleged parent is the true biological parent of the child, and Hd, an untested unrelated man of the same ethnicity is the true biological parent of the child.

The False-Positive Fallacy A declared match between the defendant and the crime scene can be either true or false. The false match happens when a forensic scientist erroneously finds “a match” between defendant’s DNA profile and the profile obtained from the crime scene when such a match does not actually exist. This type of error is called a “falsepositive error.” The probability of reporting such an erroneous match is called probability of a false-positive match. Many consider that for correct evaluation of DNA evidence, it is only important to have information about the value of the random match probability. However, due to a false-positive error, a reported match between the defendant and the crime stain can be false. So, to evaluate the evidence correctly, it is also necessary to have data on the false-positive error rate in the forensic laboratory. The false-positive fallacy happens when a scientist mistakenly assumes that if the false-positive probability (i.e., the prior probability of declaring a match falsely) is low, then the probability of a false match (i.e., the probability that a declared match is false) in the case in question must also be low (Aitken and Taroni 2004). A scientist may think that if the probability of a false-positive match in the laboratory is 0.001, then the probability of a true match reported in a particular case is 0.999. This fallacy, which is yet another version of the prosecutor’s fallacy, arises from mistakenly equating the conditional probability of a match being reported when the samples do not match (the false-positive probability) with the probability that the samples do

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not match when a match has been reported (Aitken and Taroni 2004). These two probabilities are not the same. The false-positive probability depends only on the probability of genotyping (and/or human) error in the laboratory and is the probability that a match between the accused’s sample and the crime sample will be reported when there is no match. The probability that the samples do not match when a match has been reported depends on both the probability of genotyping (and/or human) error and the prior odds that such a match will occur. The assumption that the false-positive probability is equal to the probability that the samples do not match when the match is reported is fallacious because it ignores the prior probability that the accused’s profile matches the crime profile (Aitken and Taroni 2004). In a simple case, assuming there are no false negatives, the probability Pr(M | R) of a true match M between the accused and the crime sample, given that a true match R has been reported, can be calculated using the following formula (Aitken and Taroni 2004): PrðMjRÞ ¼

1  1 þ kPrðRjMÞ

where k are the prior
odds that the accused’s profile matches that from the crime scene and Pr R j M is the false-positive probability. A more complex formula, which takes into account the probability of false negatives, can be found in the same work. Aitken and Taroni (2004) provide a compelling example illustrating the falsepositive fallacy. Let us assume that the prior odds of the accused matching the crime sample is 1 in 1000, because the accused was identified by a DNA search and appeared initially to be an unlikely perpetrator. The match between the accused and the crime stain was obtained. Further, let the probability of the false-positive match in the forensic laboratory which analyzed the samples be 0.01. The false-positive fallacy would suggest that because the false-positive error in the laboratory is 0.01, the probability of a match reported correctly in this particular case would be 0.99. In reality, this probability is: PrðM jRÞ ¼

1 ¼ 0:0909 1 þ 1000  0:01

which is almost 11 times smaller than that assumed under the false-positive fallacy.

How Not to Commit the Prosecution Fallacy The error of transposed conditional is very common. It happens to inexperienced and trained scientists, especially under immense pressure of court proceedings. The error stems from misinterpretation of the meaning of random match probability and also misunderstanding of what the expert can and cannot comment on during criminal proceedings. Very often, an incorrectly asked question confuses even a well-trained testifying scientist to commit the error. To avoid the prosecution fallacy, the scientist has to remember that:

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1. DNA evidence is circumstantial evidence and as such allows only indirect inference about the fact at issue (which can be, e.g., “the defendant is the source of biological material in the crime stain”). 2. The weight of DNA evidence (LR) only indicates the probability of obtaining evidence under two contrasting propositions (e.g., Hp, “the defendant is the source of biological material in the crime stain,” and Hd, “an untested unrelated man of the same ethnicity and not the defendant is the source of biological material in the crime stain”) and not the probability of the propositions. 3. The scientist is not the fact finder and must never comment directly on the fact at issue (i.e., express her opinion as to whether the defendant is the source of biological material in the crime stain). Only the fact finder can directly draw conclusion as to the fact at issue. This is done by taking into account all available evidence, including DNA evidence. However, the scientist can provide an indirect opinion as to the fact at issue by giving estimation of the likelihood of observing DNA evidence when comparing two contrasting propositions. 4. The scientist must not comment on which of the evaluated hypotheses is more or less likely. She provides the probability of evidence, and the fact finder deals with interpretation.

The Defendant’s Fallacy Besides the prosecutor’s fallacy, the other logical error commonly heard in the courtroom is the defendant’s fallacy (Thompson and Schumann 1987) which, as the name states, usually favors the defendant. The fallacy stems from the (erroneous) assumption that in a given population, anyone with the profile matching that of the crime stain is as likely to have left the sample as the defendant. The fallacious reasoning goes like this: “The crime was committed in the UK by a male. Suppose the total UK male population is 33 million. The random match probability reported for the case is 1 in 1 million. As there are 33 people in the UK who have the DNA profile matching that from the crime scene, the probability that the defendant is the purpertrator of the crime is 1 in 33 or only 3%.”

Some may go even further and argue that because the number of possible culprits is high, the probative value of DNA evidence is low (only 3% in the above case) and it should be ignored. Strictly speaking, the defendant’s fallacy is not a fallacy in the sense of the prosecutor’s fallacy. The logic behind the defendant’s fallacy’s reasoning would be correct if everyone indeed was equally likely to be guilty of the crime in question (which of course contradicts the main principle of criminal law – innocent until proven guilty). In practice, though, this assumption is very unlikely. Even when there is no evidence directly incriminating the defendant of the crime, there is usually enough background information related to the location and nature of the crime to

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exclude some or even most individuals from the number of potential perpetrators and make others more plausible suspects. In addition, the DNA evidence in the above example has reduced the number of possible perpetrators from 33 million down to 33 individuals, the accused being one of them, and increased the odds in favor of his guilt 1 million times. In other words, the evidence is 1 million times more likely if the accused is the perpetrator of the crime than if he is innocent.

The Uniqueness Fallacy Another fallacy which often crops up in cases when DNA evidence is used is the uniqueness fallacy. This fallacy arises when the match probability for an unrelated perpetrator is small enough to imply that no other individual with the same profile could be found in the relevant population. For example: “The crime was committed in the UK by a male. The random match probability between the defendant and the crime stain is 1 in 1 billion (billion is one thousand million). Let assume the total male population of the UK to be 33million people. Considering the size of the population it can be safely assumed that defendant’s DNA profile is unique in the UK, and he is the only source of DNA in the crime stain.”

The uniqueness fallacy misinterprets the role of forensic scientist in criminal proceedings and the role of statistics in forensic inference. It stems from taking the value of random match probability literally. Because of relatedness, the actual number of people who might have the same DNA profile as the defendant may be higher. In the above case, it is wrong to assume that there are 33 million males in the UK who are unrelated to each other. Considering the meaning of the word “unrelated” as “second cousin,” the number of unrelated males is significantly lower and is probably in the region of 3–5 million. Instead of having a literal meaning, the value of the random match probability indicates how strong the evidence against the defendant is and how much weight the fact finder should assign to it. If all 7 billion people living on the planet were not related to each other, probably, a match probability which is several orders of magnitude smaller than the 1 in 7 billion would be sufficient to judge that a profile is unique and no identical one can be found. However, this is not the case. All people are related to each other to a certain degree, especially island populations like the one in the UK. Because of this, the chances of finding another individual with an identical DNA profile are not negligible, making the claim of uniqueness statistically unsubstantiated, even when calculations of the random match accounted for co-ancestry. When the match probability is 1 in 1 billion, it does not mean that we need to test 1 billion people to find the matching profile. There is a high probability that the number of people needed to be tested to find the matching profile is significantly smaller. This is discussed in more detail in the next section. But even in the hypothetical case when all people on the planet are not

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related to each other, because of the non-DNA evidence, there seems to be no satisfactory way for an expert witness to address the question of uniqueness in court (Balding 1993). The other problem with the uniqueness fallacy is related to misunderstanding of the law of evidence, the mechanics of proof in criminal proceedings and what is the domain of the fact finder during a criminal trial. In expressing uniqueness fallacy, the scientist takes on herself the role of the fact finder to draw conclusions as to the identity of the source. In making a statement on uniqueness of the DNA profile, the scientist has to account for the size of the relevant population of potential perpetrators. Because the size of this population depends not only on demographic characteristics of the total population but on other evidence not available to the forensic scientist, in expressing her opinion on uniqueness, she will always be using the population of the wrong size. In addition, it is not in the domain of the forensic scientist to regard some sections of the population as more capable of committing the crime than the other ones.

The Probability of another Match Error The logical aw of the probability of another match error is similar to the uniqueness fallacy. This error stems from a mistaken belief that the probability of a random match is the same as the probability of finding another person in the population with the same DNA profile. The logical error would go as follows: “The random match probability between the defendant and the crime stain is 1 in 1 million meaning the probability of finding a male with the DNA profile identical to that of the defendant is 1 in 1 million.”

The logical error here is to think that the small value of the match probability between the defendant and the crime stain implies a similar small probability of finding another man with the same genetic characteristics. The mistake is to take the probability that a randomly selected person would have the profile identical to the defendant (the match probability of 1 in 1 million) for the probability that there exists someone else in the population who has the same profile as the defendant. The latter probability may be greater than the former. Following Aitken and Taroni (2004), let N denote the size of the relevant population and F the match probability. Then the probability that a randomly selected person will not match the crime profile will be 1  F, while the probability that not a single member of the relevant population with the size N will match the crime profile will be (1  F)N. It follows from this that the complement probability θ, that at least one match will be found in the population, is θ ¼ 1  (1  F)N. If the population N is 1 million and the random match probability F as 1 in 1 million, the probability of finding at least one other person out of the million people in the population with matching DNA profile will be:

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θ ¼ 1  ð1  FÞN ¼ 1  ð1  1=1, 000, 000Þ1,000,000 ¼ 0:63212or63:21% When F is 1 in one million, it may be extremely unlikely to randomly pick a person whose DNA profile matches that of the defendant out of a million people, but the probability that at least one such man exists is 63.21%. The probability of finding another person with matching DNA profile is a good argument when talking about uniqueness. In the UK, when a complete match is observed between the defendant and the crime stain, the match probability is conventionally reported to be “less than 1 in 1 billion (a billion is a thousand million).” By calculating θ we can get an estimation of how unique a DNA profile with such low match probability would be in the UK. Let us assume the crime was committed by a man; the total male population of the UK N is 33 million, and the match probability F is 1 in 1 billion. Then: θ ¼ 1  ð1  FÞN ¼ 1  ð1  1=1, 000, 000, 000Þ33,000,000 ¼ 0:03246or3:25% Can we consider a DNA profile to be unique when there is 3.25% probability of finding another man in the UK with matching genetic characteristics?

The Numerical Conversion Error Sometimes, the random match probability may be incorrectly interpreted as the number of people who need to be tested until the second identical profile is found. A conclusion that when the probability of a random match is, say, 1 in 33 million, the number of people who need to be tested until the matching profile is found is at least 33 million is fallacious and is known as the numeric conversion error. This error exaggerates the number of people who would need to be tested before a match may be expected and thus exaggerates the probative value of DNA evidence (Koehler 1993). As in the previous example, following Aitken and Taroni (2004), let N denote the relevant population, F be the match probability between the defendant and a particular crime profile, and n be the minimum number of people who would have to be tested before another match was found. The numerical conversion error is to equate N with n. As above, the probability that a randomly selected person will not match the crime profile will be 1  F, while the probability that, for n randomly selected individuals, none will match the crime profile will be (1  F)n. The probability Pr (M) that at least one match will be found is 1 – (1  F)n. For a match to be more likely than not, this probability Pr(M) has to be greater than 0.5, and so: ð1  FÞn < 0:5 Solving this inequality for n, we obtain the formula for calculating the number of people needed to be tested before we might expect the match to be more likely than not (i.e., with the probability greater than 0.5):

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n¼

815

log ð1  0:5Þ , log ð1  FÞ

and the generalized formula will be: n¼

logð1  ð1  PrðMÞÞ : logð1  FÞ

Taking F to be 1 in one million, we would expect to find a match more likely than not after testing 694,147 people. At the same time, the number of people needed to be tested to ensure that finding a match is guaranteed (say, with the probability of 0.99) is a staggering 4.6 million people!

Other Interpretation Difficulties Besides the abovementioned errors and fallacies, forensic reports sometimes contain statements that have a potential for causing misinterpretation of DNA evidence. Some of these statements are discussed below.

Relative Frequency of Occurrence Sometimes an expert statement contains, instead of the match probability, the reference to the frequency of occurrence of a particular DNA profile in the population. In such a case, the report includes a statement about the DNA evidence along the following lines: “The DNA profile of a particular type occurs in about one person in 100,000 of the population.”

There are several major objections to this approach (Aitken and Taroni 2004): 1. When the relevant population is greater than the inversed frequency of occurrence, then it is possible by this approach to estimate the number of potential perpetrators and use DNA evidence as prior odds when evaluating the remaining evidence in this case. For example, when the size of the relevant population is one million and the frequency of the DNA profile is 1 in 100,000, then the number of potential perpetrators will be 1000,000  (1/100,000) ¼ 10. However, if the inversed frequency of occurrence is bigger than the relevant population (the frequency is, say, 1 in 1 billion, while the relevant population is one million), we would not expect to see another profile like this, which makes it impossible to combine DNA evidence with other evidence that might provide the support or an alibi for the defense case. 2. When a relative of the accused is considered as a potential perpetrator, a statement like: “The DNA profile in question occurs in about one brother in 400”

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does not make sense. The scientist has to find alternative ways of expressing the meaning of this statement, which may be even more confusing to the fact finder. 3. When assessing DNA evidence, there are at least two con icting hypotheses – the prosecution hypothesis, which states that the accused is the donor of the crime stain, and the defense hypothesis, stating that the accused is not the donor of the crime stain but owing to chance his/her DNA profile matches that of the real perpetrator. This means that there is another person in the population with the profile matching that of the accused. In contrast to frequency, the concept of a match involves two profiles which permits evaluating both prosecution and defense theories. It is not the relative frequency of a particular DNA profile or the probability of finding a particular profile in the population that is essential for a criminal identification but rather the probability of finding a particular DNA profile in the population, given that the accused has this profile. It is important to emphasize that in human populations, because of relatedness and population subdivisions, the match probability exceeds the probability of finding a particular profile in the population (Balding 2005). Thus, the use of DNA profile frequency instead of the match probability favors prosecution and should be avoided in criminal trials. 4. When a mixed profile is obtained from the crime stain or in cases of paternity testing or identification of victims of mass disasters, it is impossible to use the profile frequency to state the value of DNA evidence as these types of profiles cannot be expressed in the form of “one in. . ..”

The “Could be” or “Could Have Come/Originated From” Approach Often, forensic scientists use statements regarding the source of DNA evidence of the form “. . . it is likely that the accused could be the donor of the DNA recovered from the left pocket of the trousers. . .”

or “. . . in my opinion the blood stains on the track suit bottoms could have come from the victim. . .”

and so on. This type of statement may be treated as the source probability error as they express a view on the probability of a hypothesis (the defendant is the donor of the crime stain) and can be understood as establishing a proven association between the crime and the accused. It should be noted, however, that “Could have come/ originated from” does not preclude other possible sources, and there may well be other, even more plausible, explanations as to the origin of biological material in the crime stain that have not been provided by the scientist. In addition, this approach does not give any indication of the likelihood that the defendant is the actual source of the evidence (Aitken et al. 2010).

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The “Could Not Be Excluded” Approach Another type of statement commonly found in forensic reports is: “. . .. the accused cannot be excluded from being a donor of the crime stain. . ..”

In some cases, especially when dealing with mixtures, the exclusion probabilities approach is used for evaluating DNA evidence. Under this approach, the probability that a random man or a contributor to the mix will be excluded as the donor of the DNA from the crime scene is calculated. However, such a probability only states the proportion of the population which would be excluded; it is thus the measure of efficacy of a particular test (Robertson and Vignaux 1992) as it answers the question “how likely is the individual to be the donor of the crime stain?” However, the other question is much more important in criminal identification, namely, “how much more likely is the evidence if the accused is the donor of the crime stain than a randomly taken individual?” (Aitken and Taroni 2004). The “Could not be excluded” approach does not give an answer to this question, and the probability of exclusion is not relevant for evaluating DNA evidence in such a case.

“Consistent with” Approach Sometimes a scientist during her testimony may state that the evidence is “consistent with” a particular proposition, for example: “Finding DNA matching that of the defendant on a vaginal swab is consistent with the theory that the defendant had a vaginal intercourse with the complainant.”

Again, like with “could be” approach above, this type of statement may be treated as the transposed conditional error as it could be expressing a view on the probability of a hypothesis (the defendant had a vaginal intercourse with the complainant) and can be understood as establishing a proven association between the crime and the accused. Also, to say that evidence is “consistent with” a particular proposition means only that the stated proposition cannot be excluded by the evidence and says nothing about how likely the proposition is to be true (Aitken et al. 2010).

The Association Fallacy When evaluating Source Level Propositions, the scientist often considers questions whether a particular bodily uid or cellular type can be the source of DNA. Association of a DNA profile with the tissue source could make the evidence seemingly more probative than just conformation of a DNA profile without providing the evidence as to the bodily uid origin. The “association fallacy” is the assumption that the observed DNA profile has come from a particular bodily uid (e.g., semen) on the strength of presumptive (or RNA) tests, verified by “expert opinion.” The fallacy here is that the observation of a body uid and the detection of a DNA profile are two separate tests, and it is erroneous to assume that there is a dependency between two observations or events. It cannot be implied with certainty

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that the body uid and the DNA have the same source. By extension, this fallacy also describes wrongful association of the presence of a bodily uid (e.g., sperm), with an activity (e.g., vaginal intercourse) (Gill 2014).

The Use of the Verbal Scale to Express the Strength of DNA Evidence Forensic scientists sometimes accompany the likelihood ratio with a qualitative verbal explanation of the support that should be given, in their view, to one of the competing hypotheses (usually the prosecution hypothesis). This is typically done by assigning a non-numerical weight to LR using one of several verbal scales proposed for this purpose (Buckleton 2005). For example, LR of 10,000 can be descried as showing “strong scientific support” and LR of 100,000 – “very strong scientific support” to a particular proposition. When using the verbal scale, the scientist usually provides an explanation like: “In expressing the evidential significance of my findings, I have used the following scale of scientific support: no support, weak, moderate, moderately strong, strong, very strong, extremely strong support.”

The idea behind the verbal explanation is to assist the fact finder in understanding how much weight should be given to the hypothesis that the accused is the donor of the DNA in the crime stain. By admittance of forensic scientists, this scale is arbitrary and hence subjective (Buckleton 2005). When presenting results using the verbal scale, the differences in meaning between “moderate support” and “moderately strong support” or “very strong support” and “extremely strong support” together with corresponding LR values have to be explained to the fact finder and the prosecution and defense. These meaning of the wording is not standardized and expresses either the subjective opinion of the testifying scientist or the subjective opinion of the scientist who developed this verbal scale. Last but not least, by expressing the strength of the scientific support to one of the alternative hypotheses, the scientist appears to be commenting on the issue of whether or not the defendant is the source of the crime stain. This is the prerogative of the fact finder who is guided in his decision by the statistical information provided by the forensic scientist and should not be affected by her personal opinion as to the strength of scientific support that should be given to one of the competing propositions.

Statements about Identical Twins Sometimes we come across statements which are now rarely made in court but often in the press, that “. . .only identical twins have identical DNA profiles. . .”

or “. . .no two people in the world have matching DNA profiles apart from identical twins. . ..”

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These statements are not correct. Identical twins indeed have matching DNA profiles; however, other people may have matching profiles owing to chance or because they are related. The chances of such a match happening are expressed by a random match probability and, however rare, a match between two unrelated individuals can happen.

Conclusions: Why Misinterpretation of Evidence Happens and What Can Be Done About It? Most errors of DNA evidence interpretation tend to exaggerate the probative value of the evidence. Koehler (1993) in his review of interpretation errors addressed this issue. He investigated several potential factors, such as deliberate exaggeration of the probative value by scientists who wish to “puff up” the utility of their science, or by prosecutors, determined to win their case. He concluded that the most plausible explanation for this sort of error is ignorance in the legal profession of statistics and rules for evidence interpretation. In a review of court cases conducted by Koehler, it transpired that DNA experts generally begin the interpretation part of their testimony with statements about population frequencies and comparison with a “random man.” Although, as we have seen, there may be errors in these statements, they are not deliberate, and the very use of such statements indicate that the scientist tries to give an unbiased opinion. The errors in the testimony begin to appear in most cases only after the expert re-describes her findings in response to the questions from the prosecutor. It has to be kept in mind that while, for a prosecutor, a court hearing is part of the daily job, the expert witness is often under immense pressure both because of her role in the proceedings and also because of the adversarial nature of the criminal trial. In such circumstances, even highly experienced scientists can make errors in answering a logically erroneous question. Because very few lawyers are trained in statistics and evidence interpretation, they may misunderstand the subtleties of probabilistic inference and have trouble differentiating probabilistic information from probabilistic hypotheses that the information suggests (Koehler 1993). All this indicates that the solution to the problem is specialized training of lawyers in matters dealing with evidence interpretation and probabilistic inference. The issue of poor training of members of the legal profession has long being recognized by forensic statistical community. To somewhat remedy the situation in the UK, the Royal Statistical Society (RSS) has published four excellent guides (Aitken et al. 2010; Puch-Solis et al. 2012; Roberts and Aitken 2014; Jackson et al. 2015) which are freely available on the RSS’s web site. The guides look at communicating and interpreting statistical evidence during court proceedings and are intended to assist judges, lawyers, and expert witnesses in coping with the demands of modern criminal litigation.

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References Aitken CGG, Taroni F (2004) Statistics and the evaluation of evidence for forensic scientists. Wiley, Chichester Aitken C, Roberts P, Jackson G (2010) Practitioner guide no. 1. Fundamentals of probability and statistical evidence in criminal proceedings. Guidance for judges, lawyers, forensic scientists and expert witnesses. Royal Statistical Society’s working group on statistics and the law. https:// rss.org.uk/news-publication/publications/our-research/. Accessed 22 Nov 2020 Balding DJ (1993) When can a DNA profile be regarded as unique? Sci Justice 39:257–260 Balding DJ (2005) Weight-of-evidence for forensic DNA profiles. Wiley, Chichester Buckleton J (2005) A framework for interpreting evidence. In: Buckleton J, Triggs CM, Walsh S (eds) Forensic DNA evidence interpretation. CRC Press, Boca Roca, pp 27–63 Buckleton JS, Bright J-A, Taylor D (eds) (2016) Forensic DNA evidence interpretation, 2nd edn. CRC Press, New York Emson R (2004) Evidence, 2nd edn. Palgrave Macmillan, Basingstoke/Hampshire Evett IW, Weir BS (1998) Interpreting DNA evidence. Sinauer Associates, Sunderland Gill P (2014) Misleading DNA evidence: reasons for miscarriages of justice. Academic, London Jackson G, Aitken C, Roberts P (2015) Practitioner guide no. 4. Case assessment and interpretation of expert evidence. Guidance for judges, lawyers, forensic scientists and expert witnesses. Royal Statistical Society’s working group on statistics and the law. https://rss.org.uk/news-publication/ publications/our-research/. Accessed 22 Nov 2020 Koehler JJ (1993) Error and exaggeration in the presentation of DNA evidence. Jurimetrics J 34:21–39 Puch-Solis R, Roberts P, Pope S, Aitken C (2012) Practitioner guide no. 2. Assessing the probative value of DNA evidence. Guidance for judges, lawyers, forensic scientists and expert witnesses. Royal Statistical Society’s working group on statistics and the law. https://rss.org.uk/newspublication/publications/our-research/. Accessed 22 Nov 2020 Roberts P, Aitken C (2014) Practitioner guide no. 3. The logic of forensic proof: inferential reasoning in criminal evidence and forensic science. Guidance for judges, lawyers, forensic scientists and expert witnesses. Royal Statistical Society’s working group on statistics and the law. https://rss.org.uk/news-publication/publications/our-research/. Accessed 22 Nov 2020 Robertson B, Vignaux GA (1992) Unhelpful evidence in paternity cases. New Zealand Law J 9:315–317 Robertson B, Vignaux GA (1995) Interpreting evidence. Wiley, Chichester Thompson WC, Schumann EL (1987) Interpretation of statistical evidence in criminal trials: the prosecutor’s fallacy and the defence attorney’s fallacy. Law Hum Behav 11:167–187

Part VI Non-human Case Studies

Wildlife DNA Profiling and Its Forensic Relevance

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Ulhas Gondhali and Aditi Mishra

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Identification of Species of Origin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Species Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Common Locus Used for Species Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA Repositories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Identification of the Geographic Origin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Principle of Identification of the Geographic Origin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods of Determining Geographic Origin with DNA Markers . . . . . . . . . . . . . . . . . . . . . . . . . . Individual Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Individualization Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nonhuman Paternity Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

824 826 827 828 830 830 831 832 833 833 835 836 837

Abstract

Wildlife DNA forensics is a relatively new field emerging to tackle challenges in wildlife crime investigation. It is an applied field which is a combination of conservation genetics and forensic genetics. DNA evidences are crucial and hold great evidential value in human identification. DNA evidence analysis techniques have been in focus since years. The present technological advantage is achieved through years of research and development in molecular techniques. The process of evidence analysis and reporting in DNA-based evidence in human identification is not much different than wildlife forensic analysis. There may be U. Gondhali (*) Jindal Institute of Behavioural Sciences, O.P. Jindal Global University, Sonipat, India e-mail: [email protected] A. Mishra School of Forensic Science and Risk Management, National Security and Police University of India, Rashtriya Raksha University, Gandhinagar, Gujarat, India e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 H. R. Dash et al. (eds.), Handbook of DNA Profiling, https://doi.org/10.1007/978-981-16-4318-7_41

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difference in case assessment and evaluation of scientific data, but the fundamental principles of forensic investigation remain same. The nature of wildlife crimes is different, and the laws controlling it are as well. The legality of any act is decided by a number of factors, such as the species involved, geographic location, source, and age. DNA evidence analysis helps wildlife law enforcement to answer important case-related questions, which are based on the above factors. Wildlife DNA profiling is essential to meet the increasing need of investigative tools in wildlife crime investigation. Keywords

Wildlife DNA profiling · Wildlife crime investigation · Species identification · Geographic identification

Introduction There is a global consensus on stopping the ever-increasing environmental crimes and overexploitation of the available natural resources. The international bodies such as Interpol, which is working to control such crimes on a larger scale, have also considered wildlife crimes as one of the prime issues. It has expressed its concerns on the increasing risk on the worlds of ora and fauna from criminal exploitation. The CITES (the Convention on International Trade in Endangered Species of Wild Fauna and Flora), which is an international agreement that controls the trade of wildlife ora and fauna to ensure their survival, defines wildlife crimes as the taking, trading (supplying, selling, or trafficking), importing, exporting, processing, possessing, obtaining, and consumption of wild fauna and ora, including timber and other forest products, in contravention of national or international law. Wildlife crime is unlike human-related crimes. Wildlife crimes such as poaching of rare species such as pangolins and subsequent trafficking of their body parts are complex issues. Such crimes often occur in remote places. Unlike human-related crime, there is no social taboo involved. The low risks and high rewards are also encouraging factors in such crimes. The financial rewards for such crimes can be highlighted in several examples. It includes poaching of musk deer (Moschus sp.). Male musk deer carry a secreting gland called “musk pod.” It is used in certain traditional Chinese medicine products and a variety of cosmetics because of its scent. Few grams of musk pod could fetch hundreds of dollars in international markets. The international ivory market is also one of the biggest. Often African rhinos and elephants are poached as a source of illegal ivory. The high demand for ivory products in international markets attracts huge prices for such catches. Often multiple actors are involved in wildlife crimes, and such crimes are executed at local and international levels. The involvement of local as well as international nexus is because of the intricate nature of source and consumer. Illegal wildlife trade is highly organized as sometimes it requires to bypass many

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international borders without alerting authorities. For law enforcement agencies to track such trades requires well-operated teams, international cooperation, and scientific resources including forensics. Time and time again, a common issue faced by the authorities is related to the identification of such ceased products or articles generated from illegal wildlife trade. The confirmation of the source of origin from a protected wildlife species is important to demonstrate the illegal nature of an activity. Forensic science is an applied field. The investigative tools of forensic science are end-user base. Usually, these tools are applied for human-related crimes. A specialized branch of forensic science that deals with nonhuman crimes or wildlife crime is called wildlife forensics. From an investigator’s perspective in wildlife crimes, there are often no victims to give testimony. Additionally, species identification becomes an important task. DNA analysis is performed routinely in wildlife forensic investigations, and it is often compared with conservation genetics. Although the nature of work is similar, the complexity and challenges faced by the wildlife forensic investigators are different. The investigation process from a crime scene to courtroom trial is similar in comparison to routine human-related forensic analysis. However, most well-equipped forensic laboratories and associated scientists do not perform wildlife-related sample test. This is due to the type of samples involved and analysis techniques to perform, which require different expertise to those acquired by common forensic laboratories. The type of samples analyzed by wildlife forensic scientists is vast. It ranges from a whole body of an animal in both live and dead condition, as well as in all stages of life (animals seized by customs authorities, wildlife protection cell): skin, shells, or exoskeleton (horns, skeletal remains, nails) and animal body parts (genital organs, heads, limbs, internal organs). Additionally, some pieces of evidence can also involve traces of DNA from body uids or highly processed animal body parts. The challenges are faced when samples carry mixtures of processed animal parts, such as traditional Chinese medicine (TCM). Analysis performed by labs catering to wildlife forensic analysis is directed by the investigative questions asked by the investigator concerning the case involved. The frequently asked questions can be put into the following five categories (Ogden and Jen 2015): 1. 2. 3. 4. 5.

Species of origin Geographic origin of a confiscated article/evidence Wild or captive source Individual origin of an article Age of an article

Scientists answer these specific questions by performing standardized techniques. For example, in queries related to species of origin, a mitochondrial DNA analysis for a conserved region, such as region coding for cytochrome b and cytochrome oxidase subunit I in mammalian specimens, can be performed. If a query is for an individual origin, then tests for nuclear DNA are more suitable.

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Identification of Species of Origin One of the common most queries is related to the identification of species of origin of a confiscated article or specimen. Species identification can also be considered as a primary step before performing individual identification of an animal, because the techniques performed for individual identification, such as short tandem repeats (STR) and single nucleotide polymorphism (SNP), require prior information on the DNA sample being tested. Therefore, individual identification is performed after species identification using DNA techniques if the species of origin is not clear otherwise. In some cases, individual identification is not required; only species identification is sufficient to verify the commission of a crime, for example, if a suspected consignment is tracked and confiscated by forest officials in a transporter carrying goods out of a forest area. If the suspected product contains monitor lizard parts, it does not matter which monitor lizard. It is protected under Indian Wildlife Protection Act, 1972, and internationally under CITES. Similarly, if a meat product is labeled as 100% pork and found after testing it contains an amount of beef, it doesn’t matter from which cow. It is a type of a food fraud, where a product is sold under a different misleading labeled content. A species is a group of organisms having common characteristics and usually mate in a group to produce fertile offspring. A group of organisms belonging to a species shares a large amount of their DNA. On a phylogenetic tree, closely spaced species will share more DNA than the distant ones. Species testing gains more when dealing with cryptic species and phenotypic plasticity. Cryptic speciation is a process where different organisms belonging to different species share morphological similarities. For example, the skipper butter y (Astraptes fulgerator), a widely distributed butter y that was reported in 1975, is confused for a single species for a long time. It was tested for species identification with recently invented DNA barcoding technology. It was found to be ten different species sharing similar morphological characteristics (Hebert et al. 2004). On the contrary “phenotypic plasticity,” which is also referred to as “polyphenism,” is when a single species is showing different morphological characteristics in uenced by a different environment. The European map butter y (Araschnia levana) is a good example of phenotypic plasticity: the species displays the morphological difference in spring and summer season. To apply DNA-based identification for species testing, the targeted locus of DNA should possess some qualities to qualify for species testing. A DNA test performed with such locus should be able to give constant results. That is, it should be able to identify species correctly and to be able to differentiate a species from another. To obtain such results, the interspecific variation of a targeted locus should be more for interspecific differentiation, and, at the same time, the intraspecific variation should be less to correctly identify a species. Species with a recent diversion on the phylogenetic tree would be challenging to differentiate with this method. Also, species identification can be problematic for animals that are produced on inbreeding among closely related species, especially when one species is protected and another

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is not, for example, the problems related to differentiation of wild boar from domestic pigs: There have been constant appeals from farmers and their representative bodies in India to declare wild boar as vermin, to ease the strict laws on hunting of the species. As per India’s Wildlife Protection Act (WPA), 1972, wild boars are protected under Schedule III, and anything related to its poaching is illegal without the forest department’s permission. Wild boars have been causing problems to farmers of certain states of India. In such a situation, more complication is caused by inbreeding of wild boars and domestic pigs. There is evidence that the two species have been inbreeding again (Frantz et al. 2015). Legally it gets challenging to identify a species where inbreeding is common and one species is protected and another is not. It makes prosecution challenging under WPA, 1972. Mitochondrial DNA (mtDNA) is preferred for species identification for its advantages over nuclear DNA. mtDNA is present in high copy number, and it is also robust because of its membrane protection. It also shows a greater divergence in closely related species as compared to nuclear DNA. This is because it lacks correction enzymes that are present in nuclear DNA. The absence of correction enzymes will lead to higher divergence in closely related species because of uncorrected mis-insertions. Also, the maternal inheritance of the mtDNA helps in linking members of the same species of one inheritance. There are other practical reasons to pick mtDNA as well. mtDNA has been used for phylogenetic studies for a very long time, and because of that, there is a great amount of data available in the scientific literature. There are universal primers available in the market which ease the process of identification. The high copy number of mtDNA helps in the forensic identification process since often samples are in processed or degraded forms.

Species Testing Samples seized by law enforcement personnel that require identification of source species are usually of two categories. A sample could be sourced from a single species such as ivory, hair, skin, and skeletonized remains. Any suitable gene for species identification can be selected. The method of identification depends on the type of sample.

Sequencing There are many tests available for species testing. Out of which some have relied upon more as compared to others. DNA sequencing is one such technique that is performed routinely in labs. This method is also approved by the International Society for Forensic Genetics (ISFG) (Bär et al. 2000). If a sample is suspected to be sourced from a single source, then, using a universal primer, a small section of DNA is amplified. Usually, mitochondrial DNA is preferred. The amplified products are sequenced. The sequencing will result in a form of an electropherogram, in which sequence of bases is identified. An electropherogram will be interpreted and translated using the software. It’s important to verify the sequence of a fragment; this can

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be achieved by using a reverse primer and corresponding it with the forward read. Another option is multiple time sequencing of a fragment to verify the sequence.

Species Specific Primers In cases where a mixture of DNA samples of different species is required to analyze for species identification, universal primers are not suitable. Universal primers that are applicable generally around species will fail to obtain separate profiles. At molecular level, two species are largely similar other than specific DNA sites. These minor sites or bases make all the difference. These minute differences need to be identified to separately identify species from a mixture of DNA. Separate identification of species in a mixture of DNA can be performed using speciesspecific primers. These primers are specifically designed to target unique sites of a species to identify it correctly. These primers provide an advantage over traditional sequencing methods, which include less time- and cost consuming; simultaneously identification of multiple species, species identification in mix sample, and degraded DNA samples may still provide results. Species-specific primers are developed through known sequence information about the species for identification. A variation that is particular to a species is identified, and primers are designed on the basis of this variation. The basis of this identification method is by aiming variable bases for primers, and products will not be formed unless the targeted species is present. Usually, one primer of a pair is uorescent labeled to be detected by a genetic analyzer. Species-specific primers can be used in several ways. A couple of species-specific primers can be used together. This can help to increase the specificity but can relatively increase the cost. This is because of the use of synthetic dyes, as it is difficult to synthesize labeled primers. A combination of one species-specific primer and a universal can be used, which gives an advantage of multiplexing several species-specific primer with one universal primer. By labeling only the universal primer, its relative cost can be reduced for detection of multiple species. Species-specific primers also have disadvantages like other tests. It has the ability to detect only one species; if a mutation occurs on the binding site, it may give falsenegative results due to the inability to bind to the targeted site. This can be overcome by using multiple primers. It is considered very unlikely that multiple mutations will occur, blocking both the binding sites. Multiple primers also give another advantage: if one primer accidentally binds to a non-targeted species, the other primer would not be expected to produce a product.

Common Locus Used for Species Identification The common most loci in mitochondrial DNA-based species identification in mammals are the cytochrome b (Cyt b) gene and cytochrome oxidase I (COI). Other mitochondrial genes have been used, such as the two ribosomal RNA genes, the control region (or D-loop), and subunits of mitochondrially encoded NADH dehydrogenase.

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Cytochrome b (Cyt b) Cytochrome b gene is responsible for the synthesis of protein cytochrome b. It is a part of a protein complex called complex III. This protein complex has a function of mitochondrial oxidative phosphorylation. The energy required for cell functioning in the form of ATPs is generated through oxidative phosphorylation. In this process, oxygen and simple sugars are consumed to create the end product, which is adenosine triphosphate (ATP). The length of Cyt b is about 380 amino acids, and it varies with species. It starts with conserved methionine codon and lasts with AGA stop codon. The role of Cyt b as a protein is to be a part of an electron transport chain in the mitochondria. It sits within the inner mitochondrial region and lengths it up to eight times. Within the transmembrane protein, several amino acid replacements are exchanged with diverse hydrophobic residues. Variation of amino acids, such as valine, leucine, or isoleucine, at a particular site can be observed among different species; they show similar properties. The position of the gene on the human mitochondrial genome is 14,747 to 15,887. Due to a change in the length of the HVI region of the mitochondrial genome, different species will show a change in the numbering of gene location. The presence of repetitive elements could also be the reason for a change in the gene location. The length of the gene is 1140 bp long, but only 400-bp-long initial stretch is employed (Irwin et al. 1991). Cytochrome Oxidase I (COI) Cytochrome oxidase is found in the inner part of the mitochondrial membrane. The protein complex is composed of a total of 13 protein subunits, out of which ten are encoded in the nuclear DNA and the rest three are part of mitochondrial DNA. Cytochrome oxidase I or subunit I is one of these three genes. It’s located between 5904 and 7446 in the human mitochondrial genome. An initial 658-bp-long region was proposed as a universal region for species identification in 2003 by a team of scientists and termed this region as barcode (Hebert et al. 2003). A database is maintained of a specified cytochrome oxidase I region of species around the world. Various species have been identified using DNA barcoding that includes various species of fish (Ward et al. 2009) and avian (Hebert et al. 2004). The Control Region (or D-Loop) It’s a noncoding region present on a mitochondrial genome. Its name is derived from the unique displacement loop structure created by a nascent short heavy strand that displaces the parental strand. It houses regulatory elements that are essential for replication and expression of the mitochondrial genome. The length of this region ranges from 880 to 1400 bp. The length among species varies based on the presence of the repetitive sequences. This region is sequenced for many species because of its availability its short size, and improved knowledge in replication and transcription mechanisms, it’s preferred for phylogenetic studies. The intro order comparison of the control region is specifically useful in the case of closely related species (Sbisà et al. 1998).

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DNA Repositories DNA repositories are valuable tools for species identification. Such repositories hold millions of DNA sequence database, which is entered by the researchers around the world. Although it is serving as an important tool, it holds some disadvantages. The databases available freely are nonregulated, which means the data entered into the system has chances of misinformation or errors which can lead to misidentification. For many who performed DNA-based identification, this reference database is a primary source of comparison. To overcome misidentification, ISFG suggested using a voucher specimen for comparison of unknown specimen and comment should be made if it’s not possible.

GenBank It’s a comprehensive open-source DNA database started in 1992. it is maintained by the National Center for Biotechnology Information (NCBI) in the United States. GenBank is a part of the International Nucleotide Sequence Database Collaboration, which comprises the DNA Data Bank of Japan (DDBJ), the European Nucleotide Archive (ENA), and GenBank at NCBI. GenBank can be accessed through the NCBI nucleotide database. It further gives access to associated information such as taxonomy, protein structure and sequences, genomes, and biomedical journal literature in PubMed. It has a sequence search and alignment tool called the “Basic Local Alignment Search Tool” (BLAST). EMBL The European Molecular Biology Laboratories (EMBL) also started in 1982 and maintained at the European Bioinformatics Institute (EBI) in the UK. It started with a repository of 685 entries of individual authors and genome projects groups. Individual submitters preferred the Web-based submission system Webin, while big genome sequencing centers enter data through the automatic procedure. It has a network browser called Sequence Retrieval System (SRS), which allows access to nucleotide and protein databases plus other databases. For sequence, various tools are available such as Blitz and Fasta. DDBJ DNA Data Bank of Japan is the sole Japanese database that collects DNA sequences from researchers and issues the internationally recognized accession number to data submitters. It’s mostly collected from Japanese researchers. It’s started functioning in 1986, and it’s the only DNA databank in Asia.

Identification of the Geographic Origin Wildlife crime has been a global issue. Any criminal activity related to wildlife happening in a country can be traced to a country thousand miles far. For example, an orangutan baby captured in a remote forest of Sumatra to which they are native

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could be transported to a buyer in the United States for private zoo collection (Foead et al. 2005). Wildlife trade which is rough counts for 20 billion USD per year (Alacs et al. 2009). It is leading to a rapid loss of biodiversity around the world. One of the challenges with the wildlife crime, which are precursors of illegal wildlife trade, is that it happens majorly in developing or underdeveloped countries, which are coincidently biodiversity hot spots. Such countries often lack the resources and ability to investigate and respond to such criminal activities. Identification of the geographic origin of a wildlife article gets important in some instances. Some species that are legal to trade in one country may not be legal in another. In cases where illegally sourced timber is laundered, the geological origin becomes vital to investigate. In other cases where a species trade is completely prohibited such as ivory, the source or origin can lead investigators in narrowing down the county and possibly the region of illegal activity taking place. Such information is vital to uncover international syndicates dealing in illegal wildlife trade and their pattern of working.

Principle of Identification of the Geographic Origin Determination of geographic origin is made simply through species identification. Species have distinct genetic makeup within their geographic range. Sometimes, the geographic isolation of a group of animals for several generations can lead to the creation of a new species altogether. For example, in Thailand, the trade of ivory is partially legal. The legal status of the ivory trade is only for domestic Asian elephants (Elephas maximus). However, ivory derived from African elephants (Loxodonta africana) is illegal. In a scenario where the law enforcement authorities have to differentiate one from another to test its legality, it can simply be achieved through testing for species of origin. However, establishing the geographic origin of a sample from a forensic genetics perspective requires narrowing it down to a reproductive population of origin. It is similar to the case of identifying a breed or variety of an animal or plant in food product identification (Woolfe and Primrose 2004). Identifying biological populations poses challenges due to the complexity caused by different levels of genetic variations, from stretched families to subspecies. Populations of species often have capabilities of breeding that result in exchanges of genetic materials. Mixing of genetic materials makes DNA markers less probable to differentiate among groups. Geographic origin is based on recurrently linking a sample to a particular population while successfully differentiating it from other populations of the same species. This requires a population of different geographic origin to be genetically distinct. Based on such genetic variations, genetic databases are created by identifying distinct genetic markers. Determination of geographic origin relies on the population markers associated with a particular population. The task of geographic identification is further complicated with the difference in genetic population signals and legal boundaries pertinent to law enforcement. The ability at which the spread of a population over an area and its spatial resolution can be identified further

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complicates the process. As compared to species identification where universally accepted tests and markers are available, geographic origin gets complicated with a focus on a single species location and defined investigative questions.

Methods of Determining Geographic Origin with DNA Markers Lineage Markers Traditionally mitochondrial DNA markers such as mitochondrial cytochrome oxidase I and cytochrome b genes are preferred for animals, while for plants rbcL and matK are preferred. These markers are ideal for species identification as discussed in the species identification part of this chapter. These are conserved regions of mitochondrial DNA. For identification of animals within species, it requires a hypervariable D-loop region or control region of mitochondrial DNA (Ishida et al. 2013). Similar to humans, D-loop hypervariable haplotypes in wildlife provide lineage information. Lineage information corresponds to specific geographic areas and ultimately enables to identify the geographic origin. There are several examples of the geographic origin of animals, such as African elephant ivory (Ishida et al. 2013) and cannabis (Gilmore et al. 2007). However, markers from mitochondrial and chloroplast DNA often fail to differentiate and identify samples among geographically distributed locations, which demand the use of nuclear DNA markers. Microsatellites The application of nuclear DNA tests for identification of geographic origin depends on the variation in the allelic marker frequency among populations. Microsatellites or STR markers show a high level of polymorphism. As the application of STR marker-based identification technology is being used and simultaneously developing in human identification, it is being used in the geographic identification of wildlife as well. It is used in several identifications, such as ivory (Wasser et al. 2008), tortoise (Schwartz and Karl 2008), bear (Andreassen et al. 2012), plants (Nazareno and dos Reis 2014), and fish (Glover et al. 2009). In a distinct population, a low number of microsatellite markers would give a sufficient degree of power for the identification of geographic origin. However, in a weakly structured population, it may take comparatively a greater number of markers to give confidence. In such a situation where due to the rise in the number of STR markers, it starts becoming excessive, an alternate maker technique may be employed, for example, the rhino indexing system. Rhino poaching for its ivory has been a threat to rhino conservation in India. For tackling such crimes, rhino index DNA system (RhODIS – India) has been developed by researchers of the Wildlife Institute of India in collaboration with WWF India. A DNA database is generated to build a DNA catalog of 749 individual Indian greater one-horned rhinoceros (Rhinoceros unicornis). The database is generated by analyzing microsatellite markers present in the nuclear DNA. Such 14 markers were employed to build a database that will create a baseline for linking a confiscated ivory item to the location of the crime (British Ecological Society 2020).

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Single Nucleotide Polymorphisms (SNPs) As a result of genome-wide analysis of biological samples in wildlife, several SNP markers are being identified and characterized in various wildlife species. This has given the chance to employ SNPs in wildlife forensic identification (Ogden 2011). Like STR markers, SNP markers not under selection display allelic frequency differences between different populations due to random genetic drift. Generating the same assignment power as of STR markers, a higher number of SNP is required because of their lower allelic variability. However, SNP markers hold an advantage over STR markers due to their accuracy, simple methods, and ease in interlaboratory data transfer. A big number of SNP markers can be analyzed simultaneously in a single sample. This technology is being utilized in human ancestorial investigation, which is leading its way to wildlife investigations (Helyar et al. 2007). SNP markers give the advantage of a selection of desired markers that are linked to genetic regions under selection for regional geographic conditions. These non-neutral markers provide better interpopulation divergence and relatively lower the number of markers needed for analysis.

Individual Identification DNA profiling has played a significant role in the routine forensic analysis linked with human identification. Today, it has been a gold standard test for human identification. On the contrary, wildlife forensics does not offer importance to individual identification of animals as it is in human forensics. It’s evident because individual identification of animals holds less importance in wildlife conservation barring some situations. In a certain situation, individual identification from animal processed products such as processed meat, animal parts of importance to poachers such as tusks, horns, fins could be performed. Individual identification could be performed for several reasons including curbing entry of illegally sourced meat into markets, identification of poached animals, and maintaining a DNA register for highly protected animals.

Individualization Testing Individual identification of DNA is based on the selection of variable DNA markers intraspecies and show differences among individuals. The ability of a DNA profile to differentiate or match an individual is based on statistical significance. When two samples of DNA are being compared together, a set of DNA markers are profiled, and their results are compared to declare their relationship. STR or SNP markers are used to create a profile having several alleles. The more the number of markers, the greater the degree of accuracy. If two samples are showing dissimilarity in the profiles generated, the likelihood of two samples originating from a single source is excluded. Two samples showing similar profiles suggest they may originate from a single individual.

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Short Tandem Repeats In human genetic identification, the type of STR markers used is either 4 (tetra)- or 5 (penta)-base-pair repeats; however, in wildlife genetic testing for individualization, 2 and 6 base pairs are suitable. STR loci with greater repeats are less common than loci with fewer repeats. Because of their commonness, STR loci with fewer repeats are more preferred. Many studies on wildlife genetic population reported many dinucleotide repeats for identification, but these were never intended for use in forensic analysis. In the past, each STR locus was analyzed through an individual reaction. The reported primer sets were not designed to be conducted in a multiplex. Many such previously reported STR markers are being multiplexed and employed in forensic analysis in spite of these problems. Commercial kits are available for nonhuman dinucleotide testing. These are for domesticated animals, such as dogs and cats. These kits are for commercial use and not suitable for forensic analysis. As per the recommendation of the International Society for Forensic Genetics (ISFG) commission on nonhuman genetic testing, tetra-repeats are to be used over dinucleotide repeats (Linacre et al. 2011). This recommendation is due to a problem with dinucleotide repeats. The problem is that the smaller repeats are unstable during DNA replication, which includes PCR as well. This results in the shortening of PCR products due to the loss of a repeat unit. This effect is called as “stutter” peak because now the PCR product is one repeat shorter. Such stutter peaks can be 30% or more of the size of the parent peak. If compared with tetra-repeats, it’s reduced to 15% (Butler 2010). Interpretation of results becomes difficult due to an increase in stutter products. In spite of the availability of many tests, the STR test is developed for very few species. It is necessary to develop a test for the species which are being tested commonly for forensic analysis. The application of STR in forensic analysis has got greater importance. This is due to some advantages linked with STR. STR loci are inherited from both parents if they are present on the autosomal chromosome. Due to a low mutation rate, two alleged offspring can be tested to determine their hereditary lineage. STR loci are sufficient for providing discrimination to permit variation within a population. Careful selection of STR markers is necessary to gain enough discriminatory power. An added advantage of using STR markers is that there can be multiple STR assessments at the same time. When a single STR is analyzed, it’s called “singleplex,” while analysis of two STR markers together is a duplex. In the human genetic analysis, 21 STR markers are being analyzed together, and it’s called “multiplex.” Unique genetic profiles can help to monitor the illegal trade of animal products in a commercial market. These products can be profiled even if they are processed. For example, the minke whale population is decreasing because of several reasons; one of the major reasons is climate change, followed by human-induced threats including poaching. A market survey was conducted for the sale of North Pacific minke whales (Balaenoptera acutorostrata spp.) in a total of 12 markets of South Korea from 1999 to 2003. The products collected from these markets were profiled using a 464 bp fragment of the mitochondrial DNA control region and eight STR markers. Profiles were compared together to identify a number of unique profiles. The number of unique profiles would suggest the number of individual animals killed to make

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market-ready products. A total of 827 individual animals were recorded to be sold in the market over the duration of 5 years estimated from the information generated from DNA profiling. Compared to the official number of 458 bycatch, it’s almost doubled, suggesting extensive killing of whales (Baker et al. 2007). To distinguish a legally sourced meat product from an illegally sourced, a DNA register can be maintained. A DNA profile of legally sourced meat generated in a certified laboratory can be entered into a database. In Norway, a database of minke whales having DNA profiles of 2676 has been established. The genetic profiles are created using ten STR markers, two sex-determining markers, and a mitochondrial D-loop region. This Norwegian DNA register has been successful in distinguishing between legally and illegally sourced whale meat entering the commercial market (Palsboll et al. 2006).

Nonhuman Paternity Testing Paternity testing in humans is a common practice. It’s usually performed to determine the biological parent of an offspring. In wildlife, genetic analysis paternity testing also holds importance in cases where the investigators have a question on captive breeding of an animal. If an animal is not captive bred, then there is a possibility that an animal may have been sourced from the wild, which in some national legislation is prohibited. The process of paternity testing in animals is similar to humans; it is based on STR analysis. The basis of this test is the likelihood ratio, where there are two opposing hypotheses: one hypothesis suggesting the tested sample is the offspring of the alleged parents and another hypothesis suggesting the sample is not an offspring of the alleged parents. Assuming the tested sample species is diploid and reproduces sexually, then an offspring will inherit one allele from each parent. For example, if the male genotype is M, N and the female genotype is O, P, then any offspring must be one of the four possible genotypes: M, O; M, P; N, O; or N, P. If only one parent is available for testing with an offspring and, the question is whether the animal being tested is a sibling of the already known offspring. And the question is whether the animal being tested is a sibling of the already known offspring. In between two offspring, the probability of a common allele is 0.5, while the probability of having both the alleles common is 0.25. There is also a probability of having no common allele despite being sibling, which is 0.25. Paternity testing is also having application in wildlife conservation. Captive breeding of endangered animals is a practice followed to increase the population of a species. It also serves as a valuable genetic resource. These captive-bred animals are then reintroduced in a depleted habitat. Accurate paternity testing becomes important for the management of the captive population and selection of suitable individuals of known genetic characteristics for release in the wild. A study was conducted on the captive population of giant panda. A technique that uses two highly polymorphic microsatellites and five less polymorphic markers is found to be highly effective and faster (Li et al. 2011).

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Conclusion While emerging technologies are giving tools to law enforcement agencies to fight wildlife crime, existing DNA technologies are capable of tackling most forensic questions as discussed in this chapter. The selection of genetic markers will depend upon the requirement of the case, the species being tested, its ecology, and biology. Every present DNA technology and markers have their merits and demerits, which one has to evaluate carefully before choosing it for a case. Choosing complementary markers with appropriate resolution could be the best practice to avoid demerits associated with any marker. An interdisciplinary approach is essential for the advancement of wildlife forensics. There are existing areas, such as conservation genetics and wildlife ecology, which have been dealing with knowledge of wildlife genetics, ecology, and biology traditionally. The existing knowledge of these fields could be a crucial factor in the advancements in wildlife forensics. Successful implementation of wildlife forensics is important for controlling wildlife crime, which eventually contributes to the conservation of endangered species. A collaboration between forensic scientists and conservation geneticists will benefit the overall protection of vulnerable wildlife to poaching and trade. In wildlife investigation, species identification remains the top priority. Wildlife forensic labs that perform wildlife DNA techniques are often loaded with samples requesting for species identification. Any article or evidence seized by enforcement agencies has to be first tagged with a species it is sourced from. In some cases where seized articles’ morphological characteristics are intact species, confirmation is relatively simple, for example, in the cases of tiger skin or deer antlers, where its morphology is important because its market value is depending on its looks. Some articles are deliberately processed to modify or change their function for reasons including its concealment, easy packaging, and transportation. Evidence that is a mixture of different animals is more challenging to identify. A sample containing multiple sources of DNA has to be analyzed differently. A multiplexing approach applies to such samples, as discussed in the chapter. However, the quality and quantity of the sample are crucial. Present technologies sometimes fail to successfully identify because of their limitation. The most damaging threat is the illegal international trading of wildlife articles. It takes precise information about the location of the crime or sourced articles to diminish the trade. Wildlife articles from rare species fetch more price; such species are often located in the isolated locations. Identification of such locations to stop further exploitation of wildlife becomes important. DNA techniques for geographic identification become an essential tool for such tasks. The science of association of wildlife articles based on DNA analysis to a specific location is much more complex than other areas of wildlife DNA analysis. The challenge is to identify a group within the species, which are isolated due to geographic barriers. It requires development of a test for a specific locality for a particular species. It is combined with population

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genetics and life history of the species being tested. Therefore, this type of testing requires detailed knowledge of the locality and a sizeable reference database for developing comparative standards. Forensic genetics techniques are now being employed to address various wildlife forensic issues discussed in this chapter. Wildlife DNA technology will undoubtedly see steady progress. However, it requires persistent efforts of research and development like human forensic genetics. It is also dependent on research in conservation genetics and related areas. The reliability of any forensic application depends on valid laboratory techniques, evidential security, data analysis, and result interpretation. Such issues must be looked after for the development of successful forensic tools. The development of a reliable reference database is expanding the ability to develop and apply forensic DNA techniques in wildlife.

References Alacs E, Georges A, FitzSimmons N, Robertson J (2009) DNA detective: a review of molecular approaches to wildlife forensics. Forensic Sci Med Pathol 3:180–194 Andreassen R, Schregel J, Kopatz A, Tobiassen C, Knappskog PM, Hagen SB et al (2012) A forensic DNA profiling system for Northern European brown bears (Ursus arctos). Forensic Sci Int Genet 6:798–809 Baker CS, Cooke JG, Lavery S, Dalebout ML, Yu M, Funashi N et al (2007) Estimating the number of whales entering trade using DNA profiling and capture-recapture analysis of market products. Mol Ecol 16:2617–2626 Bär W, Brinkmann B, Budowle B, Carracedo A et al (2000) Guidelines for mitochondrial DNA typing. Vox Sang 79:121–125. Birds through DNA barcodes. Plos Biol 2:1657–1663 British Ecological Society (2020) Researchers turn DNA detectives to aid rhino poaching prosecutions with forensic evidence. ScienceDaily. Retrieved December 21, 2020 from http://www. sciencedaily.com/releases/2020/12/201215164923.htm Butler JM (2010) Fundamentals of forensic DNA typing. Elsevier, Sydney Foead N, Elliott W, Ziegler S, Van Gool M, Nijman V (2005) WWF’s species action plan for the conservation of orangutans in the wild. WWF’s Species Action Plan, UK Frantz L, Schraiber J, Madsen O et al (2015) Evidence of long-term gene ow and selection during domestication from analyses of Eurasian wild and domestic pig genomes. Nat Genet 47:1141–1148 Gilmore S, Peakall R, Robertson J, Organelle (2007) DNA haplotypes re ect crop-use characteristics and geographic origins of Cannabis sativa. Forensic Sci Int 172:179–190 Glover KA, Hansen MM, Skaala O (2009) Identifying the source of farmed escaped Atlantic salmon (Salmo salar): Bayesian clustering analysis increases accuracy of assignment. Aquaculture 290:37–46 Hebert PDN, Cywinska A, Ball SL, Dewaard JR (2003) Biological identifications through DNA barcodes. Proc R Soc Lond Ser B Biol Sci 270:313–321 Hebert PDN, Stoeckle MY, Zemlak TS, Francis CM (2004) Identification of birds through DNA barcodes. PLoS Biol 2(10):e312 Helyar SJ, Hemmer J, Bekkevold D, Taylor MI, Ogden R, Limborg MT et al (2007) Application of SNPs for population genetics of nonmodel organisms: new opportunities and challenges. Mol Ecol Resour 11:123–136 Irwin DM, Kocher TD, Wilson AC (1991) Evolution of the cytochrome-b gene of mammals. J Mol Evol 32:128–144

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Ishida Y, Georgiadis NJ, Hondo T, Roca AL (2013) Triangulating the provenance of African elephants using mitochondrial DNA. Evol Appl 6:253–265 Li DS, Cui HM, Wang CD, Ling SS, Huang Z, Zhang HM (2011) A fast and effective method to perform paternity testing for Wolong giant pandas. Chin Sci Bull 56(24):2559–2564 Linacre A, Gusmão L, Hecht W et al (2011) ISFG: recommendations regarding the use of non-human (animal) DNA in forensic genetic investigations. Forensic Sci Int Genet 5:501–505 Nazareno AG, dos Reis MS (2014) Where did they come from: genetic diversity and forensic investigation of the threatened palm species Butia eriospatha. Conserv Genet 15:441–452 Ogden R (2011) Unlocking the potential of genomic technologies for wildlife forensics. Mol Ecol Resour 11:109–116 Ogden R, Jen M (2015) A review of wildlife forensic science and laboratory capacity to support the implementation and enforcement of CITES. United Nations Office on Drugs and Crime (UNODC) CoP17 Doc. 25 Annex 4 Palsboll PJ, Berube M, Skaug HJ, Raymakers C (2006) DNA registers of legally obtained wildlife and derived products as means to identify illegal takes. Conserv Biol 20:1284–1293 Sbisà E, Tanzariello F, Reyes A, Pesole G (1998) Mammalian mitochondrial D-loop region structural analysis: identification of new conserved sequences and their functional and evolutionary implications. Gene 205:125–140. https://doi.org/10.1016/S0378-1119(97)00404-6 Schwartz TS, Karl SA (2008) Population genetic assignment of confiscated gopher tortoises. J Wildl Manag 72:254–259 Ward RD, Hanner R, Hebert PDN (2009) The campaign to DNA barcode all fishes, FISH-BOL. J Fish Biol 74(2):329–356 Wasser SK, Clark WJ, Drori O, Kisamo ES, Mailand C, Mutayoba B et al (2008) Combating the illegal trade in African elephant ivory with DNA forensics. Conserv Biol 22:1065–1071 Woolfe M, Primrose S (2004) Food forensics: using DNA technology to combat misdescription and fraud. Trends Biotechnol 22:222–226

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Gianmarco Ferri, Beatrice Corradini, Francesca Ferrari, and Enrico Silingardi

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Forensic Botany and DNA Barcoding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Forensic botany refers to the study of plants and how they can relate to law and legal matters. Unfortunately, while widely known as a science, this discipline has few professionally trained botanists. Law enforcement workers and forensic scientists are no more informed about the science of botany with the consequence that few individuals understand the importance of plants in criminal investigations. Therefore, important plant evidence is frequently overlooked. Plants or their parts can place a person or object at a crime scene, verify or refute an alibi, and help determine the cause and the time since death, the time of a crime, the place where a crime occurred, or the reason for an illness. Plant DNA barcoding identifies species using recent advances in genetics, genomics, and bioinformatics using short DNA sequences, known as the DNA barcode. This concept was established almost 20 years ago, and it’s based on the comparison of sequences obtained from unknown species against a reference database with the purpose of identification.

Gianmarco Ferri and Beatrice Corradini contributed equally with all other contributors. G. Ferri (*) · B. Corradini · F. Ferrari · E. Silingardi Department of Biomedical, Metabolic and Neural Sciences, Institute of Legal Medicine, University of Modena and Reggio Emilia, Modena, Italy e-mail: [email protected]; [email protected]; [email protected]; [email protected] © Springer Nature Singapore Pte Ltd. 2022 H. R. Dash et al. (eds.), Handbook of DNA Profiling, https://doi.org/10.1007/978-981-16-4318-7_42

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This universal and highly standardized method has also proven its benefits in forensic investigations both for wildlife and plants, as several studies and real caseworks have proven over the years. However, especially for plants, some challenges in the implementation of this system still exist, mainly related to the current status of the global reference sequence databases. Efforts must be encouraged to enrich data engendered by barcoding projects so that forensic botany can benefit from it. This is particularly critical in countries with extraordinary biodiversity. Keywords

Forensic botany · DNA barcoding · Non-human DNA

Introduction The use of nonhuman DNA analysis in forensic science has seen a very rapid growth in recent years. Applications range from investigations of rape and murder of humans to cruelty and poaching toward domestic or wildlife animal species and analysis of plant evidence, bacteria, and viruses to aid the resolution of legal cases. Nonhuman biological specimens are often part of the physical evidence from crime scenes or involved in most forensic caseworks, and results have been used as evidence in court in a variety of cases (Miller Coyle et al. 2001). Forensic botany, otherwise known as plant forensics, is the study of plants and how they can relate to law and legal matters, and it’s a relatively recent young application of an old science. The kidnapping and death of Charles Lindbergh’s young son in 1932 was the first modern era case to use such botanical evidence in court (Bock and Norris 1997). This discipline regards the analysis of plant and their parts, such as leaves, owers, pollen, seeds, wood, fruit, and spores, plus plant environments and ecology. Unfortunately, it remains a little considered discipline in the forensic context today. Despite its poor implementation, the correct identification of plant species can be very relevant in forensic investigations, and the importance of the botanical evidence is amply demonstrated (Brown 2006; Stambuk et al. 2007; Margiotta et al. 2015). Traces of plants can be associated with crimes and could represent a valid aid in the process of environmental reconstruction as plants could act as indicators of a geographical place or an ecological environment. Also, plants can help experts in the temporal reconstruction giving information on the postmortem interval, the exposure time of an evidence, and the date of an hidden burial. Moreover, as highlighted in a recent works (Aquila et al. 2014, 2019), botanical traces can help the forensic pathologist to answer fundamental questions about a case, including the dynamics of the event, and the cause of death (suicide, homicide, or accident) or when autopsy findings are not sufficient to ascertain the exact circumstances in which death occurred (Aquila et al. 2014).

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Plants can be used in food traceability and quality control, illegal logging and trade, and investigations of poisoning with products derived from plants, among others (Coyle 2005). Another important field is the study of diatoms in drowning cases. The peculiar silica wall that resists to the acidic environment of the human body and survive for many years makes these golden brown algae as an important tool in forensic investigations especially when it’s necessary to determine if it’s a case of antemortem or postmortem drowning (Liu et al. 2020a; Zhou et al. 2020). Moreover another area of interest is the particular threat to some taxa that comes from the overexploitation for commercial trade in plants and their products, which has dramatically increased in recent decades. International conventions like CITES (Convention on International Trade in Endangered Species) are designed to combat illegal trades for endangered and threatened species, but the effectiveness of their governing rules and measures is highly dependent upon the rapid and accurate identification of the species of interest. However, the reasons why botanical evidence is still underused and mostly ignored in the forensic context are different and linked to a variety of reasons. A relevant reason concerns the first part of an investigation, that is, the recognition of pertinent plant evidences at crime scene and the subsequent collection, transportation, and preservation. This is a complex matter, requiring trained personnel, but unfortunately, very few professionals involved with law enforcement have an adequate background or training in botany. Even if properly collected and stored, a botanical evidence can be fragmentary or could be a portion of the organism, preventing the accurate species identification based upon traditional morphological and anatomical characters, and often requires the intervention of experienced taxonomists or specialized forensic botanists, which are very rare. Furthermore, the forensic reality is very different from that in which taxonomists are used to working as a complete intact and fresh specimen is collected very rarely by investigators. Typically, legal investigators should seek a botanist with well-rounded experience who possesses knowledge of the various specialties within the botanical field in question. Some of the various botanical specialties involve systematics (plant names), anatomy (plant cells), morphology (plant structures), ecology (relationship of organisms within the environment), and physiology, chemistry, and genetics. However, in common forensic scenarios where the specimen is incomplete, traditional morphological methods appear to be ineffective in the taxonomic identification the organism of origin. Forensic botany tried to implement molecular biology techniques developed firstly for phylogenetics and botanical taxonomic studies which are fast, more accessible, and affordable. The first criminal case that used plant DNA typing to gain legal acceptance was a homicide that occurred in 1992 in Arizona’s Maricopa County using a PCR-based fingerprinting technique of randomly amplified polymorphic DNA (RAPD) (Mestel 1993).

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In this case, a woman’s strangled body was found near some Palo Verde (Cercidium oridum) trees. During the investigation, in the back of the suspect’s truck, detectives found a few seed pods from a Palo Verde tree, which were sent for DNA analysis. RAPD analysis was carried out, and it was found that the banding pattern seen in these seed pods was identical to that obtained from the tree under which the woman’s body was found and had shown signs of abrasion thought to have resulted from the suspect’s truck. Moreover, each tree in the area was found to show unique banding patterns. This was the first instance for the admission of plant DNA evidence in court, and the jury convicted the suspect of first-degree murder. However, since RAPDs are not considered sufficiently reliable, their use in forensic science has since diminished. In another homicide case, three species of bryophytes (mosses) were found on the suspect and identified to species. DNA fingerprinting analyses of traces bryophytes demonstrated that they likely originated from the crime scene (Korpelainen and Virtanen 2003). Bryophyte material found on the suspect’s car tires, shoes, and clothes was successfully linked with bryophyte patches located at crime scene. This study further demonstrated the great importance of botanical evidence by virtue of the ubiquitous presence of plants and their parts in the surrounding environment. Plant fragments, e.g., bryophytes, can easily become attached to shoes and clothes, and they can be analyzed quite long after the plant has been fragmented. Another homicide was investigated using leaves of sand live oak, Quercus geminata, collected from three trees at the site where the victim was found and those found in the trunk of a suspect’s car (Craft et al. 2007). Four STR loci were analyzed, and the results showed that alleles from the plant material on the suspect’s car did not match those obtained from the three trees near the burial site across four loci. Although in this specific case it did not provide the required physical evidence to link the suspect’s car to the crime scene, the potential offered by the use of plant DNA evidence in forensic science was demonstrated. AFLP is another technique used to create a DNA profile for plant varieties and has been applied to marijuana samples by some authors to link growers and distributors of clonal material and the subsequent individualization of a sample (Miller Coyle et al. 2003). Those whole-genome DNA fingerprinting methods were used until a few years ago because they were the only ones available by the scientific community. They are quite adequate as a means of rapid screening, but the problem of reproducibility especially from degraded specimens and the need for high amounts of quality DNA and the difficulty of interpreting electrophoretic profiles in mixture prevent the standardization of results across laboratories, and subsequently their use in routine forensic investigations was abandoned after a short time. The fact of not having a standardized and reproducible molecular methods for many years, especially having to consider compromised forensic samples, has contributed to the poor consideration of forensic botany over time and to the fact that it remained an elitist subject accessible only to a restricted circle of few experts in the world.

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Forensic Botany and DNA Barcoding Almost 20 years ago, Hebert and colleagues of the University of Guelph (Ontario, Canada) launched the ambitious idea of creating a universal molecular system for the identification of every living species, reliant on the analysis of sequence diversity in small segments of DNA, called DNA barcoding (Hebert et al. 2003a). The ability of DNA barcoding to distinguish species from a range of taxa and also to reveal cryptic species has really revolutionized the taxonomic world, facilitating species identification by using short, standardized portions of the genome. As stated by the authors, in order for a region of DNA to be effective as a barcode, it must simultaneously contain enough variability to be informative for unique species identification; work easily on all group of animals, land plants, and fungi; be short enough to sequence in a single reaction; and contain conservative regions which can be used to develop universal primers. This ambitious project has been supported from the beginning by an international consortium (Consortium for the Barcode of Life, CBOL) of major natural history museums, herbaria, and other organizations (https://ibol.org/). Arguably, the greatest beneficiaries from this system will be the many professionals whose work involves solving real-world problems with broad impacts on all areas in which society interacts with biodiversity but whose job is not to carry out taxonomy, among which there are forensic scientists. A DNA sequence of nearly 648 bp of the mitochondrial gene cytochrome c oxidase 1 (COI) is commonly accepted as the universal barcode for the animal kingdom because it meets all the features of universality, reproducibility, high level of standardization, and high discrimination to the species level (Hebert et al. 2003b). The barcode approach demonstrated to have great potential for the identification of animal species, providing rapid and accurate recognition of unknown samples including partial fragments and compromised or old tissues, whose DNA barcodes have already been registered in a DNA sequence library. In agreement with forensic quality procedure and standardization requirements, the barcode identification system for animals has been widely applied in many forensic cases involving animals as the abundant scientific literature on the subject demonstrates (Dawnay et al. 2007; Wilson-Wilde et al. 2010; Johnson et al. 2014). In casework situations, DNA degradation and recurrent sample inhibition may complicate the recovery of a full-length 648 bp barcode. Although longer sequences give greater resolution, also shorter sequences unexpectedly provide excellent resolution at the species level (Dawnay et al. 2007; Hajibabaei et al. 2007). Therefore, for highly compromised material, shorter COI fragments can be sequenced with an alternative set of primer pairs developed to enhance the robustness of the test (Ferri et al. 2009). As is well known, the species discrimination capacity strictly depends on the quantity and the quality of data available in a reference DNA library. Then, one of the primary goals of DNA barcoding focuses on the assembly of a reference library of barcode sequences for known species recovered from multiple voucher specimens using standardized protocols.

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CBOL developed the Barcode of Life Data Systems (BOLD), an online freely available workbench that aids the collection, management, analysis, and publications of DNA barcode records together with morphological and distributional data. Furthermore, the BOLD platform provides an identification system, namely, BOLD Identification System (IDS), a dedicated species diagnosis tool, which accepts sequence queries for animal, plant, or fungal species identification to be compared with specimens already registered in the repository. While DNA barcoding enjoyed a remarkable success for animal identification, attempts to identify the barcode loci for plants have been more tortuous. DNA barcoding in plants presents challenges that are not encountered in animals. In plants, as a consequence of their evolutionary history, species boundaries are less defined. Added to this, there are logistic difficulties of undertaking identification of something like more than 400,000 land plant species known to date. Moreover, the mtDNA of plants is not suitable for species identification procedures since it is usually slowly evolving, resulting in the absence of interspecific variation, and has high intramolecular recombination and pseudogenes. Therefore, the standard barcode markers for animals could not be applied to the plant kingdom. The search for the corresponding DNA barcode focused on the plant chloroplast (cp) genome, which is an alternative to the animal mitochondrial genome. The cp genome could contain suitable barcoding markers because of its presence in each plant cell in a high number of copies and consists of conserved gene sequences. The downside of the chloroplast genome is its relatively low rate of evolution. Focus has been placed upon identifying those regions that evolve quite rapidly but still slowly enough to be present in all land plants and that are good candidates for robust, universal primers. After years of research, in 2009 the Plant Working Group of CBOL promoted a multilocus solution comprising portions of the two plastid coding genes rbcL and matK as the core system for land plant identification, remarking that the discriminatory power is to be expected lower than for the animal kingdom, with a discrimination value of 70–80% to the species level (CBOL Plant Working Group 2009). Even if the percentage of success doesn’t equal that of animals, this system could be useful for specific applications where the resolution to the species level is not always required, as in forensic casework investigation. Following the DNA Barcoding Consortium final decision on core barcoding markers for plants, our group tested the selected regions on some samples of local ora in order to evaluate the possible application to forensic botany (Ferri et al. 2015). Our efforts have focused on developing a DNA-based identification system that provides criteria to progressively identify an unknown plant sample to a given taxonomic rank also by any nonspecialist botanist. Our results, based on the official CBOL land plant barcode (matK and rbcL), as well as on two alternative intergenic plastid loci (trnH-psbA and trnL-F) tested individually and in combinations, showed as expected that a two-loci synergic

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approach best addresses the needs for species testing of plant material also for forensics in the context of local ora, and it must also be explored in other contexts to extend and further evaluate its usefulness. The selection of rbcL+trnH-psbA was driven by the availability of well-tested and robust universal primers for both loci which facilitate the reproducibility of results and the implementation of the method in forensics together with a large volume of sequence data available in public sequence databases. In the implementation of this multistep model, a less variable coding locus (rbcL) complements the resolution given by a highly variable and therefore potentially more discriminating noncoding marker such as trnH-psbA. The use of trnH-psbA had been well documented in the literature, and also the Consortium proposed it as an optional supplementary marker, together with the advantage of possessing greater sequence variability than the proposed core barcode loci. The results obtained confirmed the effectiveness of this assay based on barcoding principles in forensic investigations also for the plant kingdom with a higher success in identifying the correct species if the two loci were both analyzed, equal to nearly 70% of the samples of local ora analyzed. Depending on the matter at hand, this core barcode system could be supplemented with additional loci such as the noncoding region trnL-trnF, for example, in cases involving highly degraded tissue. Moreover, given the complexity and the great variety of the plant kingdom, it is to be expected that for some specific taxon groups particularly difficult to distinguish, the use of alternative markers may be required. As an example of this, Liu et al. reported a work in the peculiar forensic context of biosurveillance, conservation management, and policing illegal trades (Liu et al. 2018). They particularly focused on the genus Taxus whose species are part of the protected ora in China but critically endangered due to deforestation and land-use change, as well as illegal exploitation. This is a notoriously taxonomically difficult genus with ongoing uncertainty and disagreement about its classification as well as with a broad distribution across temperate of the northern hemisphere, covering North America, Europe, North Africa, and Asia. As they stated, policing this illegal trade requires an accurate species identification system. However, morphological characters tend to vary greatly within species of this genus and often with overlap among species, leading to ongoing taxonomic controversy which in turn causes uncertainty about the distribution range. In the mentioned study, three data sets, with a total of 4,151 individuals representing all the 15 currently known Taxus species worldwide, were used to determine the ideal DNA barcode and construct a structured species identification system for the genus considered. Five data analysis methods were tested for species discrimination power. Based on the performance of single barcodes and their combinations, they recommend trnL-trnF as the best single DNA barcode for Taxus and trnLtrnF + ITS as the best combined barcode to reach identification at species level.

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For plants, more than for animals, a correct identification at species level strictly depends on the state of the available databases (Meiklejohn et al. 2019; Ekrema et al. 2007). For trnH-psbA, the successful use of this marker depends on the number and the quality of sequences only in the GenBank repository, as the BOLD database does not accept queries from this marker but only from the standard accepted barcodes, rbcL and matK (http://www.barcodinglife.org/index.php/IDS_OpenIdEngine). Therefore the scientific community is kindly invited to submit the highest number of reference sequences of proven quality in order to minimize the risk of misidentifications or ambiguous and incorrect results and aid the implementation of this marker in the species identification process in the forensic field. Therefore the growth of the reference barcode library BOLD and the improvement of the bioinformatic platform to better support a multimarker barcoding in plants are undoubtedly basic requirements to encourage the implementation of the barcoding approach even in the forensic botany routine. Furthermore, the scientific community should be encouraged to produce more studies on the levels of inter- versus intraspecific variation in different taxon groups for the chosen barcode fragments, in order to enhance the confidence of results, for example, in such cases where there is not a 100% homology with the best closest match in the database search, a situation that is quite common for plants. After choosing the best markers for forensics, in order to facilitate the implementation and simplify its use by forensic laboratories, we developed an operating protocol based on a specific DNA extraction, amplification with universal primers, Sanger sequencing analysis of selected markers, and a description of the main steps for database search considering the latest suggestions of the Plant DNA Barcoding Project (Ferri et al. 2012). Few years ago, Meiklejohn and colleagues developed a particular and promising DNA barcode protocol suitable for processing complex forensic-type biological fragments recovered from surface soil samples. Its utility and applicability was broadly tested in the mentioned study with a wide variety of different types of fragments (e.g., seeds, leaves, bark, head, legs) (Meiklejohn et al. 2018). Actually, the Barcode of Life Data Systems (BOLD) and GenBank are the main public repositories of DNA barcode sequences (Fig. 1). A broad taxonomic barcode coverage has been achieved for certain groups in recent years, but this success has so far always been limited mainly to animal groups and restricted to specific geographical regions. A comprehensive barcode library could be defined as one that captures 95% of genetic variation, and this has been estimated to require a minimum of individuals per species (Bergsten et al. 2012); this is also affected by the geographical scale of sampling as well as the population structure of the species sampled. However, it is often difficult or impossible to obtain material from the full distribution range of a species, especially for plants. Therefore, many existing libraries are incomplete, introducing bias and possible misidentifications. These issues could cause serious problems for conservation and especially law enforcement regarding endangered species or species protected by international conventions (e.g., the Convention on the International Trade of Endangered Species of Wild Fauna and Flora, CITES).

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Fig. 1 The BOLD web platform interface and the specific identification engine page for the plant kingdom

Ideally, all barcode sequences contained in either database should have been derived from a vouchered specimen, initially identified by a taxonomic expert. However, given the inherent nature of any public database, it is inevitable that some erroneous data will be present. The generation and submission of incorrect

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sequences likely occurs due to misidentification of the original material, poor isolation techniques, contamination of cultures, endoparasites in insects and plants, and PCR-based errors. It is also known that sequence availability in GenBank varied greatly among taxa and markers (Kolter and Gemeinholzer 2021). Thanks to the barcoding project, a carefully validated library of reference sequences from voucher specimens of all eukaryotic life is available. BOLD already contains barcode sequences for 319,715 formally described species covering animals, plants, fungi, and protists (with ~11.9 million specimens), and it is still in continuous expansion (http://barcodinglife.org/index.php/TaxBrowser_Home, last access to data 4th January 2021). The BOLD Identification System (IDS) is the dedicated species diagnosis tool and accepts queries also from the plant kingdom for the selected barcoding genes rbcL and matk. Supported by an integrated bioinformatics platform, the use of BOLD-IDS is a very important source and could allow for a rapid, easy, and reliable identification even by nonspecialists who paste their sequence record into the web window. Actually, a forensic expert can rely on the availability of two reference databases to attempt to assign the unknown botanical evidence to the correct species to which it belongs (Liu et al. 2018; Pentinsaari et al. 2020). While GenBank aims for comprehensive coverage of genomic diversity, the Barcode database would aim for comprehensive taxonomic coverage of just a single gene or at most few genes per kingdom, but it ensures tighter controls over the sequences deposited. In fact, to be able to deposit a sequence data in BOLD, gene sequences must derive from a designated gene region, and they must meet quality standards and derive from a specimen whose taxonomic assignment can be reviewed, ordinarily through linkage to a specimen that is held in a major collection (Table 1). Integration of search with different databases and implementation of a multilocus barcode approach may improve the chance of reliability of results and come to aid in the decision process when making forensic species identifications. In a recent study, Meiklejohn et al. demonstrated that both databases perform comparably for plants and macro-fungi (~81% and ~57%, respectively). Moreover, their results illustrated that using a multilocus barcode approach increased identification success (Meiklejohn et al. 2019). However, beyond the theory, some challenges in the implementation of DNA barcoding in the forensic laboratory still last. As pointed out by Liu et al. referring to a group of organism of particular forensic relevance as the diatoms are (Liu et al. 2020b), they noted that a high percentage of incorrect sequences are deposited in GenBank, compromising the database searching. Reportedly, the original materials, from which the sequences were extracted, were not correctly identified. Thus, even if the barcode sequence was successfully obtained from the diatom evidence, it’s very difficult to verify all these sequences without correct reference sequences in the repository.

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Table 1 Scenarios that may be encountered in species identification searching in sequence databases (GenBank and BOLD) Situation The query sequence doesn’t already exist in the database

The query sequence exists in the database: a) Single best match with the exact sequence b) Multiple best matches with the exact sequence c) Best match with different unrelated sequence(s)

Description The genomic region for a given barcode is not yet available for comparison

Outcome Ambiguous or incorrect (falsepositive) ID, as comparison, is only achievable at higher taxonomic ranks (genus, family, order)

Query sequence is returned as the unique best hit with the highest statistical values Query sequence plus one or more identical sequences is returned as best hit with highest statistical values The true species is present in the reference alignment but the method failed to assign as the best hit

Correct ID at species level

Correct ID only at genus or family level (based on the taxonomic category giving the best match) Incorrect ID

ID Identification

Another important field of forensic botany that can benefit from the implementation of the barcode project and its high degree of standardization are the complex discipline of forensic palynology. Forensic palynology refers to the study of pollen and spores to prove or disprove a connection in criminal cases. Pollens have been utilized by experienced researchers for at least the last three decades to provide forensic evidence and knowledge in certain legal circumstances (Chen and Shi 2020). Pollen grains are utilized in forensic applications because they are exceptionally impervious to chemical attack (Schield et al. 2016). They can remain at the crime scene for long time after that the event under investigation happened. Forensic palynology has been particularly useful in cases where there is a suspected movement of evidence or where a crime has occurred in a location with distinct plant species. Analysts could tie individual criminals’ travel histories together based on finding a similar pollen species composition on seized evidence, possibly linking their crimes and providing direction for further investigation. Officials could determine illegal imports’ country of origin. One of the most famous cases, following the Bosnian war, regards the uncovering of mass graves where bodies had been moved from different locations. Pollen was one of the lines of evidence used to trace bodies to their original burial sites (Brown 2006). Traditionally, forensic palynology is done by examining pollen grains under a microscope and comparing them to known pollen morphology. This is a highly specialized skill, and

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there are few experts able to identify plant species based on the size, shape, and color of the pollen grains. After all, researchers estimate more than 400,000 species of plants living on our planet today. Forensic palynology is further limited by the labor intensiveness of morphological identification. Frequently it’s impossible to determine the exact species present; identification is typically to a genus or family of plants, a group of species, in other words. This limits the technique’s utility, because while many plant species occur in a small geographic range, the genus or family to which they belong may cover a much broader area. In a work of Bell et al. (2016a), authors revealed how identifying pollen through DNA barcoding could be a practical alternative although not without challenges. As underlined by authors, DNA barcoding has the potential to greatly increase the efficiency and accuracy of pollen identifications, allowing forensics for the first time to unlock the potential of pollen as a key geographic and temporal marker. However, compared to the classic barcode work ow, in order to better distinguish the mixed-species pollen samples, they underlined the need to implement a “nextgeneration” or “high-throughput” sequencing (HTS) method that generates multiple reads per sample of the set of DNA barcoding markers, allowing the simultaneous identification of several species from a single mixed-species forensic sample. High-throughput DNA sequencing (also known as DNA metabarcoding) is the methodological advance that could make pollen DNA barcoding more feasible. This method allows researchers to sequence multiple pieces of DNA at the same time, without separating them first. It’s a key innovation because forensic pollen samples typically contain a mixture of species. Without high-throughput sequencing, these species would first need to be painstakingly separated, and the same efficiency problems of traditional morphological analysis come back. With high-throughput sequencing, the whole mixture of pollen grains can be ground up in one sample, and the DNA is isolated and sequenced and matched to a database. To date, five markers have been employed for pollen DNA metabarcoding, including rbcL, matK, ITS2, trnL, and trnH-psbA with relative success (Bell et al. 2016b). The relative advantages and limits of different barcodes are mostly the same for pollen DNA barcoding as for general plant DNA barcoding. As underlined by Bell et al. (2016a), there are two main benefits of using a DNA barcoding method in forensic palynology field. First, this method is able to identify multiple taxonomy groups, and secondly, it more efficiently identifies parts of the organism that do not appear in the morphology. Even unlinked, as in a DNA metabarcoding context, multiple markers can generate better discriminatory power than one. Additionally, amplicon fragment size is important in HTS DNA metabarcoding, as the read lengths in many platforms are limited. In this regards, ITS2 and trnL have been successfully sequenced via HTS with sufficient overlap for paired-end reads. The long amplicon length generated by standard rbcL and matK primers, as well as the length hypervariability of trnH-psbA, poses a technical limitation, necessitating a redesign of primer pairs for shorter amplicon lengths. Future improvements in sequencing technology are likely to increase read lengths of HTS technologies and may alleviate this issue in the future. However, even after more than 10 years of the identification of the core barcoding

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regions for plants and the impressive work done by the Barcoding Consortium to collect sequences from as many species as possible, there are some challenges in the implementation of a validated species identification method for plants in forensic science. Difficulties in training qualified personnel primary involved in the collection of crime scene evidences are still an obstacle to overcome in order for forensic botany to become familiar to the forensic experts. Moreover, when analyzing plant evidence, forensics should be aware of the intrinsic limitations inherent in the identification of plant species with respect to the animal one. Among these, we remember the larger numbers of plant species globally, the complexity of molecular evolution of plant genomes that makes resolution at the species level more difficult to achieve, the poorer coverage of species in the sequence databases, and the need to use a multilocus system including at least two markers instead of being able to use only one as for vertebrates. In these years, forensic geneticists are more and more realizing the benefits deriving from the introduction of the DNA barcoding technique in assigning taxonomic names to unknown trace botanical specimens from a casework, even for the untrained user. The features of this easy but reliable DNA-based identification system might be able to bridge the current lack of a highly standardized identification process in forensic botany.

Conclusion In a short time, forensic science has seen an exponential growth of nonhuman genetics, and the contributions of new research findings to investigations in this emerging field are enormous. As widely demonstrated, forensic botany is a powerful tool for crime scene investigation. Even a small piece of plant may reveal important information for the court, and botanic materials may connect a suspect with a crime scene or reveal the manner and the time (PMI) of death (Hall and Byrd 2012). But only if an investigator is aware of the potential existence of that evidence any efforts will be made to search for it. It must be highlighted that a botanical evidence can be present in a crime scene at the microscopic level (such as grains of pollen) and that careful collection, documentation, and preservation of botanical evidence are critical to the evaluation of plant evidence. Unfortunately, it appears that despite the tremendous potential offered by plant DNA found as trace evidence in solving crimes, there has been little progress to date. Until recently, plant identification has been largely dependent upon morphologybased approaches, which in turn depended upon taxonomical specialists, who are generally the only experts on some specific groups of plant. Furthermore, traditional taxonomic approaches can rarely be scaled up for high throughput, making it inconvenient for routine forensic applications in species identification.

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DNA based-approaches have come to meet the limits related to botany classical and forensic use of identifying morphology. The introduction of DNA barcoding firstly in the taxonomic and systematic field with the aim of creating a practical, costeffective tool to assign any unidentified specimen to the correct species based on sequence diversity has really revolutionized the field. The current popularity of DNA barcoding relates to its potential power to allow quick identification of organisms for the widest group of practitioners and potential societal benefits to various disciplines of the methodology which must be accessible and easily carried out by multiple users. In recommending rbcL+trnH-psbA as a forensic two-loci barcode system for plants when species identification of an unknown specimen is necessary, some challenges must be kept in mind by users. Species discrimination with plant barcodes is typically lower than with COI in animals, being anyway limited by a discriminatory power near 70–80% even if adding more markers [23]. Some particular plant species belonging to complex taxonomic groups may require the use of additional and specific barcode markers to identify plant specimens or parts of them (Liu et al. 2018). However, the lack of solid, easy, and reproducible protocols prevents the routine use of these analytical techniques in forensic laboratories, especially for plant species identification even if in forensic casework it is highly relevant to be able to deduce the species origin of an unknown biological sample. The attractive features rely in the high standardization of the identification process which is rapid, practical, accurate, simple, and easily carried out by multiple users, especially nonspecialists of wildlife/botany. In addition, it is applicable to a vast range of animal and plant species in a universal manner and must, therefore, prove a valuable tool for establishing species identity for forensic application. Even if identification to the species level is not always possible, identification to higher taxonomic ranks can be helpful in forensic investigations as in many of the situations in which this method would be applied; the application of a broad species concept is accepted. While past works have validated the ability of COI sequences to diagnose species in most taxonomic groups of animals, plant species are harder to discriminate, and it is straightforward that a multilocus approach will be necessary. We emphasize that actually some technical difficulties in the sequence similarity database search, such as the in uence of the registered sequence length and quality, as well as the number of ambiguities, affect the results and the success of speciesspecies identification (Pentinsaari et al. 2020). This is a fundamental point on which we must commit to work in the near future for the quality and reliability of the data of the identification process, as admissibility of any evidence in court clearly requires a certain set of quality standards. However, when a complete global reference library of DNA based on international guidelines will be available in the public domain, and universal data standards are applied to DNA barcode records, the scientific community will be able to obtain reliable information concerning the barcode sequence for unknown nonhuman

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specimens, and this will modify in a relevant manner the approach on non-human forensic genetics. The expansion of the reference databases is an urgent priority of the scientific community, to include more species that might be of interest to forensics specialists. Moreover, it would beneficial if researchers submit to the literature more cases implementing the analysis of plant specimens to add evidence in solving the cases. In conclusion, sequence analysis of the DNA markers (COI gene for animals and the multilocus regions for plants) with a barcoding strategy proved to be a very sensitive and powerful technique for specimen detection and, together with its unambiguous and direct identification capacity, becomes a useful tool in routine forensic issues. It shows powerful universality and versatility at the species level and can sometimes provide insights beyond those obtained through morphological analysis alone. In the presence of a well-established reference library, an unknown sample can theoretically be identified to species using its DNA barcode sequences. Some more complex disciplines of forensic botany such as forensic palynology will greatly benefit from further development of advanced molecular technologies of next-generation methods of sequencing as they are the only ones that can guarantee a better resolution in cases of mixed environmental samples in which the single species component is difficult to separate. However, to realize the full potential of DNA barcoding for plants, and particularly its application to metabarcoding for samples consisting of mixtures of different species, systematic sequencing of reference collections in the future is required using an augmented set of DNA barcode loci, applied according to agreed data generation and analysis standards (Alotaibi et al. 2020).

References Alotaibi SS, Sayed SM, Alosaimi M, Alharthi R, Banjar A, Abdulqader N, Alhamed R (2020) Pollen molecular biology: applications in the forensic palynology and future prospects: a review. Saudi J Biol Sci 27(5):1185–1190 Aquila I, Ausania F, Di Nunzio C, Serra A, Boca S, Capelli A, Magni P, Ricci P (2014) The role of forensic botany in crime scene investigation: case report and review of literature. J Forensic Sci 59(3):820–824 Aquila I, Sacco MA, Ricci P, Gratteri S (2019) The role of forensic botany in reconstructing the dynamics of trauma from high falls. J Forensic Sci 64(3):920–924 Bell KL, Burgess KS, Okamoto KC, Aranda R, Brosi BJ (2016a) Review and future prospects for DNA barcoding methods in forensic palynology. Forensic Sci Int Genet 21:110–116 Bell K, de Vere N, Keller A, Richardson R, Gous A, Burgess K, Brosi BJ (2016b) Pollen DNA barcoding: current applications and future prospects. Genome 59(9):629–640 Bergsten J, Bilton DT, Fujisawa T, Elliott M, Monaghan MT, Balke M, Hendrich L, Geijer J, Herrmann J, Foster GN et al (2012) The effect of geographical scale of sampling on DNA barcoding. Syst Biol 61(5):851–869 Bock J, Norris D (1997) Forensic botany: an under-utilized resource. J Forensic Sci 42(3):364–367 Brown AG (2006) The use of forensic botany and geology in war crimes investigations in NE Bosnia. Forensic Sci Int 163(3):204–210

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CBOL Plant Working Group (2009) A DNA barcode for land plants. Proc Natl Acad Sci U S A 106: 12794–12797 Chen YH, Shi HF (2020) Research Progress on forensic palynology and its application in forensic science. J Forensic Med 36(3):354–359 Coyle HM (2005) Forensic botany principles and applications to criminal casework. Boca Raton: CRC Press Craft KJ, Owens JD, Ashley MV (2007) Application of plant DNA markers in forensic botany: genetic comparison of Quercus evidence leaves to crime scene trees using microsatellites. Forensic Sci Int 165:64–70 Dawnay N, Ogden R, McEwing R, Carvalho GR, Thorpe RS (2007) Validation of the barcoding gene COI for use in forensic genetic species identification. Forensic Sci Int 173:1–6 Ekrema T, Willassen E, Stura E (2007) A comprehensive DNA sequence library is essential for identification with DNA barcodes. Mol Phylogenet Evol 43(2):530–542 Ferri G, Alù M, Corradini B, Licata M, Beduschi G (2009) Species identification through DNA “barcodes”. Genet Test Mol Biomarkers 13(3):421–426 Ferri G, Corradini B, Alù M (2012) Capillary electrophoresis of multigene barcoding chloroplast markers for species identification of botanical trace evidence. Methods Mol Biol 830:253–263 Ferri G, Corradini B, Ferrari F, Santunione AL, Palazzoli F, Alu M (2015) Forensic botany II, DNA barcode for land plants: which markers after the international agreement? Forensic Sci Int Genet 15:131–136 Hajibabaei M, Singer GAG, Hebert PDN, Hickey DA (2007) DNA barcoding: how it complements taxonomy, molecular phylogenetics and population genetics. Trends Genet 23(4):167–172 Hall DW, Byrd J (2012) Forensic botany: a practical guide, 1st edn. UK: Wiley-Blackwell Hebert PDN, Cywinska A, Ball SL, de Waard JR (2003a) Biological identifications through DNA barcodes. Proc R Soc Lond B 270:313–321 Hebert PDN, Ratnasingham S, deWaard JR (2003b) Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc Biol Sci 270(Suppl 1):S96–S99 Johnson RN, Wilson-Wilde L, Linacre A (2014) Current and future directions of DNA in wildlife forensic science. Forensic Sci Int Genet 10:1–11 Kolter A, Gemeinholzer B (2021) Plant DNA barcoding necessitates marker-specific efforts to establish more comprehensive reference databases. Genome 64(3):265–298 Korpelainen H, Virtanen V (2003) DNA fingerprinting of mosses. J Forensic Sci 48(4):804–807 Liu J, Milne RI, Moller M, Zhu GF, Ye LJ, Luo LH et al (2018) Integrating a comprehensive DNA barcode reference library with the global map of yews (Taxus L.) for species identification. Mol Ecol Resour 18(5):1115–1131 Liu M, Zhao Y, Sun Y, Li Y, Wu P, Zhou S, Ren L (2020a) Comparative study on diatom morphology and molecular identification in drowning cases. Forensic Sci Int 317:110552 Liu M, Zhao Y, Sun Y, Wu P, Zhou S, Ren L (2020b) Diatom DNA barcodes for forensic discrimination of drowning incidents. FEMS Microbiol Lett 367(17):fnaa145 Margiotta G, Bacaro G, Carnevali E, Severini S, Bacci M, Gabbrielli M (2015) Forensic botany as a useful tool in the crime scene: report of a case. J Forensic Legal Med 34:24–28 Meiklejohn KA, Damaso N, Robertson JM (2019) Assessment of BOLD and GenBank – their accuracy and reliability for the identification of biological materials. PLoS One 14(6):e0217084 Meiklejohn KA, Jackson ML, Stern LA, Robertson JM (2018) A protocol for obtaining DNA barcodes from plant and insect fragments isolated from forensic-type soils. Int J Legal Med 132: 1–12 Mestel R (1993) Murder trial features trees genetic fingerprint. New Sci 138(1875):6 Miller Coyle H, Ladd C, Palmbach T, Lee HC (2001) The green revolution: botanical contributions to forensics and drug enforcement. Croat Med J 42(3):340–345 Miller Coyle H, Shutler G, Abrams S, Hanniman J, Neylon S, Ladd C, Palmbach T, Lee HC (2003) A simple DNA extraction method for marijuana samples used in amplified fragment length polymorphism (AFLP) analysis. J Forensic Sci 48:343–347

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Pentinsaari M, Ratnasingham S, Miller SE, Hebert PDN (2020) BOLD and GenBank revisited – do identification errors arise in the lab or in the sequence libraries? PLoS One 15(4):e0231814 Schield C, Campelli C, Sycalik J, Christopher Randle C, Sheree Hughes-Stamm S, Gangitano D (2016) Identification and persistence of Pinus pollen DNA on cotton fabrics: a forensic application. Sci Justice 56(1):29–34 Stambuk S, Sutlović D, Bakarić P, Petricević S, Andelinović S (2007) Forensic botany: potential usefulness of microsatellite-based genotyping of Croatian olive (Olea europaea L.) in forensic casework. Croat Med J 48(4):556–562 Wilson-Wilde L, Norman J, Robertson J, Sarre S, Georges A (2010) Current issues in species identification for forensic science and the validity of using the cytochrome oxidase I (COI) gene. Forensic Sci Med Pathol 6:233–241 Zhou Y, Cao Y, Huang T, Chen L, Deng K, Ma K, Zhang T, Zhang J, Huang P (2020) Research advances in forensic diatom testing. Forensic Sci Res 5(2):98–105

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James M. Robertson, Natalie Damaso, and Kelly A. Meiklejohn

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . History of DNA-Based Analysis of Plant Evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sample Types of Botanical Evidence, Collection, and DNA Isolation . . . . . . . . . . . . . . . . . . . . . . . . Sample Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Collection of Plant Materials and Soil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Isolation of DNA from Plant Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Applications of Analyzing Plant Material Associated with Forensic Evidence . . . . . . . . . . . . . . . Taxonomic Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Geographic Origin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Individualization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Case Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Applications of New Sequencing Technologies to Plant Evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . Guidelines and Standards for DNA Analysis of Plant Evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alternatives to DNA for Analyzing Plant Material Associated with Forensic Evidence . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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J. M. Robertson Research Support Unit, Federal Bureau of Investigation Laboratory Division, Quantico, VA, USA e-mail: [email protected] N. Damaso Biological and Chemical Technologies, Massachusetts Institute of Technology Lincoln Laboratory, Lexington, MA, USA K. A. Meiklejohn (*) Department of Population Health and Pathobiology, North Carolina State University, Raleigh, NC, USA e-mail: [email protected]

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Abstract

Plant material has been used in traditional forensics to reveal and support investigative leads and establish the geographic origin. Identification of plant poisons in a rapid timeframe may be crucial in the treatment of accidentally or intentionally exposed victims. The establishment of the time or season of death may be estimated from plant evidence. Genetic analysis of evidentiary plant material can aid in regulatory forensics in the elucidation of illegal trade of endangered species, to monitor the trade of illegal substances, as well as provide information on the adulteration of foods, medicinal and herbal products. Yet, despite the several advantages of having plant identification capability, most forensic laboratories do not offer this service. There are numerous reasons for this deficiency in operational offerings. Case examiners who are experts in human DNA identification generally have no experience with non-human DNA analysis. External experts would have to be used to provide botanical knowledge for an investigation. Examiners would have to be trained for provision of plant DNA evidence in court. Another issue is the perception of a lack of standard procedures to perform botanic investigations that include field collection of evidence and reference samples, DNA isolation techniques, and genetic analysis methods. The aim of this chapter is to provide information on the genetic analysis of plant material that may alleviate concerns to induce a more proactive approach to forensic botany. Keywords

Taxonomic identification · Individualization · Geographic origin · DNA typing · Forensics · Case studies · Plants

Introduction Traditionally, forensic laboratories rely on morphological examination of pollen, plant parts (e.g., leaves and owers), and physical characteristics of materials (e.g., sawdust and plant fragments). However, this approach fails with powders, seed mash, medicinal and herbal products, and plant fragments. It may be difficult to visually identify timber, which makes up a substantial proportion of the 35,000 plant species categorized as endangered by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). Likewise, there may be no morphological characteristics of plant material in the stomach contents of a deceased individual. Plant fragments in questioned soil usually cannot be identified using morphology with confidence. However, in all these situations where morphological examination fails, it may be possible that genetic analysis of the plant material will provide information on (1) taxonomy (e.g., genus, species, etc.), (2) individual matching, and (3) geographic origin. Forensic botany can be divided into two categories: traditional (e.g., human criminal cases) and regulatory (e.g., illegal trade, mislabeling). Table 1 lists several genetic methods that have been applied to plant material of forensic interest. A brief description of each of the

Method RAPD RAPD and PCR-RFLP AFLP AFLP Microsatellites (SSRs) Microsatellites (SSRs) Microsatellites (SSRs) Microsatellites (SSRs) Microsatellites (EST-SSRs) STR Multiplex by length analysis STR Multiplex by MPS SNPs SNPs and Indels SNPs and Indels STRs, SNPs, Indels Barcodes Barcodes Barcodes

Illegal substance Conservation Trace evidence Conservation Illegal substance Poisonous plants Adulteration Illegal substance

Forensic use Homicide case Adulteration Illegal substance Homicide case Homicide case Conservation Adulteration Conservation Illegal substance Illegal substance

√ √ √ √

√

√

√

Application Taxonomic identification

√

√

√ √

√ √ √

Individualization √

√

√

√

√

√

√ √

Geographic origin

DNA-Based Analysis of Plant Material in Forensic Investigations (continued)

Houston et al. 2018 Finch et al. 2020 Ward et al. 2009 Dormontt et al. 2020 Roman and Houston 2020 Bruni et al. 2010 Stoeckle et al. 2011 Carrier et al. 2013

Reference Carita 2005 Um et al. 2001 Carita 2005 Koopman et al. 2012 Craft et al. 2007 Jolivet and Degen 2012 Bosmali et al. 2012 Vlam et al. 2018 Vašek et al. 2020 Young et al. 2020

Table 1 Summary of the progression in the use of DNA-based methodologies to analyze plant material associated with forensic evidence. Exemplar references are provided for each method and are not listed in chronological order. Abbreviations are as follows: RAPD, random amplified polymorphic DNA; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; AFLP, amplified fragment length polymorphism; SSR, simple sequence repeats; STR, short tandem repeats; EST, expressed sequence tags; MPS, massively parallel sequencing; SNP, single nucleotide polymorphisms; Indels, insertions and deletions; BAR-HRM, barcode DNA high-resolution melting; eDNA, environmental DNA

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Forensic use Medicines and conservation Medicinal herbs Conservation Adulteration

Illegal substance Trace evidence Trace evidence Trace evidence Trace evidence

Barcodes STRs, Barcodes BAR-HRM

BAR-HRM Metabarcoding Metabarcoding; eDNA Metabarcoding; eDNA Metabarcoding

Method Barcodes, MPS

Table 1 (continued)

√ √

√

√ √ √

Application Taxonomic identification √

√

Individualization

√ √ √ √

√

Geographic origin

Cui et al. 2020 Ng et al. 2020 Osathanunkul and Madesis 2019 Solano et al. 2020 Giampaoli et al. 2014 Fløjgaard et al. 2019 Boggs et al. 2019 Timpano et al. 2020

Reference Coghlan et al. 2012

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methods used in forensic botany investigations are listed from top to bottom to depict chronological introduction, starting with random amplified polymorphic DNA (RAPD) in the late 1980s, continuing to metabarcoding in the 2010s. References to articles are provided that present examples of the use of the techniques in addressing forensic questions.

History of DNA-Based Analysis of Plant Evidence The first DNA typing methods did not require knowledge of the DNA sequence. RAPD involves the use of primers of random sequence that bind to analogous binding sites dispersed throughout the genome. After amplification, a group of fragments is produced that can be separated by gel electrophoresis, producing distinct patterns. RAPD analysis was the first technique to be applied in a forensic case for a homicide in 1993, in an attempt to link plant evidence in a suspect’s vehicle to trees at the location where the victim was found (discussed by Carita 2005). In this case, there was insufficient collection of reference material to rule out a match by chance. However, RAPD did provide sufficient reproducibility to distinguish ginseng roots originating from Korea and China by using long primers (20-mers) (Um et al. 2001). Another technique, called amplified fragment length polymorphism (AFLP) analysis, also provides characteristic fragment patterns on gel electrophoresis. It leverages species-specific variations in restriction enzyme recognition sites to produce restriction fragments that can be amplified after ligating adapters to the sticky ends of the fragments (Carita 2005). AFLPs were used to fingerprint a clonally propagated, common weed known as knotgrass, relevant to a homicide. Since knotgrass is ubiquitous, it was expected to be in the vicinity of many areas surrounding and including the crime scenes. In this case, unique genotypes could be reported due to careful reference material collection and analysis of genotypes of the knotgrass plants in the area around the crime scene (Koopman et al. 2012). The major issues involving the use of RAPD and AFLP methods are the requirement for intact non-degraded DNA and the often poor reproducibility between laboratories and analysts (Carita 2005). A gel analysis technique using simple sequence repeats (SSRs), randomly repeated 1–6 base pair units also referred to as microsatellites, has been used to distinguish plant materials such as timber, food adulterants, pollen, illegal substances (Table 1), and leaves in a homicide (Craft et al. 2007). Several articles involving microsatellite loci have been reported for successful timber tracing, and each uses multiple loci to provide statistical significance for discrimination. For example, five nuclear SSRs were used to discriminate timber and reportedly could distinguish the species only 14 km nearby as well as 836 km distant from the origin of the investigation (Vlam et al. 2018). In a modification of the technique, high-resolution melting of microsatellites and a DNA barcode were used to identify admixtures of 50% in lentils (Bosmali et al. 2012).

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Another type of molecular analysis that produces length-based patterns is the amplification of short tandem repeat (STR) sequences, which can be discriminatory among individual plants. For example, a panel of 19 STRs was found useful not only for individualization of the opium poppy but also for determining the geographic origin between eight countries (Young et al. 2020). In a modification of this technique, another group reported using massively parallel DNA sequencing (MPS) (also known as next-generation sequencing (NGS) and high-throughput sequencing (HTS)) of STR fragments to provide more information on allelic variants than a length-based approach provides (Houston et al. 2018). To accommodate challenging samples, such as those with degraded DNA, single nucleotide polymorphisms (SNPs) and short insertions or deletions of DNA bases (known as indels) have been targeted. These assays have smaller amplicons and are thus optimal for plant samples with low molecular weight DNA such as from processed, milled, or powder materials as well as from old or degraded samples. Additionally, a large panel of SNPs may be used to provide sufficient discrimination. For example, a panel of 17 indels and 68 SNPs was formulated and shown to work with degraded and trace amounts of DNA for molecular identification of grass, often an element of forensic evidence (Ward et al. 2009) and possibly difficult to discriminate morphologically. In the first report that describes a developmentally validated protocol for forensic botany, Dormontt et al. (2020) used an array of SNPs and indels for individualization of timber samples. A combination of STRs and SNPs has also been reported for individualization and geographic tracing of timber species (Ng et al. 2016, 2020). In the early 2000s, a supplemental molecular analysis invoked sequencing informative regions of the genome for species taxonomic identification. DNA barcoding involves sequencing a small segment of either plastid (i.e., chloroplast), mitochondrial, or nuclear DNA. The resulting unknown sequence is searched against known sequences in a public or private DNA sequence database, to determine the taxonomic identity based on the most similar sequence (Bell et al. 2019). DNA barcodes must have sequence conservation to provide primer binding sites and enough variability within the amplified barcode sequence to allow discrimination between species. In 2009, a few well-researched barcodes were selected as being optimal for plant identification (reviewed by Hollingsworth et al. 2011). A useful application of barcoding for the identification of poisonous plant species used two plastid barcodes (Bruni et al. 2010). Another application of barcoding is food authentication and adulteration testing, whereby the presence of unlabeled ingredients may be revealed (Stoeckle et al. 2011). One must be careful in designing a multiple barcode system, because bias can be introduced by preferential amplification of one barcode over others (Timpano et al. 2020). While barcodes may be capable of distinguishing different species and sample populations, they might not be capable of classification of crop type or geographic origin (Roman and Houston 2020). Barcoding is currently performed using Sanger sequencing and MPS, whereas metabarcoding, a spin-off of barcoding, is performed exclusively on MPS instruments. Metabarcoding is used to simultaneously characterize multiple taxa present in a bulk sample. For example, a questioned soil sample will contain DNA from a community of species (bacteria, plants, and other eukaryotes) that may be diagnostic for the habitat and thus provide

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information on its geographical origin. Associating reads generated during metabarcoding to both a target taxon and sample is achieved by adding short tags to the 50 -ends of generated amplicons (known as indexing or barcoding) and downstream bioinformatic processing. This technique allowed the separation of soil samples from 11 different habitats in Denmark (Fløjgaard et al. 2019). In contrast to identifying a species definitively with barcoding, it is sufficient in some scenarios to classify the plant components of the sample to the family or order level with metabarcoding. Additional discussion on the use of plant barcodes and metabarcoding, with several case descriptions for botanical forensics, will be presented later in “Taxonomic Identification” and “Applications of New Sequencing Technologies to Plant Evidence” sections of this chapter. Traditional barcoding has also been combined with high-resolution melting analysis of the amplicons (identified as BAR-HRM), to simplify taxonomic identification. The principle is to leverage differences in sequence length, GC content, and nucleotide variation that result in different melting curves and indicative melting temperature (Tm) values for the amplicons. The decrease in uorescence as the double strand amplicon melts is measured by real-time instruments commonly found in many forensic laboratories. The technique was successfully used for certification of timber in a theft and for discriminating drugs (Solano et al. 2016, 2020). BAR-HRM was described as another strategy to analyze low molecular weight DNA, especially when DNA degradation is suspected as shorter amplicons (i.e., mini-barcodes) can be used instead of the larger barcode amplicons (Osathanunkul and Madesis 2019). There are a number of books and review articles that provide comprehensive general knowledge on the DNA-based analysis of plant material that cannot be included in this short chapter. Forensic Botany, A Practical Guide is a collection of ten chapters that include basic botany, plants as evidence, and retrieval and analysis guidelines. Plant DNA Fingerprinting and Barcoding is part of the Springer Methods in Molecular Biology Series (Sucher et al. 2012) and provides protocols for the techniques and the necessary equipment for analysis (listed in Table 1). Kress (2017) provided a review covering the history of plant DNA barcoding, phylogenetics, taxonomy, and the applications of barcoding to forensics. Knowledge required to select the appropriate barcode for a specific botanical question is available (Hollingsworth et al. 2011). Issues in the application of DNA barcoding to forensic botany questions, such as commercial product authentication, are reviewed by Mishra et al. (2016). The use of barcoding in forensic palynology, in which pollen can provide information on the time of death, crime scene location, and suspect association with the crime scene, is discussed by Bell et al. (2016). Three reviews are available that focus on DNA metabarcoding. One presents metabarcoding issues to distinguish CITES-listed endangered species (Staats et al. 2016); another one describes bioinformatics challenges, amplification, and sequencing errors (Coissac et al. 2012); and a third specializes in analysis of soil evidentiary samples (Young et al. 2017). Referral to these references on forensic botany will provide answers to questions of the novice law enforcement officer, as well as of the experienced biologist.

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Sample Types of Botanical Evidence, Collection, and DNA Isolation Questioned botanical samples often include the familiar parts of a plant such as leaves, stems, roots, bark, seeds, and pollen; however, when encountered in a forensic investigation, these common parts may be in modified forms such as timber, powders, and herbal and medicinal derivatives (Fig. 1). Considering the different physical matrices and textures, metabolites, chemical inhibitors of PCR, and possible differences in the freshness and age of evidentiary plant material, no standard protocol has been adopted for all questioned samples. Instead, analysts have reported specific protocols tailored to the different forms this evidence can take. The information provided in this section is meant to familiarize the reader with botanical evidence types, best practices for their collection, DNA isolation techniques, and preservation and storage practices.

Fig. 1 Examples of plant material associated with forensic investigations: (a) wooden beads; (b) plant roots associated with soil; (c) grass seeds associated with soil; (d) leaf fragment associated with soil; (e), chainsaw; (f), wood shavings; (g), shipping container of timber; and (h), tea and spices. (Pictures kindly provided by Dr. Ed Espinoza (US Fish and Wildlife Service, National Fish and Wildlife Forensics Laboratory) and Microtrace LLC (Elgin, IL, USA))

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Sample Types Botanical evidence samples can be classified in two groups: macro, or visibly identifiable, and trace, which are identified via microscopy or other molecular and chemical methods. The macro sample group primarily includes CITES-protected, endangered whole plants and trees, plant fragments and wood, roots, fruits-seeds, dried and fresh mature leaves, owers, and algae. This could include leaves taken from a suspect’s vehicle (Craft et al. 2007) and blades of grass or seeds collected both near to and distant from a crime scene (e.g., roadway shoulder) (Ward et al. 2009; Koopman et al. 2012). Wood samples can include processed and fresh timber (Rachmayanti et al. 2009; Ng et al. 2016; Dormontt et al. 2020), saw dust, branches, pieces from tree trunks, crosssectional disks (Paranaiba et al. 2020), growth rings (Solano et al. 2016), bark, and dry shavings. There are multiple descriptions of chemically processed and heated plant material, sometimes presented as a mash from toxic seeds or moassel (hookah) from tobacco (Carrier et al. 2013) or hemp (Houston et al. 2018), along with visible amounts of teas (Stoeckle et al. 2011) and herbal, medicinal, and culinary powders (Mishra et al. 2016; Staats et al. 2016; Kress 2017; Osathanunkul and Madesis 2019; Cui et al. 2020). Articles describe analyzing various forms of opium including a) dried sap and poppy seeds (Young et al. 2020); b) uncooked, cooked (with gummy texture), and chemically processed (Marciano et al. 2018); and c) culinary poppy and pharmaceutical opium (Vašek et al. 2020), along with the derivative heroin (white and brown powder, along with black tar) (Marciano et al. 2018). Also included in the macro group are commercial food grains (Bosmali et al. 2012), fraudulent or misleading products (https://www.fda. gov/consumers/health-fraud-scams/fraudulent-coronavirus-disease-2019-covid-19-prod ucts), and residues from stomach contents obtained during autopsy. Reference samples are available for this group in the form of vouchers containing whole leaves and seeds that are obtained from local herbaria and xylaria for woods. Trace evidentiary botanical sample materials include small plant and wood fragments, owers, pollen, powders, and seeds trapped in clothing, shoes, hair, dust, textiles, ropes, and tape used to bind a victim but also can be adhered to car undercarriages and tires (Hardy and Martin 2012; Hall and Byrd 2012; Schield et al. 2016). Evidence may include samples from tape lifts, dust (Craine et al. 2017) collected from unexploded IEDs (Wilks et al. 2017), and personal articles. These items may contain distinctive pollen grains that can be used to limit where the explosive was assembled or places a suspect has visited. Small wood fragments from a door frame on a knife or crowbar may be presented as evidence for a break-in. Soil samples collected from shoes, shovels, tools, rugs, and bulk soil from specific habitats may have traces of rare or geographically isolated plant material (Giampaoli et al. 2014; Boggs et al. 2019; Fløjgaard et al. 2019). Trace plant materials can be used to link the presence of the suspect to a location or habitat, or to eliminate a location as a possible source of the material. However, caution is warranted in making this assumption because microscopically small plant material can also be transferred by secondary contact with another person or sharing transportation used by others, for example.

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Collection of Plant Materials and Soil Collecting visibly large evidence such as intact leaves, owers, pinecones, woody tissues, timber, and grass at a crime scene may seem like a simple operational task; however, an evidence collection team is advised to contact a local botanist or palynologist to guide them in what items to collect (Hardy and Martin 2012; Hall and Byrd 2012; Wiltshire 2016). Consultation is especially important in planning collection of appropriate and sufficient comparator materials, so that subsequent population genetic analysis of the questioned sample is statistically sound. To avoid clonality, the local specialist will be able to advise appropriate areas associated with the crime scene for collection (Craft et al. 2007). In addition, fragmentary botanical material associated with a crime scene may go unnoticed without a botanist present to identify it. Botanists use specialized equipment for field collection of large samples, and these items are commercially available or can be assembled from parts obtained from a home improvement retailer. For morphological examinations, leaves are not put in ziplocked bags or plastic food containers, but rather laid at between two paper sheets or field-pressing cardboard or pressed at in a special box designed for transport and storage to inhibit morphological changes such as curling. The plant parts either are allowed to dry naturally at ambient temperature during transport or are placed on ice (Craft et al. 2007). An archival article provides practical suggestions to collect plant species in the field based on convenience, speed, and simplicity (Nickrent 1994). For DNA analysis, leaves, cambria, and bark are protected from bacterial and fungal degradation by placing them in ziplocked bags with a drying agent. Recent guidance on field collections that are suitable for DNA analysis has been provided by the Global Timber Tracking Network (https://globaltimbertrack ingnetwork.org/2019/02/07/new-gttn-sampling-guide/). Collection of fresh wood samples includes not only the obvious bulk materials, but also small samples such as 1 cm patches of bark and sapwood that are stored with silica gel (Vlam et al. 2018). Freshly collected wood tissue may be placed in 2 mL tubes with silica gel for transport (Jolivet and Degen 2012). The sources listed in section “Guidelines and Standards for DNA Analysis of Plant Evidence” provide in-depth information on the methods of botanical material collection and references of accepted guidelines. Soil samples may be collected at the origin of a crime scene to provide information on plant material of the habitat for comparison to other habitats. The taxonomy determined for the plant material in the sample may be useful for predicting geographic origin, especially if rare taxa and rich biodiversity are revealed. The depth of soil collection varies in reports of several authors, depending on the questions being asked. For example, samples have been collected from the surface to a depth of 3 cm for mock crime scene evidence (Meiklejohn et al. 2018; Boggs et al. 2019) and from cylindrical cores 5 cm in diameter by 15 cm long to capture the complete biodiversity of meadow and heath habitats (Yoccoz et al. 2012). To link soil to ecological habitats, one report described using a large volume of bulk soil, about 10 L, collected at a depth of 0–15 cm and from cores made in a 40 by 40 m site (Fløjgaard et al. 2019). Soil samples typically are sieved to remove

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debris, roots, insects, and stones before transport in a collection container. Prior to storage, samples should have been either air-dried or heat-dried at 60  C (Giampaoli et al. 2014). For trace evidence sample collection, several techniques are used. Pollen and small plant fragments can be tape-lifted from the surface of fabrics, vehicles, and tools. The tape-lift method has the benefit of minimizing sample loss during transportation. The samples can be removed from the adhesive using organic solvents, such as hexane (Itamiya et al. 2020), or recovered using sterile forceps. Surface dust samples may be collected with a vacuum filter sock attached to a portable vacuum system available from several manufacturers. A portable vacuum device was described to collect pollen (Schield et al. 2016) as well as filter cassettes (Wiltshire 2016). To minimize contamination during trace evidence collection, it is common practice to use gloves, facemasks, and DNA-free devices. Another component of evidence collection is the use of a validated method to preserve the samples during transport to the laboratory. However, preservation of the collected plant material does not necessarily follow a standard method. The reviewed reports described methods that were convenient for field collection. Depending on its type, samples have been air-dried, placed in containers with desiccating agents, or stored wet over ice. The use of alcohol, DMSO, or formaldehyde to prevent fungal growth may be acceptable for preserving morphology of the tissue, but their use may decrease the PCR efficiency following DNA isolation. While no report was found that recommended immediate freezing to preserve the sample for genetic analysis, freezing is used for long-term storage once the sample is in the laboratory.

Isolation of DNA from Plant Materials The steps involved in obtaining DNA from plant material generally include (1) isolating and cleaning of the tissues, (2) grinding, (3) combining the sample with appropriate reagents and extracting, (4) measuring the DNA yield and purity, and (5) performing an additional purification if warranted, to remove remaining PCR inhibitors. In cases involving macro samples, one already has sufficient material to process. However, with trace evidence, the plant material may need to be screened under a microscope first to separate and isolate each putative plant sample one-byone (Meiklejohn et al. 2018). Plant fragments isolated from soil typically are cleaned with 5% bleach and rinsed with water before beginning the extraction procedure to remove possible fungal contaminants; this step is also recommended for voucher specimens (Meiklejohn et al. 2018). If the plant material had been preserved in alcohol and glycerol, it should be washed off before processing, as any residual preservative could decrease the extraction efficiency. Next, the DNA is released from the plant tissue via grinding. Given plant samples can be dense, fibrous and have thick and non-pliable cell walls, grinding to a powder in liquid nitrogen is often used to efficiently lyse cells and subsequently release the DNA. Samples can be ground manually with a mortar and pestle, but mechanical grinding is rapid and may produce a more homogenized material (Carita 2005). Seeds can be ground in a

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commercial blender. Hard tissues may require mechanical shearing. In the literature reviewed, the amount of sample to grind varied with the tissue type: fresh or dried leaves (5 mg–0.5 g), seeds (one to ten whole seeds depending on size or 0.1 g ground powder), fresh sawdust (5 g), and gummy material (e.g., 100 mg of tobacco). This variation in input amount is most likely due to the variety of matrices and various amounts of inhibitors and metabolites specific to each plant species. Thus, it is recommended to consult the relevant published literature to choose the appropriate ratio of plant material to volume of extraction buffer, to ensure successful DNA isolation. The first popular DNA extraction protocol for plant material used cetyltrimethylammonium bromide (CTAB). This protocol removes polysaccharide and tannin containments in the extract that inhibit restriction nucleases and other DNA processing enzymes (Murray and Thompson 1980). Commercial plant extraction kits became available in the 1990s, some of which were based on the CTAB procedure. Their popularity is due to the provision of an extract free of metabolites, convenience, and reasonable cost. Out of the papers we have referenced in this chapter that described extraction procedures, 42% used the same commercial kit, 23% used either the original or modified CTAB method, and remaining papers used a variety of kits or methods (Fig. 2). Following extraction, DNA quantity is typically measured either by UV measurement or uorescence, with purity established using the A260/A280 ratio (for protein and phenolic contaminants of plant origin) and A260/ A230 ratio (for protein contaminants). If the quantity is considered too low, it can be increased by whole-genome amplification using a commercial kit (Finch et al. 2020). If inhibitors such as phenolic compounds are co-isolated, the extract will be insufficiently pure and will reduce the success of downstream DNA analysis (Meiklejohn et al. 2018). These inhibitors can be removed by adding 1% (w/v) polyvinylpyrrolidone (PVP) to the extraction buffer (Jhang and Shasany 2012) or by Nucleospin Plant II Kit, 6% PrepFiler Express BTA Forensic DNA Extraction Kit, 4% DNeasy PowerMax Soil Kit, 2% DNeasy PowerSoil HTP 96 Kit, 2% EuroGold Plant DNA Mini kit, 2% FastDNA SPIN Kit for Soil, 2%

Other, 35% DNeasy Plant Mini Kit, 42%

Maxwell 16 Tissue LEV Total RNA Purification Kit, 2% Nexttec 1-step DNA Isolation Kit for Plant, 2% NucleoSpin Soil kit, 2% Patented Timber Extraction Method, 2% Plant DNA Isolation Mini Kit, 2% Plant Genomic DNA Kit, 2% PowerMax Soil kit, 2%

CTAB, 23 %

PureLink Plant Total DNA Purification Kit, 2% QIAamp DNA Stool Mini Kit, 2%

Fig. 2 Summary of plant DNA isolation methods employed in chapter references. Pie chart illustrates the relative proportions of the two popular plant DNA isolation methods with the remaining kits listed in the table

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using a polymerase designed to work effectively in the presence of such inhibitors (Meiklejohn et al. 2018). However, for wood samples, PVP is added to the lysis buffer due to their relatively high tannin content. When further purification was required, two reports described the use of two different template cleanup kits. A filtration method was also used to concentrate DNA in cases of low yield (Marciano et al. 2018). In summary, one can generally conclude that more than half of the scientific community prefers to use a variety of kits and procedures for DNA isolation, thought to be tuned to their particular plant samples. There is no standardized isolation protocol for all plant tissues because of the encountered biodiversity but also because the extraction efficiencies of different tissues may vary considerably. An all-encompassing protocol may bias taxa detection and genotyping, especially where there is insufficient DNA from one taxa or uninhibited extracts (Young et al. 2017). For example, wood is very dense, making extractions from a dry sample challenging (Ng et al. 2016). A step-by-step extraction protocol for DNA from heartwood is available (Vlam et al. 2018). It has been demonstrated that chloroplast DNA quantity decreases as samples are taken from the outer sapwood region toward the inner heartwood region (Rachmayanti et al. 2009). The quantity of samples available to isolate DNA is also a consideration against a standardized procedure, since it varies with the type of sample under investigation. To reduce the hands-on time and improve consistency, an automated protocol was developed for wood in which multiple sawdust samples were pretreated via cryogenic maceration and ground to a powder while wet (Paranaiba et al. 2020). Protocol selection may be dependent on the ultimate use of the DNA. For example, extraction without grinding was recommended for obtaining high molecular weight template suitable for whole-genome sequencing (Jagielski et al. 2017). Another issue is extraction from preprocessed samples, because the treatment may reduce the DNA quality and lower the yield. One example is the extraction of DNA from some heroin samples, including those that have been preprocessed. To obtain inhibitor-free DNA from such samples, commercial extraction kits including those that use magnetic bead or silica column technologies were evaluated for heroin samples. The resulting extract was subjected to membrane filtration to concentrate the template (Marciano et al. 2018). A nondestructive extraction method may also be needed in instances where sample morphology needs to be preserved. One such protocol was developed for pollen grains, where ethanol in a sealed capillary tube held at 60  C permitted DNA isolation without grain lysis (Kelley et al. 2020). Finally, researchers developed a protocol to reduce DNA yield variability from soil samples and recommended it for soil metabarcoding studies (Minich et al. 2018). When isolating DNA from plants, the same working conditions implemented to reduce contamination when processing human DNA samples should be followed. Surfaces and pipettes should be cleaned with bleach followed by alcohol to remove any existing contaminants in the workspace (e.g., residual biological materials). Basic hygiene principles must be deployed, such as wearing gloves and lab coat. Researchers performing highly sensitive procedures need to take special precautions to prevent contamination, particularly with low biomass samples. Tubes should be handled without touching the lid and opened one at a time, touching the small tip end

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and not the underbelly of the cap (Bell et al. 2019). One report recommended extracting in single tubes instead of plates to avoid cross contamination (Dormontt et al. 2020). The same authors warned about producing aerosols from bead beating and transfer of powders. Working in an uncluttered laminar ow hood and organizing both pre- and post-amplification space are necessary. Hood filters should be replaced regularly because they could trap seasonal pollen in the laboratory air and become a source of contamination or become ineffective after reaching filtration capacity. The same is true for laboratory coats, which can also trap plant debris and pollen and thus should be replaced or laundered regularly.

Applications of Analyzing Plant Material Associated with Forensic Evidence The loci and methods employed to analyze plant evidence are highly dependent on the questions being asked. Generally speaking, analysis falls into one of three key areas: taxonomic identification, geographic origin, or individualization (Fig. 3). In this section we will explore the commonly used loci and typing methods implemented in each scenario, along with examples of their use in forensic practice.

Taxonomic Identification Taxonomic identification to a particular species or higher taxonomic level is a crucial first step in analyzing botanical evidence to aid in forensic investigations. Traditionally, identifications have been conducted using physical characteristics that require largely intact material and expert taxonomists. However, in typical forensic samples, diagnostic characteristics are often absent or small, limiting classification only to higher taxonomic levels. DNA-based methods have been demonstrated to be a fast, easily implementable, affordable, and reproducible approach for taxonomic identification of forensic-type samples that are often fragmented, degraded, and compromised (e.g., food processing conditions). Plant identification can aid in forensic investigations by providing linkages between crime scenes and individuals, as well as determining cause of death. For instance, identification of vegetable matter in a victim’s stomach contents can be used to verify suspect’s alibi or constrain time since death (Bruni et al. 2010; Lee et al. 2020). Further, the identification of small plant fragments associated with soil such as grass seeds and pollen can provide useful investigative information (e.g., location of the crime scene and time of the year) due to their ubiquity in the environment, spatial and temporal variability, and transferability (Ward et al. 2009; Bell et al. 2016, 2019; Meiklejohn et al. 2018). Taxonomic identification using DNA-based analysis has also demonstrated to be a powerful tool to aid the investigation of illegal drugs (Carrier et al. 2013; Paranaiba et al. 2019), authentication of food and herbal medicines (Stoeckle et al. 2011; Coghlan et al. 2012; Osathanunkul and Madesis

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Plant Material

Taxonomic Identification

Geographic Origin

Wet-lab work

OR

Amplification of informative gene regions with taxon-specific or universal primers (rbcL, matK, ITS2, trnL, psbA-trnH)

Data analysis

Application

Sample Extraction & Quantification

Sequence and search reference database for taxonomic identification

Individualization B A C

Amplification of taxon specific STRs or SNPs

Comparison of Q to K

Generation of RMP or LR

Fig. 3 Simplified owchart for plant DNA analysis for taxonomic identification, geographic origin, and individualization. Abbreviations are as follows: STRs, short tandem repeats; SNPs, single nucleotide polymorphisms; Q, questioned sample; K, known sample; RMP, random match probability; LR, likelihood ratio

2019; Uncu and Uncu 2020), and enforcement of illegal trade of endangered or protected species (Ng et al. 2016, 2020). DNA barcoding is the most common method for taxonomic identification by targeting short yet informative regions of DNA that permit species discrimination. DNA barcoding ideally targets one or more standard loci that are usually highly conserved and easily amplifiable, produce high-quality sequence data (i.e., limited homopolymer stretches), and have high discriminatory power (Hollingsworth et al. 2011). To date, the search for a unique barcode for universal plant identification has been challenging as no single region meets the requirements in all taxonomic groups. Lower success of species discrimination for plants using DNA barcoding has been attributed to multiple factors such as hybridization, polyploidy, life history, breeding

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system, species history, level of taxonomic splitting, and seed dispersal mechanism (Hollingsworth et al. 2011). In 2009, the Consortium for the Barcode of Life (CBOL) Plant Working Group recommended the two-loci combination, ribulose 1,5-bisphosphate carboxylase (rbcL) and maturase K (matK), as the plant barcode given these loci together permitted 70–75% species level discrimination (Hollingsworth et al. 2009). Additional supplemental markers (trnL, psbA-trnH, and ITS2) were recommended in instances where species resolution is not possible with the two-loci approach and have been widely used as supplementary loci (Cui et al. 2020; European Network of Forensic Science Institutes 2015). Consideration of the appropriate markers based on question of interest and application is vital. For instance, rbcL mini-barcodes and psbA-trnH (~200 bp) may be sufficient for identification of highly degraded DNA and have been used for higher-level identifications (e.g., genus, family, etc.) for CITES-listed plant species (Dormontt et al. 2015; Staats et al. 2016). Even though matK can permit species-level discrimination, amplification and sequencing are less consistent than with rbcL considering a) DNA degradation can limit the recovery of the ~850 bp region typically targeted, and b) universal primer design is difficult and therefore class-specific primer pairs are needed at a minimum. With a forensic unknown, DNA degradation can impact data recovery, and selection of an appropriate primer pair for PCR is difficult. Understanding the limitations of the barcode markers is equally important. For instance, ITS2 primers are highly similar to the fungal ITS sequences, leading to off-target amplification and possible misidentification (Mishra et al. 2016). Implementing a tiered approach to barcoding can be useful. As an example, amplification and sequencing of rbcL is simpler and can still permit taxonomic identification to a higher level (e.g., family or order). The information gleaned from rbcL can subsequently assist in the selection of most appropriate DNA region(s) and associated primer pairs to verify the presumptive identification. Such a multi-locus approach for plants has been demonstrated to greatly improve the identification capability and reliability (Meiklejohn et al. 2019). A DNA barcoding database is a crucial component for accurate and reliable taxonomic identification. Currently, Barcode of Life DataSystems (BOLD) and GenBank are the two main public databases of DNA barcode data. Ideally sequence databases used for taxonomic identifications should consist of sequences generated from high-quality vouchered specimens housed in museum collections and herbaria, which were quality checked (e.g., vector contamination, proper translation for coding regions, correct bibliographic citation, and correct taxonomy) before inclusion. In public sequence databases, incorrectly labeled taxonomic sequences have been reported, which are primarily the result of poor-quality DNA sequences, contamination of the DNA sample (e.g., fungal endophytes), PCR-based errors (e.g., unintentional sequencing of pseudogenes), and obsolete or dated nomenclature (de Boer et al. 2014). Searching algorithms, such as GenBank’s basic local alignment search tool (BLAST), provide distance measures between two sequences and reports which sequence in the database are most similar to the unknown. These fast distance-based algorithms are limited to the database entries and therefore can provide misleading or ambiguous identifications. For instance, a false positive can

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result if a sequence for the true source species is not present in the database. Moreover, ambiguous identifications can result when there is overlap in the intra(within) and interspecific (between) genetic divergence, such that multiple species will share the same barcode sequence. Bell et al. (2019) found a small number of false positives due to contamination or misidentification, many of which could be eliminated with more stringent laboratory and data analysis methodologies, along with a more comprehensive reference database. Meiklejohn et al. (2019) observed ambiguous correct matches (i.e., multiple records with different taxonomic names that have the same top match statistics) when searching against both GenBank and BOLD and reported that multi-locus barcoding approaches provide greater precision and reliability. Overall, careful interpretation and reporting of search results is vital and will be discussed in section “Guidelines and Standards for DNA Analysis of Plant Evidence.”

Geographic Origin Somewhat akin to determining human ancestry, pinpointing the origin of plant evidence at either a population or regional scale is a question often posed in forensic casework. Geographic origin is primarily used to identify whether a violation or crime occurred (e.g., determining whether the species was sourced from a traderestricted area) and for investigative leads for illegal activity (e.g., the DEA’s Heroin Signature Program focused on determining where the poppy was grown). A modified application of geographic origin is for plant material associated with geologic materials (soil, dust), which can be used to constrain the circumstances of a crime. Identification of the plant community present in such a sample could serve to limit the search area in a homicide investigation. For example, Craine et al. (2017) characterized plants associated with outdoor dust and reported that samples from urban lower latitudes with hotter climates had a higher proportion of moss and grass, whereas those from rural northern latitudes with cooler climates had a higher proportion of pine and conifer. In a real forensic scenario, interpretation is likely not as straightforward given that a murder victim could have been transported or dragged over various soil types. A plant community recovered for origin prediction in such a scenario would likely be a mixture. Morphological examination of the trapped pollen grains on the victim’s clothing or hair may indicate a certain pathway was taken to a possible burial site. Machine learning models of the genetic analysis results could assist with such interpretations (Boggs et al. 2019), enabling the use of plants to predict sample origin even for highly mixed samples. For forensic purposes, a population is defined as a “group of individuals of the same species that live and breed together in a given geographic region, which gives rise to allele or haplotype frequency differences among populations” (Moore and Frazier 2019). Given this, assigning an individual to either a population or region depends on specific regions of the genome that exhibit spatial genetic structure. Given that plants are immobile, a “breeding” population will largely be defined by the mechanism in which a species achieves fertilization. For example, species that

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use insects, mammals, or birds for pollen dispersal can successfully breed with individuals several miles apart, whereas species that rely on wind or water for dispersal typically are restricted to individuals within a 500 m radius (Shivanna and Tandon 2014). Additionally, both natural and man-made barriers can restrict the ability for individuals to successfully interbreed, thus creating population structure that can be exploited for determining source origin. Population or region assignment of an individual plant is commonly achieved using nuclear STRs or SNPs, or SNPs from either the chloroplast or mitochondrial genomes (Table 1). To ensure assignments have statistical confidence, a comprehensive database comprised of genotypes from individuals across a species known distributional range is necessary, with a minimum of 500 samples per population recommended (Carracedo et al. 2013). Thus, while there are published examples of plant origin assignment for forensic applications with fewer samples in the genotype database, this analysis is typically only implemented in specific cases given the substantial investment needed to create a large database. Origin assignments also rely heavily on specialized statistical software (most commonly STRUCTURE) to assign an individual to a specific population and provide statistical weight to the assignment (Moore and Frazier 2019).

Individualization As discussed in previous chapters, forensic laboratories globally have used DNA-based approaches to permit individual matching of human biological materials, such as blood, saliva, and sexual uid, associated with forensic evidence since the 1990s. Similar to human casework, individualization of plant evidence has been pivotal for prosecution and investigative leads associated with human and wildlife forensic casework. For instance, plant individualization can be used to link a suspect to a crime scene (Craft et al. 2007; Koopman et al. 2012), link seized wood material back to the stumps of illegally felled trees, and verify chain of custody (Dormontt et al. 2015). Completing individualization comparisons with plant evidence comes with a range of challenges not encountered when dealing with human biological evidence. First and foremost, the reproduction strategy of the plant will determine whether individualization is possible (Koopman et al. 2012). For plants that self-breed (10–15% of seed plants (Wright et al. 2013); e.g., orchids, rice) or are clonally propagated, the resulting progeny will be very similar or identical (except for rare mutations), respectively. Plants that reproduce via crossbreeding will produce genetically variable offspring (Koopman et al. 2012). The life span of the plant, annual or perennial, can also impact the success of individualization. If the evidence in question is from an annual plant, timely collection of reference samples to permit questioned-to-known comparisons or the generation of a population database is paramount before die off (Hall and Byrd 2012). An additional challenge when collecting known samples from the crime scene for comparison is the size and density of the plant species (Hall and Byrd 2012). For instance, if the size of the

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questioned species is small and there are hundreds of individuals at the crime scene (e.g., grasses), sufficient sampling to identify the source individual with certainty may not be feasible. Alternatively, if the plant is large (such as a tree) and sparsely distributed (i.e., acres to hectares), adequate sampling within proximity to the crime scene might be logistically difficult. Another key challenge is the vast number of different plant species that could be encountered in casework. Unlike human biological evidence which only deals with a single species, there are approximately 374,000 plants globally which could in theory be associated with forensic evidence (Christenhusz and Byng 2016). Highly polymorphic loci targeted for individualization typically are species-specific, meaning that typing will only be successful in closely related taxa (Koopman et al. 2012). Thus, initially determining taxonomic identity is imperative to ensure appropriate loci/markers are typed for individualization. For species more commonly encountered in casework, such as those used to make drugs (e.g., cannabis and poppy) and CITES listed timbers, informative loci that permit individualization have been identified (Ward et al. 2009; Houston et al. 2018; Roman and Houston 2020). However, the associated genotype databases are typically small (70,000 sequences, respectively, consisting of >500 bp length, while ITS has >15,000 sequences consisting of >100 bp size. A database is a structured collection of records or data that is stored on a computer so that it can be consulted by a program to answer queries, i.e., information generated through known analysis are used to predict the unknown (Berrington 2017). Every data recorded in databases has a label and a value. In case of DNA barcoding databases, sequences of different plant species at different barcode regions such as rbcL, matK, etc., are stored, which can be used to identify the plant species in question. Therefore, name of the plant species is a label and the sequence of specific barcode is the value (Tnah et al. 2019). A database is similar to an excel sheet as it contains rows (records) and columns (fields). Each row will represent sequences for different DNA barcodes for a single species, whereas, each column will represent sequences for a single barcode for different plant species. However, there are significant differences too. Firstly, DNA databases have a powerful data-manipulating feature, i.e., they can retrieve, search, match, and identify the added data from the previous recorded data. Secondly, it has the feature of cross-reference from each column or different tables as well. Thirdly, it is important for a database to have unique entries. It means that each entry in the database must be unique in itself for it to be considered as a new entry or else, the database will register it as an old entry (Berrington 2017; Tnah et al. 2019). However, there are certain problems which are faced when using DNA barcoding in the case of plants. To perform species identification using DNA barcodes, it is imperative to have reference sequences from authentic, reliable, and unadulterated reference materials. In the case of protected and medicinal plants, it is often very difficult to procure such reference samples. In other words, there is limited reference data to compare results (Hebert and Gregory 2005; Hollingsworth et al. 2011). Even in cases where such reference material is available, it might be difficult to include them in databases because of lack of cooperation between agencies. Although, BOLD has solved this to an extent, but work is still to be done so that more and more plant species are included in BOLD. Another issue arises due to the lack of standardized protocols/procedures to generate DNA sequences of uniform quality. It has been observed that more than 22% studies available for species identification have some kind of problem with the methodology used in the studies (Raja et al. 2017). Therefore, the aforementioned aspects must be accounted for to ensure fool-proof usage of DNA barcodes for the plant species identification in forensic casework.

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Conclusion Plant DNA barcoding is still in its development stage. From the first plant genome project till now, a lot has changed, new markers have been developed and new techniques have come into play to proceed with the work more precisely and that too in a less amount of time. In near future, a more exciting advancement is coming that is “metabarcoding” which can be helpful in identifying the species even if the plant materials are found in soil, water, or reef. More or less there has been an agreement on some of the markers to be utilized as standardized DNA barcodes in plant species identification (rbcL, matK and ITS2). It has been in high demand to update the database of plant DNA barcodes and the studies should have the correct and reliable data so that no further complication is faced. Number of organizations are working on finding a single universal plant DNA barcode, and the coming future might hold the answer to all the queries of the present. With the great combination of molecular genetics, current sequencing techniques, and bioinformatics; DNA barcoding will be helpful in solving criminal cases which involve evidentiary material of plant origin.

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Neeti Kapoor, Pradnya Sulke, and Ashish Badiye

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bioterrorism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Individualistic Microflora for Person Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Postmortem Examination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Detection of Body Fluids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microflora of Soil and Water for Forensic Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epidemiology and Microbial Forensics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sample Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Detection Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interpretation of Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Microbial forensics is a field that has attracted tremendous interest off late. Bioterrorism, biowarfare, and weaponization of the microbiome have terrified the world. While microbial forensics deals with the study of microbes for legal purposes, its applicability in the other subfields of health and science is irrefutable. It has its application not only to bio-crime investigation but also for clinical and toxicology purposes. There are several risks associated with the handling and analysis of microbes. Hence, it is essential to follow the established guidelines to prevent contamination, cross-contamination, and accidental infections with limited or widespread drastic effects. These guidelines mainly ensure the safety, N. Kapoor · A. Badiye (*) Department of Forensic Science, Government Institute of Forensic Science, Nagpur, Maharashtra, India e-mail: [email protected]; [email protected] P. Sulke Government Institute of Forensic Science, Nagpur, Maharashtra, India © Springer Nature Singapore Pte Ltd. 2022 H. R. Dash et al. (eds.), Handbook of DNA Profiling, https://doi.org/10.1007/978-981-16-4318-7_44

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collection, preservation of microbial forensic samples. This chapter covers a broad overview of epidemiology and microbial forensics, the critical elements of microbial forensics, the sample collection methods and guidelines, the various detection methods (molecular), and the result interpretation. Keywords

Microbial forensics · Microbial evidences · Processing and detection methods · DNA-based methods · Massive parallel sequencing · STR · VNTR · PCR

Introduction Microbiology refers to “the study of microorganisms, i.e., the organisms that exist as single cells or cell clusters and must be viewed individually with the aid of a microscope” (Nema 2018). These diverse communities of microbes are manipulated by humans and are used as biological warfare agents. With the increase in tools and technologies to manipulate these organisms, their use or abuse has also increased. Thus, the need to investigate such acts has also increased; herein, forensic microbiology or microbial forensics plays a vital role. Microbial forensics is the scientific discipline that analyzes evidence related to bioterrorism and bio-crimes, hoax, or inadvertent microorganism/toxin release for attribution purposes (Budowle et al. 2005b). Microbial forensics involves the characterization of microbial evidence with the help of microbiological methods to determine the source and assist in identifying incidences of bioterrorism, bio-attack, bio-crime, outbreaks and transmission of pathogens, or accidental release of a biological agent or a toxin (Budowle et al. 2003, 2005a; Oliveira and Amorim 2018; Schmedes and Budowle 2019; Smith 2019). Microbial forensics has its application not only to bio-crime investigation but also for clinical and toxicology purposes. The human skin is believed to have a unique microbiome that may be individualistic to a person. Therefore, microbial forensics also encompasses identifying a person from their leftover microbial traces on the materials they were in contact with. In addition to this, it also has its application in determining the cause of death, helps to investigate drowning cases, toxicological cases, estimation of postmortem interval, etc. (Oliveira and Amorim 2018).

Bioterrorism Bioterrorism is emerging as a global threat and one of the deadliest means used to cause mass disasters. This affects not only the economic condition but also the workforce of the country. The usage of bioweapon was prevalent for thousands of years. For example, the Romans were known to contaminate the water resource by decaying animal carcasses to harm their enemies. During the war of Kaffa, Tatar soldiers threw diseased bodies on the city’s walls to spread plague among enemies.

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Even in many wars such as WWI, WWII, French and Indian war, biological weapons were reportedly employed (Schmedes and Budowle 2019). There are other known incidences of potential bio-attacks. One such example is the bubonic plague attack in the middle of the fourteenth century in the Siege of Caffa (Budowle et al. 2005b; Barras and Greub 2014). Another classic example is the use of plague as a biological weapon by the Japanese during the Sino-Japanese war in the 1930s–1940s (Budowle et al. 2005b; Barras and Greub 2014; The College of Physicians of Philadelphia 2018). Salmonella was used to contaminate the salad bars in the Dalles (Török et al. 1997; Schmedes and Budowle 2019). In 1996, Dallas, TX, hospital technician intentionally infected the muffins with Shigella and placed them in the eating area. Due to this, 12 people were infected, and 4 were hospitalized (Kolavic et al. 1997; Schmedes and Budowle 2019). In 1993, anthrax produced by bacterium B. anthracis became the most potential bioweapon disseminated in Tokyo by the Aum Shinrikyo Japanese cult (Schmedes and Budowle 2019). The intentional use of pathogens and microorganisms to create biowarfare, the threat of terrorism, and detection of West Nile virus in New York City in 1999 was the major concern of the United States. In 2001, a 63-year-old employee of American Media in Boca Raton, Florida, suffered from fever, deprived sleep, emesis, and confusion. Later he was diagnosed with anthrax. The detection was confirmed by Laboratory Response Network, Department of Health, Florida. Meanwhile, at the commencement of the twentieth century, most cases of inhalation of anthrax in the United States were due to occupational exposure to infected animal skins or products. Thus, an urgent need was felt to improve the capabilities to collect, examine, and investigate the scene of incidence involving potential bio-crime acts (Morse and Budowle 2006). Letters laced with anthrax (Bacillus anthracis spores) were sent through United States Postal Service along the eastern seaboard, to two senators, to news anchor of NBC News, and the New York Post, each containing B. anthracis spores. These spores infected 22 persons causing 5 deaths and disruptions (Barras and Greub 2014; Schmedes and Budowle 2019). As a result, the use of the US postal system for spreading endospores of B. anthracis became a national threat, and the FBI began the investigation. Over the decades, the number of epidemic outbreaks has been increasing, such as H1N1 virus-swine-origin influenza A 2009, (Smith et al. 2009); Mycobacterium tuberculosis (Gardy et al. 2011); Vibrio cholerae (Hendriksen et al. 2011); MERS coronavirus, 2012, Saudi Arabia (Assiri et al. 2013); H7N9 virus, 2013, China (Kageyama et al. 2013); Escherichia coli O104:H4, 2011, Europe (Grad et al. 2012); Ebola virus 2014, Sierra Leone (Cenciarelli et al. 2015); Zika virus 2015, Brazil (Faria et al. 2016; Oliveira and Amorim 2018); Nipah virus, 2018, Kerala India; Ebola, 2018–2020, the Democratic Republic of the Congo and Uganda; Coronavirus disease 2019 COVID-19, worldwide 2019 to present; and Black fungus, 2021, India (Wikipedia Contributors 2021). The devastating effect of such epidemics, pandemics, and global bio-attacks led to the official launching of microbial forensics (Budowle et al. 2005b; Barras and Greub 2014).

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Individualistic Microflora for Person Identification The massive collection of microbes that reside on or inside a human body is termed as human microbiome. Personal identification is primordially made using DNA and fingerprints with a great degree of accuracy and validity. Nevertheless, some attempts have been made to individualize person based on the unique microbiome each human is believed to have (Fierer et al. 2010; Tridico et al. 2014; Meadow et al. 2014; Lax et al. 2015; Schmedes et al. 2017; Oliveira and Amorim 2018; Robinson et al. 2020). Several different microbial colonies reside in or on different parts of our body. To identify persons, skin microbiome or hair microbiome are potentially used. Oral microflora also has great significance for identification purposes (Robinson et al. 2020). Each individual has a unique skin microbiome composition, which can be collected from the items they came in contact with. Many studies are conducted on establishing the use of skin microbiome for individualization purposes (Fierer et al. 2010; Schmedes et al. 2017). As stated by Sir Edmond Locard in principle of exchange, “when two objects or entities or surfaces come into contact, there is always a mutual exchange of traces.” Similarly, when an individual touches any surface, they transfer their unique microbiome to that surface. This can be principally utilized in criminal identification. When a suspect touches any surface, it leaves traces there. Surfaces such as mobile phone screens, tablet screens, laptop screens, keyboards, mouse, floor, doorknobs, etc., act as an excellent source for harnessing the microflora of the person using it. Similarly, suspect’s unique microflora can be extracted from floors, victims clothing, or anything probable of being touched by him. Appropriate examination of the crime scene could assist in getting unique skin microbiome of the suspect (Meadow et al. 2014; Lax et al. 2015; Nema 2018; Oliveira and Amorim 2018; Robinson et al. 2020). Additionally, this can assist in finding geological locations of suspects based on the identification of microbes that are found explicitly in particular areas; however, this study is still in its stage of infancy (Oliveira and Amorim 2018; Robinson et al. 2020). When individualizing a person using skin microbiome, one should be aware and cautious of the fact that whole human skin is not supposed to have a uniform microflora. The human body is heterogeneous and consists of different substances in or on the body. Likewise, the skin has various compositional changes moving from hair to toes every part having slightly different microflora. Thus, when using skin microflora for identification, the place where it is generated should be looked upon. Hair is a shred of persistent evidence found on several crime scenes. Some approaches also made to use scalp microflora (also found on hair) for individualization (Tridico et al. 2014; Robinson et al. 2020).

Postmortem Examination PMI estimation: Determination of postmortem interval (PMI) is an integral part of postmortem examination. PMI estimation is predominantly done using sequential changes occurring in cadavers or entomological evidence. Many approaches are now

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directed to estimate PMI using microbial techniques (Metcalf et al. 2013; Hauther et al. 2015; Javan et al. 2016; Nema 2018; Oliveira and Amorim 2018; Zhang et al. 2019; Metcalf 2019). It is well known that microbes carry out decomposition, which is one of the postmortem changes in human cadavers. Decomposition occurs in several sequential steps, affecting the colonization of microbes on or in the body. This successional colonization of microbes can be used for the determination of PMI. This can be done using an ecological succession of microbes of skin or in internal organs of the body defined as thanatomicrobiome. Studies have been conducted to estimate PMI based on changing microbial community on the skin (Metcalf et al. 2013). Meanwhile, other approaches are based on the thanatomicrobiome, which harnesses the microbial community of internal organs and natural orifices, including gastrointestinal tracks, intestines, stomach, eyes, vagina, ears, lungs, etc. (Hauther et al. 2015; Javan et al. 2016; Metcalf 2019). Estimation of PMI using microbiology has great advantages and can also be potentially applied to calculate submersion interval by studying successional colonization of marine microbes with great accuracy. Further, based on statistical regression models, there are also approaches to estimate PMI (Zhang et al. 2019). There are external factors affecting sequential colonization of microbes on the corpse, which includes that till what time after death PMI can be accurately determined, the weather conditions (sunlight, wind, humidity, etc.) the body is present in, whether the body is covered or open or is buried in soil (Metcalf 2019). These factors also need consideration and also standardization through further research. Other postmortem examination: It includes determination of cause and manner of death. The cause and manner of death can also be potentially determined using microbial community. There are five manners of death, i.e., natural, accidental, suicidal, homicidal, and undetermined. Some studies show a predominance of a specific community of microbes for a particular manner of death, also some studies have shown that the abundance of specific taxa in different internal organs is also affected by the manner of death, which could be positively used to infer the manner of death (Oliveira and Amorim 2018; Robinson et al. 2020). However, the gender, age, and sex can also interfere in the interpretation of results. Thus, this cannot be used as a sole method of determination of cause and manner of death. It would require proper validation to be admissible in the court of law. There are numerous causes of death. Death can occur due to prolonged infection, drowning, poisoning, etc. Microbial evidence cannot provide the definitive cause of death but has excellent potential in determining the cause of death. In death cases due to drowning, examination of diatoms is the gold standard in forensic investigations (Díaz-Palma et al. 2009; Oliveira and Amorim 2018). Some microbial communities can be utilized to investigate these cases (Oliveira and Amorim 2018). In this order, some studies establish the use of microflora in toxicological cases and hospitalacquired infections (Castle et al. 2017; Oliveira and Amorim 2018). In addition to determining the cause and manner of death, microbiome can also assist in determining the sex of the cadaver (Bell et al. 2018). Some approaches are being made to differentiate the postmortem changes in the body based on sex to be employed potentially for sex determination (Bell et al. 2018).

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Detection of Body Fluids Biological fluids such as saliva, semen, synovial fluid, blood, vaginal fluid, menstrual blood, urine, etc., are common evidence that forensic investigators encounter at crime scenes. Several preliminary and confirmatory tests achieve identification of these fluids. However, the microbial diversity also has its part in the identification of different body fluids (Zou et al. 2016; Hanssen et al. 2018; Oliveira and Amorim 2018; Robinson et al. 2020). Different body fluids have characteristic microbial profiles where some microbial communities are found in abundance and others in traces, thus can be used as bioindicators confirming the presence of a particular body fluid (Hanssen et al. 2018; Oliveira and Amorim 2018; Robinson et al. 2020). For instance, a study conducted on the Han Chinese population for detection of a particular microbial profile of saliva, vaginal fluid, and feces suggested that the presence of Lactobacillus crispatus, Lactobacillus gasseri, Bacteroides uniformis, Bacteroides thetaiotaomicron can be used as identification microbes for vaginal fluid and feces (Zou et al. 2016). Similarly, many approaches are being carried out and many more are needed to establish several biomarkers specific to a particular body fluid that can be positively used for fluid identification.

Microflora of Soil and Water for Forensic Use Soil and water microflora can ultimately be used to identify geological locations as the microbial profile in soil and water changes over a few meters. Soil has its unique microflora, which can act as bioindicators to confirm two soil samples originating from the same site. Microflora of soil traces found on a crime scene, compared with the suspected soil site, can confirm the location (Oliveira and Amorim 2018; Robinson et al. 2020). In the same way, water microflora can also assist in determining geographical locations. Water microflora has its prime role in investigating drowning cases where diatoms are primarily employed for the purpose. The species of diatoms present in water and the composition of diatoms present in the lungs and internal organs of drowned bodies can confirm whether the person was dead or alive at the time of the drowning. Also, if some different diatom species or different microflora are seen in the body’s internal organs, it generates the line of doubt that the person was initially killed in some different environment. Other microbes can also be used as biomarker while investigating drowning cases (Levkov et al. 2017). Besides, there are studies conducted to examine the microflora of freshwater and seawater (Kakizaki et al. 2009). This application also needs some advancements and validation so that it can be potentially used to prove or disprove legal questions. Apart from diatoms, some other microbial markers should also be potentially included in such investigation.

Epidemiology and Microbial Forensics Epidemiology is defined as the manifestation, topographies, and causes of disease among populations. Epidemiologic methods to investigate the infectious outbreak by examining the possible evidence of intentional and criminal behavior as

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contributing factors are termed forensic epidemiology (Goodman et al. 2003; Morse et al. 2019). Similar principles of epidemiology were being utilized to investigate the bio-crime, which involved the bioagent in creating a threat (Flowers et al. 2002). Microbial forensic epidemiological investigation deals with the legal system that involves examining a crime scene, sustaining the chain of custody, validating the methods, and understanding of results and evolution of new methods to investigate bio-crimes. The investigator must use techniques that match the standard legal system, such as Daubert Standard, which will positively defend the crossexamination (Morse et al. 2019). There are important factors that need to be considered while investigating the outburst of contagious diseases, such as: • • • •

Occurrence of the outbreak Identifying the population and community at risk Mode of transmission and medium used for transmission Illustrating the agent(s) responsible for the dissemination of infectious disease

An epidemiologic investigation would try to recognize the contributing agent and its source of disease outbreaks. Various molecular techniques are used to examine, identify, and characterize pathogens involved in deadly bio-crimes. However, in microbial forensics, the investigation is used for legal purposes (Morse et al. 2019). Numerous indicators of outbursts can be identified in the epidemiological investigation of infectious diseases. The below-listed factors are the potential clue to indicate the signs of an epidemic (Morse et al. 2019). • Disease caused by unknown agents and without explanation of epidemic. • The presence of uncommon strain and its antibiotic resistance pattern. • Higher rate of illness and death due to uncommon disease and patients showing failure in responding to the treatment. • The distribution of uncommon diseases affected by the season and geographical conditions like influenza spreading in the Northern Hemisphere in the summer season. • Transmission of disease through different mediums such as air, food, water, and aerosol, etc. • The presence of one or more strains of the disease in one patient and its reason remains unknown. • The transmission of the disease affecting a significant heterogeneous population. • The unfamiliar pattern of morbidity among animals is caused by the unexplained agent responsible for causing the same effect in humans. • The unexplained and uncommon illness and death occurring in humans by the agent responsible for causing illness and deaths in animals. • Origin of agents of illness from the source having the same genotype. • Dissemination of uncommon illness to the noninfectious area either, domestic and foreign. • A large number of senseless deaths and diseases.

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The two essential aspects of microbial forensics are determining the reason for the release of pathogens, whether caused intentionally or due to some negligence and studying the application of protocols for monitoring the pathogens to distinguish between the unprompted and destructive spread of pathogens microorganisms. The bio-agents are classified based on the hazards on humans. This classification was given by the National Institute of Health (NIH) in 2019 (Department of Health and Human Services, National Institutes of Health 2019). In 2013, they classified these microbes into three categories, but in 2019, these were divided into four main categories below in Table 1. In addition, the Centre for Disease Control and Prevention

Table 1 NIH classification of bio-agents (2019) Risk Group 1 (RG1) Risk Group 2 (RG2)

These are the agents that do not have any impact on humans These are the agents associated with humans but are not severe and preventive, and therapeutic interventions are available

Risk Group 3 (RG3)

These are associated with serious or lethal diseases for which preventive or therapeutic interventions may be available

Risk Group 4 (RG4)

These are the reagents associated with serious or lethal diseases in humans for which the preventive or therapeutic interventions are usually not available

Bacillus subtilis, Bacillus licheniformis, Escherichia coli, etc. Actinobacillus, Bacillus anthracis, Clostridium botulinum, Dermatophilus congolensis, Erysipelothrix rhusiopathiae, Francisella tularensis, Haemophilus ducreyi, H. in uenzae, Leptospira interrogans, Mycobacterium, Pseudomonas aeruginosa, Staphylococcus aureus Fungal agents such as Blastomyces dermatitidis, Cladosporium bantianum, C. (Xylohypha) trichoides, Cryptococcus neoformans, Dactylaria galopava, etc. Parasitic agents such as Entamoeba histolytica, Enterobius, Fasciola including F. gigantica, F. hepatica, Giardia including G. lamblia, Heterophyes, Hymenolepis, etc. Viruses such as Alphavirus, Adenovirus, Coronavirus, Arenoviruses, Hepatitis A, B, C, D, and E, etc. Bartonella, Yersinia pestis, Coccidioides immitis, Orientia tsutsugamushi, Bunyaviruses, Corona virus, Rhabdovirus, Retroviruses, Orthomyxoviruses, etc. Arenavirus, Bunyaviruses, Ebola virus, Herpesviruses, Equine Morbillivirus, Herpesvirus simiae, etc.

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Table 2 CDC classification of biological agents (2018) Category A

Category B

Category C

These are the highest priority agents Can be quickly disseminated or transmitted Results in a high mortality rate and have the potential for public health impact May cause public panic and social disruption and may require special action for public health preparedness These are the second highest priority agents Can be moderately easily disseminated or transmitted Result in moderate morbidity rate and low mortality rate Requires specific enhanced surveillance Third highest priority agents Availability Less easily disseminated as category A and B Potential for high morbidity and mortality rates and significant health impact

Bacillus anthracis, Clostridium botulinum toxin, Yersinia pestis, Variola major, Francisella tularensis, Filoviruses, Arenaviruses, etc.

Brucella, Clostridium perfringens, Escherichia coli, Burkholderia mallei, Burkholderia pseudomallei, Chlamydia psittaci, Coxiella burnetiid, Ricinus communis, Staphylococcal enterotoxin B, Rickettsia prowazekii, alphaviruses, Vibrio cholerae, Cryptosporidium parvum, etc. Emerging Nipah virus and hantavirus

(CDC) classifies these biological agents into three categories: Category A, B, and C, given in Table 2 (Centres for Disease Control and Prevention 2018).

The Elements Microbial forensics adopt genomic, microbiological, and epidemiological methods to characterize and determine biowarfare weapons and ascertain the intentional or unintentional release of destructive pathogens and toxins (Morse et al. 2019). Microbial forensics is an emerging field dedicated to the depiction, examination, and elucidation of evidence found during the act of biological terrorism, biowarfare, and involuntary release of endospores and microorganisms for the attribution purpose (Murch 2003; Morse and Budowle 2006). The significant elements include: (i) Detection and identification: This is the first and the foremost step that incorporates detection and identification of the attack and the causing microbe behind it. For this, there should be proper collection and preservation of samples for analysis. Furthermore, for accurate analysis, powerful tools and techniques are needed to improve the sensitivity, accuracy, and specificity of

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results. These techniques can be categorized into three broad groups that are: (a) molecular techniques, (b) analytical techniques, and (c) physical analysis techniques (Budowle et al. 2005b; Varun et al. 2012). Information and database: The availability of information and databases, of course, increases the accuracy of results. Thus, the available database should be enhanced and expanded such that they contain the bioagent genomic sequence data, the whole genome of the agent used in the past, etc. This can be achieved by establishing the record systems at the national and international levels (Budowle et al. 2005b). In addition, interagency sharing of information and database must be encouraged. Development of strain repository: It is imperative to house these pathogens and near neighboring microorganisms in a strain repository. This information may prove essential in determining and identifying these near neighbors’ broad and narrow classes using different robust and sophisticated techniques (Budowle et al. 2005b). However, the security of such a repository would remain an active concern as any potential lapse may have devastating outcomes. Need for validation: This emphasizes the need for proper validation of new and existing techniques used in microbial forensics. All the methods used to analyze microbial samples must be accepted and validated (Budowle et al. 2005b; Varun et al. 2012). Furthermore, they must be robust enough to provide accurate results even in mutations and changes in the known strain. Quality assurance guidelines: Safety and quality assurance must be practiced using the appropriate guidelines and norms. These guidelines are important to be followed by microbial forensic laboratories to guarantee reliable results and maintain safety during and after the analysis (Budowle et al. 2005b; Varun et al. 2012).

Sample Collection Given the exchange principle, every contact leaves a trace; an investigator needs to collect evidence adequately; otherwise, it will lose its evidential value. Therefore, to avoid human error, the National Institute of Justice issued specific guidelines to minimize the chance of error in collecting microbial evidence. The guidelines mainly ensure the safety, collection, and preservation of microbial forensic samples. The main postulates of guidelines are mentioned below: • Valuation of the actual situation at a crime scene. • Planning related to sample collection, which includes assessing safety protocols for personnel, acquiescence with all guidelines and legal requirements, and discussing the prioritization of samples. It also mentioned selecting personnel and equipment utilized to collect and preserve samples with a proper time frame.

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• Documentation of place, area, subjects, whether human and animal. The possible and source need to find out and maintain the proper chain of custody of possession of evidence. • Mention the proper method and equipment that will utilize to collect the microbial forensic evidence. • Method of proper preservation of samples (Smith 2019). The techniques for collecting microbial forensic evidence involves strategic planning, logistic support, and statistic data in collecting microbes that will not produce a toxic effect on other microorganisms. Some principles and guidelines need to be followed to properly handle, collect, and preserve microbial forensic evidence. Tools used to collect the microbial evidence should be validated first and should not react with the sample of interest (Schutzer et al. 2011). The preservation procedure is also based upon the same principle that it will not affect the targeted sample. After the preservation process, samples are packaged and sent for analysis to the laboratory (Schutzer et al. 2011). This is the primary step of the analysis as the quality of the result may vary if the sample is not collected and preserved well. In addition, contamination and cross-contamination should be avoided. The sampling process includes collection and preservation, but several other steps need to be followed given in Fig. 1 (National Research Council 2014; Smith 2020).

Assessment of scene/ situation

Planning and designing sampling

Application of collection techniques

Documentation before and during collection

Storage, preservation, packaging, labeling and transportation

Fig. 1 Steps in collection and preservation of sample

Setting protocols for quality assurance and controls

Logistics and preparations

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Sampling can be achieved by one of the two strategies mentioned below (National Research Council 2014; Smith 2020). Choosing the sampling strategy varies depending on the location, type, and the extent of symptoms in clinical and agricultural settings (Budowle et al. 2006) (i) Targeted sampling: This is the sampling procedure where a sample is collected from a targeted area. In this process, the sample is collected from an area that is believed to be contaminated based on past knowledge. It is also known as judgmental sampling (Budowle et al. 2006; Sego et al. 2007; Smith 2020). (ii) Random sampling: This is the type of sampling in which the sample is collected from random areas without any prior knowledge (Budowle et al. 2006; Sego et al. 2007; Smith 2020). Actual sample collection is the process of taking the sample for analysis. Sample collection can be carried out using three approaches that are: (i) Collecting the whole item (is also termed as a bulk collection): In this, the whole item is collected and transported to the laboratory for analysis. This approach reduces the extra time required for the collection, but this can only be used if the object or the evidence can be easily removed from the scene (Budowle et al. 2006; Smith 2020). (ii) Collecting a portion of an item: This applies to immovable objects that cannot be transported to the laboratory. This includes methods such as vacuuming using high-efficiency particulate air vacuums, filtration, etc. (iii) Swabbing or wiping surfaces: This approach is best suited for trace pieces of evidence. It can be achieved using relevant sample collection devices. For this purpose, dry swabs, premoistened swabs or wipes can be employed (Budowle et al. 2006; Smith 2020). There are three main issues with these collection methods: (a) many of these collection methods and devices are not rigorously validated; (b) some of the methods are validated, but some security restrictions hamper sharing of this validation data to authorities who need them; (c) the collector must be well acquainted with the analyte or target signatures that are to be analyzed and accordingly the method should be chosen. Once the evidence is collected, they are packed appropriately and labelled along with the tags indicating the biohazard materials. The preservation of these samples can be achieved using preservative or transport media such as buffered tryptose broth, buffered glycerine, phosphate-buffered sucrose, etc. (Budowle et al. 2006). These media should be chosen based on the type of pathogen to not interfere with the analysis. In some cases, postmortem sampling is also required. Table 3 shows the viscera sample and quantity to be collected related to some common pathogens (Fernández-Rodríguez et al. 2019).

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Table 3 Postmortem microbial samples to be collected for forensic analysis Pathogen suspected Mandatory specimen

Pneumonia and other respiratory infections

Sample Blood Serum Spleen A portion of the affected lung part Urine Pleural exudate Swabbing the affected mucosa

Flu/viral respiratory infection

Nasopharyngeal swab

Invasive fungal infection

Lung, heart, brain, kidneys, large intestine, cerebrospinal fluid (CSF) Brain, liver, lung, myocardium, blood Feces Exudates Tissues

Malaria and other parasitoses Botulism

Quantity 5–10 ml 3–5 ml >1–2 cm3 >1–2 cm3 3–5 ml >1–2 cm3 Two swabs Two swabs

>1–2 cm3 >/¼ 1 ml Two swabs >1–2 cm3

Detection Methods Microbial forensic evidence encompasses various samples such as food, water, air, swab, soil, animal food, tissue, and clinical samples like blood, urine, stool, tissue, sputum and saliva, etc. The microbial samples are analyzed using various methods such as culture, microscopy, immunoassays, mass spectrometry, realtime PCR, microarray, genetic typing, whole-genome sequencing, and targeted sequencing. Meanwhile, the culturing method is considered the gold standard, but sometimes there is a delay in growth, and it may compromise the safety of an individual. The culture method also suffers problems when dealing with novel and uncharacterized microorganisms (Schmedes and Budowle 2019). Microbial analysis can be carried out using three methodologies: (a) molecular methods, (b) analytical methods, and (c) physical analysis (Budowle et al. 2005b; Varun et al. 2012). Molecular techniques can be either protein-based, such as microarray assays, immunoassays, etc., or DNA-based techniques (Fig. 2) such as PCR, SNP, etc. (Budowle et al. 2005a; National Research Council 2014; Nema 2018; Blondeau et al. 2019; Kieser and Budowle 2020). Analytical techniques include the use of various instrumental techniques such as matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), gas chromatography-mass spectroscopy (GC-MS), and liquid chromatography-mass spectroscopy (LC-MS) (National Research Council 2014; Nema 2018; Blondeau et al. 2019). The physical analysis includes microscopic analysis of materials such as soil, water, etc. (National Research Council 2014; Nema 2018; Schmedes and Budowle 2019).

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Fig. 2 DNA-based techniques used in microbial forensics

Polymerase Chain Reaction (PCR) and Real-time PCR

DNA based Techniques

Single Nucleotide Polymorphism (SNP)

Variable Number of Tandem Repeats (VNTR)

Massively Parallel Sequencing (MPS)

Metagenomics

Polymerase chain reaction (PCR) and real-time PCR: PCR-based techniques are the easiest to perform and require a minimal sample quantity. This includes in vitro amplification of DNA carried out in a specified instrument (Budowle et al. 2005a). The instrument performs programable cycling at controlled temperatures to generate millions of copies of targeted DNA that can be detected using different techniques such as hybridization or electrophoresis (Budowle et al. 2005a). These assays can screen many samples with a specific target DNA sequence (Budowle et al. 2005a). Real-time PCR (RT-PCR) is employed for those microbes having RNA as genetic material; this is because PCR can only be proceeded on DNA, and therefore in RT-PCR, reverse transcription is carried out to generate DNA from RNA to procced with PCR. With real-time PCR, amplification and detection of a specific variant of a microbe can be done simultaneously. This is generally done using fluorescent chemistries (Budowle et al. 2005a). Pyrosequencing PCR can also be helpful in many ways. This process is based on detecting luminescence by releasing pyrophosphate on nucleotide addition into the strand (Kieser and Budowle 2020). PCR and RT-PCR show tremendous significance in forensic microbiology (Bauer et al. 1999; Power et al. 2010; Wang et al. 2013; Park et al. 2014; Rajalakshmi 2017; Aqeel and Omran 2018; Jung et al. 2018). There are numerous species for which PCR markers are readily available, i.e., Lactobacillus, Gardnerella vaginalis,

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Mycoplasma hominis, etc. (Rajalakshmi 2017). Some studies have positively used PCR markers for the identification of fluids such as blood, saliva, menstrual blood, other vaginal secretions, etc. (Bauer et al. 1999; Wang et al. 2013). Studies have been carried out on messenger RNA (mRNA) profiling using PCR and RTPCR. SPTB, PBGD, HBB, HBA, ALAS2, CD3G, ANK1, PBGD, SPTB, AQP9 can be potentially used as RT-PCR mRNA markers for identification of blood form dried as well as wet stains (Bauer et al. 1999; Wang et al. 2013). Some other attempts are also made using streptococcal bacteria present in saliva as a marker to identify expirated blood (Power et al. 2010). Similarly, matrix metalloproteinase (MMP) and protamine mRNA markers can be used to detect menstrual blood (Wang et al. 2013). One study used oral bacteria Streptococcus salivarius, Streptococcus sanguinis, and Neisseria sub ava to identify saliva (Jung et al. 2018). Other fluids such as semen, vaginal fluid, saliva also have such identification mRNA biomarkers (Wang et al. 2013). PCR and RT-PCR markers are also available for the skin microbiome (Hanson et al. 2012; Aqeel and Omran 2018). These are some standard PCR and RT-PCR markers that can be potentially used in microbial forensics. Other than those mentioned here, there are several other PCR and RT-PCR markers available for different species as well. Single nucleotide polymorphism (SNP) array: This technique detects the variation at a single DNA site. It is a multiplex analysis of SNP (Budowle et al. 2005a; Schmedes and Budowle 2019; Kieser and Budowle 2020). After the PCR product is achieved, it is subjected to SNP primer and fluorescently labelled terminator nucleotide. The polymerase reaction is ceased by terminator nucleotide. This product is then separated by capillary or slab electrophoresis (Budowle et al. 2005a). This microarray can be a highly efficient screening technique with species to strainlevel detection (Schmedes and Budowle 2019). SNPs (single nucleotide polymorphisms) and other genetic signatures, which can be highly efficient screening and characterization tools, have been used precisely for bacterial and viral detection and can attain species to strain-level identification. Whole-genome shotgun sequencing (WGSS) is based on the sequencing method, requiring any previous sequence information to be determined. It can analyze any number of genetic markers such as SNPs, insertion, duplication, deletion, rearrangement, genetically and engineered genomes. Previously the WGSS used Sanger sequencing but is required to use cloning vector, was time-consuming, had low output, and was more expensive (Sanger et al. 1977). PCR-based assays are one of the less expensive and easy methods to perform being utilized to analyze the strain. Although real-time PCR allows analysis of the microbial sample, this technique is limited to detecting few microbes variants (Schmedes and Budowle 2019). SNP has the potentials of providing strain-specific information about the bacterial or any microbial genome being examined. A study conducted on Bacillus anthracis Ames strain for its detection by SNP concluded that among 88 other B. anthracis strains, they successfully found 6 different SNP characteristics to Ames strain, and 5 of them were even capable of differentiating Ames strain from its close isolates. Thus, this shows that SNP can be utilized to gain strain-level information about the microbial community (Van Ert et al. 2007). Similarly, another study on

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Mycobacterium tuberculosis using IS6110 SNP and some other analyses showed that SNP assays have also concluded its use for establishing ancestry in the microbial community (Faksri et al. 2011). While SNP assays having application in providing information about these disease-causing microbes, it also has potential in providing species or strain-specific information in 16s rRNA gene which is the most used gene for forensic analysis (Gu et al. 2017). One other such approach was also made to discover potential SNP of Cutibacterium acnes (Propionibacterium acnes) 16S rRNA, which is the most used microbial marker from the human skin microbiome to establish a contact of a person. It would assist in identifying different strains of C. acnes and could be incorporated for determining ownership. This resulted in discovering many such SNPs that can be utilized for the defined purpose (Yang et al. 2019). SNPs can be used as informative markers in Microbial forensic. Variable number of tandem repeats (VNTR): Multilocus VNTR can be performed to analyze polymorphism at the minisatellite region of DNA (Budowle et al. 2005a; National Research Council 2014; Schmedes and Budowle 2019). This polymorphism is unique to a species and can be used to screen samples for a particular species or strain. This technique encompasses amplification of fragments of DNA that differ in size by the number of repeat units present within the sample (Budowle et al. 2005a). These are then separated using electrophoresis and are viewed under fluorescently labelled primer incorporated during PCR. This ensures the separation of pathogen DNA but has limitations in phylogenic isolation (Budowle et al. 2005a). MLVA (multilocus variable number tandem repeat analysis) is another method to detect polymorphisms found in minisatellite regions found in bacterial genomes. It is found to affect the discrimination of strains of highly monomorphic species such as B. anthracis (Schmedes and Budowle 2019). MLVA also has a great species to strain identification capacity as SNP and thus can differentiate distantly related isolates and closely related isolates (Klevytska et al. 2001; Noller et al. 2003; Keim et al. 2008; Thierry et al. 2014). Varied approaches are being made to generate different VNTR markers that can be used to identify microbes. MLVA markers for microbes causing disease outbreaks such as Bacillus anthracis and for Yersinia pestis are studied. A study on the genome of B. anthracis using 31 VNTR loci showed that this technique, combined others, can potentially differentiate different strains of B. anthracis (Thierry et al. 2014). Similarly, many such studies have shown positive discrimination of B. anthracis using multiple locus VNTR (Keim et al. 2008). Another study on Yersinia pestis using 42 VNTR loci analysis from one chromosomal and two plasmid pMT1 and pCD1 DNA sequences also suggested the potential use of MLVA for differentiation of closely and distantly related isolates (Klevytska et al. 2001). Another similar study on Escherichia coli O157:H7 suggested that MLVA is as potent a method as pulsedfield gel electrophoresis (PFGE) (Noller et al. 2003). The studies mentioned here shows that multilocus VNTR analysis can also be positively utilized for microbial analysis. Massively parallel sequencing (MPS): Massively parallel sequencing (MPS) is another genetic tool available in the hand of a scientist. MPS is the technique used

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to analyze gigabase sequences of data in a short period. MPS is the technique in which millions of sequencing reactions can be carried out in a massively parallel way in a single run (National Research Council 2014; Kieser and Budowle 2020). This technique offers a complete characterization of the viral or bacterial genome. Much deeper genetic information can be achieved using this technique (Schmedes and Budowle 2019). It provides a high outturn, culture-independent method for whole genome sequencing (WGS). MPS has been a potent tool to detect and identify various disease outbreaks (Schmedes and Budowle 2019). Also, it provides an advantage that it has no requirement for enriched DNA for sequencing. MPS technique detects SNP and other genetic variants among the selected agents such as B. anthracis and Y. pestis. This technique can detect and differentiate among four different variants of both the microbial agent in a single sequencing run (Schmedes and Budowle 2019; Wetterstrand 2020). Metagenomics: Metagenomics involves the application of sequencing genetic material collected from an environmentally generated source such as water (Biers et al. 2009), soil (Mocali and Benedetti 2010), and human linked samples (Huttenhower et al. 2012; Schmedes and Budowle 2019). Metagenomic samples are applied in the determination of various forensic aspects such as to cause of death (Kakizaki et al. 2012), identification of human (Fierer et al. 2010), time since death (Hyde et al. 2013), characterization of biological fluids (Benschop et al. 2012), and pathogenic outburst investigation (Loman et al. 2013). In the forensic investigation, the microbial samples encountered are variable, mixed profile with other organisms, and low quantity samples. Forensic metagenomics is used to analyzed target microorganisms from complex matrices (Schmedes and Budowle 2019). These were the commonly used DNA-based techniques that can assist in the investigation of microbial attacks. As a result, these techniques are widely used in microbial forensics.

Interpretation of Results Interpretation is a crucial step that validates the findings and provides confidence to withstand trial and scrutiny. Forensic science is based upon the comparative study between the questioned and specimen/reference samples. Three types of interpretation are generally acceptable viz., inclusion, exclusion, and inconclusive result. Inclusion is the similarity between the compared samples and shows the exact antecedent beyond a reasonable doubt. Exclusion show dissimilarity between the compared samples and different origin beyond a reasonable doubt. An inconclusive result signifies the insufficient information is obtained to summarize interpretation and reach any specific conclusion. The result should be statistically strengthened by using various tools to validate and concrete scientists’ observations and should be able to endure strongly before the legal system. When dealing with microbial data, interpretation of results is critical as it requires high knowledge about sequencing and other techniques. Different techniques have their interpretation guidelines. For instance, in the case of analysis using commercially available RT-PCR kits, the

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presence of a particular microbe is or can be confirmed using fluorescence which confirms its presence. This is relatively easy and does not require much knowledge about sequencing data. However, when dealing with more sophisticated techniques such as SNP, VNTR, RFLP, MPS, metagenomics, etc., prior knowledge about sequencing phenomena and other basics of technique is needed to interpret results accurately. While interpreting results, the other thing that should be looked upon is reviewing sampling technique, sample condition, external factors affecting samples such as geographical location, temperature, humidity, etc., affecting microbial community colonizing at the site of colonization. These are how interpretation can be interfered by different factors needed to be kept in hand while interpreting results.

Conclusion Microbial forensics is the emerging branch of forensic science. It plays a vital role in investigating bio-crimes. The increasing use of microorganisms as war agents has also increased the need to have a body that separately analyses such outbreaks. Microbial forensics deals with the collection, preservation, storage, transport, and analysis of microbial forensic evidence. Forensic analysis of such evidence incorporates many techniques, among which molecular techniques may be helpful for analysis. Different molecular techniques used in forensic microbiology are discussed in the above sections. Further standardization is needed to strengthen the system to make it more robust and enabling high throughput outcomes.

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Part VII Quality Control and Challenges in Forensic DNA Analysis

Touch DNA Analysis

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Sourabh Kumar Singh, Amarnath Mishra, and Akanksha Behl

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . History of Touch DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Techniques for Collection of Touch DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Swab Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cutting Out Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Scraping Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tape Lift Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Techniques of Touch DNA Examination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Extraction and Isolation of Touch DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evidentiary Value . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Limitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Case Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A Valid Example: Charging the Waldo Attacker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Touch DNA examination is regularly used to pick up data from natural materials to help examinations related with criminal offenses, catastrophe casualty recognizable proof and missing individual’s examinations. As the noteworthiness estimation of DNA profiling to scientific examinations has expanded, so too wants to create this data from more modest measures of DNA. Touch DNA tests might be characterized as any example which falls beneath suggested edges at any phase of the investigation, from test discovery through to profile translation, and cannot be characterized by an exact picogram sum. The assortment, DNA extraction, intensification, S. K. Singh Amity Institute of Forensic Sciences, Amity University Uttar Pradesh, Noida, India A. Mishra (*) · A. Behl Amity Institute of Forensic Sciences, Amity University Uttar Pradesh, Noida, Uttar Pradesh, India e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 H. R. Dash et al. (eds.), Handbook of DNA Profiling, https://doi.org/10.1007/978-981-16-4318-7_45

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profiling, and translation of touch DNA tests, tainting, and move issues are additionally quickly examined inside the setting of touch DNA examination. Keywords

DNA · Helix · Swabbing · Scaping · Tape lifting · PCR · Gene analyzer

Introduction Touch DNA usually denotes the DNA particulates left behind by a carrier while coming in the physical contact with any surface. It is encountered and collected from any biological matter and is transferred from a carrier to any surface or an individual during a contact. This evidence acts very importantly in any case seeking for a forensic lab examination and is also taken as the important tool for proceeding with the investigation. Till date there have been a lot of studies reported but only a few suggests and reports the investigation and retrieval of touch DNA from any garment. These are also referred as invisible DNA as it is not visible to the naked eye and are usually present in a very small quantity as compared to the amount of DNA present in any blood sample, and so are also very difficult to encounter because of its invisibility (Burrill et al. 2019). This technique increased dramatically the amount of evidence which might be used for the detection of DNA. As these are invisible to the naked eyes, so one does not need to look for anything specific for DNA. It just needs a few cells (7–8) from epidermal layer of our skin (Mishra et al. 2015). STR profiles extracted from touch DNA can be analyzed for the purpose of solving crimes and the extraction process can determine the recovery of genetic material obtained from different origins (Mishra et al. 2020). Individualization of people has for some time been a challenge confronting law authorization. An ideal framework incorporates distinguishing attributes novel to every person, with highlights that do not change over the long haul, which can be recorded with the end goal that suspects examples can be looked at against a bunch of realized reference test. Taken with regards to current legal science, in 1910 Edmund Locard set up the main crime lab as an educator of legal medication at University of Lyons, France, also is most popular for his proof exchange hypothesis in criminological science, “The Locard’s Exchange Standard.” In 1918, he additionally first recommended the 12-point coordinating framework for positive unique mark distinguishing proof. With the innovation of today, the straightforward demonstration of getting an item or touching a surface can prompt the recognizable proof and anxiety of a crook. Touch DNA is too called as contact trace DNA. It alludes to the DNA that is recuperated from skin (epithelial) cells that is abandoned when an individual touches or meets things, for example, garments, a weapon, or different articles. As a criminal touch the weapon or then again any article at the crime scene, skin cells left behind on surface prompts individual recognizable proof of the lawbreaker. An individual sheds around 400,000 skin cells every day, except it is

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Cell free DNA Endogenous nucleated cells Fragment-associated residual DNA

Transferred exogenous nucleated cells

TOUCH DEPOSIT

Anucleate corneocytes

Fig. 1 Potential source of touch DNA

the lower skin cells that will give the best DNA profile. These cells are normally recuperated when power is utilized, for example, on the casualty’s garments or at a crime scene after a battle has happened. “Touch DNA” is not to be mistaken for “Low Copy Number” DNA, or improved PCR techniques. Different touch DNA testing procedures have been utilized at the crime scene and in criminological labs worldwide for longer than 10 years. The quantity of cells moved to touched items is exceptionally factor, and regularly results in under 300 picograms of DNA. Touch DNA otherwise called contact trace DNA is recuperated from epithelial cells of skin that are moved to surfaces when an individual interacts with any surface. Human creatures shed a few skin cells consistently and these cells might be moved to surfaces of our skin co-crime is carried out; the culprit of the crime may store adequate number of skin cells on things found at crime scene like homicide weapon, dress, casualty, addressed archive, and so on. These confirmations whenever gathered and DNA removed may help in connecting the culprit to the crime scene (Daly et al. 2012) (Fig. 1). Ordinarily, the lower skin cells are expected sources of DNA bringing about a decent DNA profile. These cells are commonly recuperated when power is utilized, for example, on casualty’s garments or at a crime scene after a battle has happened. This opened conceivable outcomes and prompted the assortment of DNA from a more extensive scope of displays (counting: instruments, attire blades, vehicles, guns, food, bedding, condoms, lip makeup, wallets, adornments, glass, skin, paper, links, windows, entryways, and stones). Broad endeavors have been made to gather touch DNA from various substrates, for example, human skin, insides of latex gloves, lip prints, controlling wheels, entryway handles, and device holds and shafts. Among potential substrates, dress is regularly thought to be a wellspring of significant proof for criminological DNA examination in criminal examinations, for example, in rape cases. It is realized that touch DNA can be recuperated from the bodies and apparel of assault casualties, however helpful profiles are hard to obtain since touch DNA is typically saved in limited quantities contrasted with bloodstains or other organic liquids (Table 1).

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Table 1 Potential evidence and types of cases Potential evidence Firearm, knife handles, and weapon handles Fired cartridges Steering wheel and other vehicles Fingerprints on victim Ligatures, hand cuffs, and shoestrings Hand swabs from suspects Face swabs from child victims (slapping, hitting) Swabs from limbs removed from animal carcasses Cell phones swabs Victim neck swabs Swabs from torn or forcibly removed clothing Airbags Tools Baggies Paper demand notes Clothing items, hats, masks, gloves, and glasses

Type of case Any Any Carjacking etc. Any Strangulation, kidnapping, rape, etc. Strangulation, rape Child abuse Poaching Robbery, etc. Strangulation Rape, assault DUI cases and others Burglary Drug possession Bank robberies Any

History of Touch DNA In 1997, Van Oorschot and Jones observed and reported possibility of recovering the touch DNA through the epithelial cells, found on an exhibit. This method turned into developed early inside the 2000s and “lets in evaluation of simply ‘seven or eight’ cells from the outermost layer of pores and skin.” Fifty-one touch DNA is likewise referred to as epithelial DNA. It makes use of the equal approaches to study bodily uids as conventional DNA makes use of, however the trying out is on those closing epithelial cells. When a person touches an item, epithelial cells are frequently left at the back of. The quantity left in the back of is frequently less than a hundred picograms and is also called low copy DNA. This is proof with “no visible staining” that might possibly incorporate DNA because of the switch of epithelial cells from the pores and skin to an item. Since then, there are numerous research studies to be had inside the literature in which DNA has been expounded to be recovered from treated objects. Such objects include handbags, garb, rings, guns, and automobile steering wheels. While this would appear the herbal development of DNA and pores and skin hint analysis, it is far vital to understand the distinction among the abilities of touch DNA and chemical evaluation of skin traces and conventional DNA. They are twofold and raise widespread problems as to whether or not the criminal framework surrounding conventional DNA analysis is sufficient for this new shape. Due to this development, decrease quantities of human DNA can be detected and, probably, a complete or partial STR profile may be generated.

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Techniques for Collection of Touch DNA Numerous crime scene examiners and labs test for touch DNA utilizing either the wet/dry cleaning or cutting techniques. At the point when the cleaning strategy is used, the outside of the thing is typically scoured with a wet q-tip, followed by a dry q-tip with an end goal to gather conceivable skin cells. The wet/dry cleaning strategy is suggested for hard, nonpermeable things, for example, metal, glass, or plastic, and can without much of a stretch be performed at the crime scene with restricted danger of defilement with exogenous DNA (for example, from the individual gathering the example, or from close by surfaces/objects). The cutting strategy might be utilized for delicate things, for example, dress, in which texture from zones of revenue is sliced to gather potential cells (Kirgiz and Calloway 2017). These two methodologies can be effective on numerous things of proof; nonetheless, the two of them have the constraint of putting super uous substrate (the q-tip itself or the texture cuttings) into the little DNA preparing tube. There is a restricted measure of substrate that can be set in a cylinder, and the substrate itself may “trap” a few cells during handling, diminishing the probability of acquiring results. Notwithstanding the ordinarily utilized cleaning and cutting techniques, a few labs additionally utilize the scraping also, tape lift strategies, in which the outside of delicate/permeable things are either scratched with a sterile surgical tool sharp edge, or tested with a little bit of scotch tape, or the glue part of a post-it note, to gather conceivable skin cells. The examiner using the scratching or tape lift technique will center their testing to a region of harm, or the zone where the culprit is accepted to have had the most contact. Using these testing techniques, a territory roughly the size of a grown-up’s hand can be examined (Hanson and Ballantyne 2013). The scrapings/tape/post-it notes are then positioned straightforwardly in the extraction tube. Labs utilize pretreated tape (generally presented to an UV crosslinker) and will likewise deal with a clear bit of tape close by the proof example to guarantee that no DNA has been presented by means of unusual pollution from the producer. It should be noticed that glues can be hazardous during the DNA extraction method and all things considered, the examiner should guarantee that their lab of decision has an approved extraction system that can effectively eliminate the glue without in uencing the DNA yield. The scratching and tape lift strategies permit a bigger surface territory to be tested rather than the cutting strategy. An expansion in surface zone builds the odds of recuperating more skin cells, which expands the odds of getting a DNA profile (Templeton et al. 2013). As referenced, the scratching/tape lift strategies are ideal in circumstances where the researcher can find zones on the things which are well on the way to contain the culprit’s skin cells. On the off chance that dress was left at the crime scene by the culprit, pressure focuses on the garments, for example, the inside neck of a shirt or within headband zone of a cap are fantastic possibility for these inspecting techniques. In a rape case in which the casualty’s attire had been taken out by the culprit, regions, for example, the belt may contain adequate cells having a place with the culprit to create a profile.

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The initial phase in gathering follow tests is to distinguish which regions to target. All things considered, follow tests on surfaces are not promptly recognizable. While finger printing specialists are utilized to distinguish touched regions on certain displays, numerous shows are cleaned, or tape lifted dependent on suppositions about where the DNA-containing material is found. The utilization of nonobtrusive discovery frameworks would be ideal. One such framework is the utilization of light sources, for example, the Poliliaght. Be that as it may, the utilization of these is not as far and wide as it very well may be. This might be because of an absence of consciousness of their handiness, or their apparent difficulty, or their nonideal presentation for explicit assignments or that further insightful and approval work is needed to characterize their degree and impediments (Singh Sankhla and Kumar 2017). Touched surfaces that have been uncovered utilizing fingerprinting techniques are generally those surfaces on which fingerprints are looked for as the need, instead of surfaces where DNA will be examined. Albeit many fingerprinting techniques do not antagonistically in uence the nature of recovered DNA, a few approaches may do as such. Furthermore, others may diminish the amount of recovered DNA. While thinking about the downstream utilization of the unique mark, more accentuation should be given to the effect of the fingerprinting procedure utilized on ensuing DNA recovery and quality. Upgrades in the techniques for distinguishing the natural wellspring of follow tests (not simply fingerprints) on show surfaces, and their application throughout criminological examinations, should assist with improving example assortment. Cleaning an accepted follow test region that is more modest than the real affidavit territory will imply that a portion of the example goes uncollected. Cleaning a territory more noteworthy than the genuine region of store may imply that example is spread over a more extensive region and that less is gathered. The two methodologies additionally can possibly give a wrong perspective on where the genuine example was found. It is, thusly, not just important to know about the exact area of the material being focused on yet additionally to gather from the zone fittingly (Aditya et al. 2011). An important thing that when a person collects the touch DNA is that everything or every individual present at the crime scene his/her DNA could also be contributed while the collection, because touch DNA is something that is invisible to our naked eye. So, an appointed personal for the collection of touch DNA is recommended to where appropriate personal protective equipment.

Swab Technique For the collection of touch DNA from fingerprints or other skin cells a swab is often taken. While collection of touch DNA one must always choose the specifically mentioned DNA-free or controlled swabs. This means the swabs are free of human touch and any contaminations like DNase, RNase, and DNA, which possibly may contaminate a sample.

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While swabbing, the swab first needs to get wet using only a single drop of distilled water and not by dipping the swab into water. When the swabbing method is utilized the surface is usually rubbed with the wet cotton swab and then is followed by a dry cotton swab in an effort to collect the possible skin cells. And the wet of the dry swabbing method is recommended for the hard nonporous items such as metal, glass plastic, etc. and it can be easily performed at the scene of crime with a limited risk of contamination with exogenous DNA (Sessa et al. 2019). Apply the tip of the swab to the sample area and using gentle pressure rub the same while making sure to make the contact of all the surface of swab with the sample surface by rotating it. Do not rotate the swab more than once to avoid the redeposition of sample on the surface. Air dry the swab and place it into the designated vial. After labeling the vial needs to be packed and sent to the lab for the analysis. To improve the quality of the resulting DNA profiles a double swabbing technique is usually been applied.

Cutting Out Technique It is the frequently used method in many forensic laboratories and the area of interest is usually been the soft items which have been left over in the crime scene. In this technique the most common sample are cloth samples. If a forensic expert considers the cloth samples, they are collected from the clothing leftover at the scene of crime by the perpetrators. The pressure points on the clothing such as the interior neck of a shirt or the inside headband area of a hat are excellent area for the sampling (Sessa et al. 2019). In the sexual assault case in which the victim’s clothing has been removed by the perpetrator, areas such as the waistbands may contain sufficient cells belonging to the perpetrator to produce a particular profile.

Scraping Technique In the scraping technique the surface of the porous items is either scraped with a sterile scalpel or blade to get the touch DNA. The scraping technique allows a larger surface area to be sampled as compared to the cutting technique or the swabbing technique. An increase in the surface area increases the chances of recovering more skin cells, which increases the chances of obtaining a DNA profile.

Tape Lift Technique In this technique adhesive tapes are used to carry out the deposited touch DNA on the suspected area. The analyst will mainly focus their sampling to the area of damage or the area where the perpetrator is believed to have had the most contact and using this sampling technique an area approximately the size of an adult’s hand can be sampled. So, the analyst may tape lift the sample from almost an area from the

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adult’s hand. The sampled tape may directly be placed for the extraction procedure. Forensic labs use pretreated tape (usually exposed to a UV cross-linker) and will also process a blank piece of tape alongside the evidence sample to ensure that no DNA has been introduces via adventitious contamination from the manufacturer (Bonsu et al. 2020).

Techniques of Touch DNA Examination The analysis of touch DNA evidences follows the same general practices of forensic DNA except an utmost care to avoid contamination in the sample. Most of the touch DNA samples tend to contain trace amount of DNA, facilitating the contamination from the exogenous DNA in them. Hence, it is advisable to process such evidences separately without mixing them with the high DNA content samples. The routine forensic DNA analysis is a multistep process, starting with the collection of DNA evidences such as saliva, blood, semen, hair, skin cells, or any biological material containing cells. The next step is extracting the DNA, where the cells are broken open using a combination of chemical and heat to release the contents of the nucleus where the DNA is stored. The material then goes through a cleanup process, so the sample is pure DNA (Verdon et al. 2014). The next step is the process to determine how much DNA was retrieved through a process called quantitation. Quantitation is important because the next step requires a specific amount of DNA to achieve optimum results. Too much or too little requires the concentration to be adjusted before the next step. After quantification comes the amplification process where specific regions inside the DNA molecule are copied. During the amplification process uorescent tags are placed on regions of the DNA which will be used in the last step. The last phase is the detection process where the copied fragments of DNA are separated by size and are passed through a laser, when this laser hits the uorescent tags a camera detector determines what color the tag is and how much there is (Comte et al. 2019). A DNA profile is a set of numbers at the different regions tested called locus. One of the numbers come from the mother and another from father linking the subject to each one.

Extraction and Isolation of Touch DNA To extract the DNA from surfaces such as metals, different extraction buffers were used. These buffers utilize protease which accounts for DNA binding by the process of opening of cells, which is essential for touch DNA recovery. The procedure of isolation and extraction of DNA can be conducted through various methods such organic extraction, Chelex extraction, and silica-based extraction. The former two methods can cause a loss of a part of DNA while the extraction process. The silicabased extraction includes silica-coated magnetic beads to seize DNA from the disintegrated cell. DNA is generally obtained from epithelial pores and skin cells; these strains are assessed for the best quantity. Some techniques such as quantitative

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PCR assay, dot blot technique, capillary electrophoresis, and uorescent dye assay are utilized for isolation of DNA inside the touch samples. As contact DNA is to be had in trace amounts, the DNA is amplified to provide numerous copies which can be then assessed for duration polymorphism and sequence polymorphism. Following the PCR amplification, out of 13 STR loci as depicted in Fig. 8 are identified which showcase polymorphism among one-of-akind individuals. In addition, the DNeasy ® plant mini package (QIAGEN ®) while in comparison with the QIAamp ® mini kit turned into found to decorate DNA restoration from paper with the aid of over a 150%. The nature of substrate from which the DNA has been recovered may want to have an in uence on DNA extraction. However, the DNA extraction manner can result in a loss of about 20% to 90% of the initial template quantity depending at the extraction method used, as well as the accuracy of the quantification approach.

Evidentiary Value When pondering testing for DNA, the examiner needs to think about the likely evidentiary estimation of the DNA. The investigator must consider the connection between the person in question and the suspect (if one exists), and any chance of “innocent transfer” of DNA that may have happened before the alleged crime. For instance, if the suspect is a relative, and either lived with, or had ongoing contact with the person in question, at that point finding the speculator’s DNA on the proof might be of restricted probative worth. Touch DNA can undoubtedly be transferred all through the family by means of everyday connections, contact with furniture things/bedding, or through the clothing (Hefetz et al. 2019). Touch or trace samples usually have only a certain quantity of DNA, which can get affected while its collection procedure and examination. The position where such DNA is lost cannot be determined due to the fact that there is not an availability of authentic positive sample which can lead to track the quantity of DNA by the examination process. When collecting touch DNA the different methods of collection can have a direct effect on the amount of DNA that has been collected from a crime exhibit. It is recommended for the crime scene investigators to choose accurate collection methods so as to procure the samples in the best possible manner. Usually in the recent time, trace DNA profiling was also conducted to extract DNA profiles from touched objects. Touch DNA can also be called as low copy number DNA or low template DNA. However, due to its amount, trace or touch DNA would be an appropriate term. It comprises of the minute level of DNA present in the small quantity of biological exhibits, and the process of extracting it. During the process of amplification, the term low template is used, where the use of low content of DNA will not generate proper results. Trace DNA can also be referred to the exhibit that consists of low levels of DNA at any stage of the examination process such as detection, collection, extraction, amplification, and interpretation. Touch DNA usually includes scanty biological exhibits and less than 100 pg, meagre content of DNA (Figs. 2 and 3).

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Fig. 2 Touch DNA sources

surfaces

cough, sneeze

touch DNA

skin cells

fluids

Fig. 3 Touch DNA transfer

crime scene

person

object

According to one such case, touch DNA is referred to as a “kind of pseudoscience.” It takes samples that incorporate a mixture of DNA from surfaces touched via an unknown wide variety of human beings and makes a wager as to the chance that a criminal suspect’s DNA is a number of the haystack of unknowns the lab is inspecting. The standards and suggestions for “touch DNA” forensics are also a multitude. There’s no unified set of requirements governing use, so analysts are loose to create their very own baselines. Guesswork continues to be guesswork, even though it involves technical software program and educated analysts. Guesswork is not evidence. Considering what’s at stake, touch DNA evaluation is not always specific enough to fulfill the evidentiary fashionable needed to lock human beings up. Journal articles regarding the switch of DNA have proven that DNA is not always transferred via contact on its own. In addition to the complications which can rise up from contact DNA on the technical side, there are many prison issues which can be implicated due to the wrong use and application of DNA. The maximum enormous is that it is able to lead to wrongful incarceration. The purpose touch DNA is the least favored DNA

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source is because the man or woman has to touch the object for a prolonged period of time and/or time and again touch the object several times to go away a sufficient quantity of skin cells to generate a usable DNA profile. Touch-switch DNA “should falsely link someone to against the law” and forensic scientists counting on current excessive-sensitivity gadget ought to “falsely conclude that DNA left on an object is a result of direct contact.” Their findings found out that it is impossible for scientists to determine whether or not the tiny bits of DNA got here. Touch DNA can get transferred from skin to object or from skin to skin and then to the sample. Secondary transfer of DNA may be from skin to skin to object or from skin to object to skin. A systematic analysis of secondary DNA transfer observed that underneath their experimental conditions secondary switch of complete profiles changed into no longer located, however those occasionally minor peaks in a few samples were located. Secondary switch of DNA became determined in some research and additionally located in DNA recovered from bedding. Research suggests a person may also slough off thousands of lifeless pores and skin cells in line with day. Studies have shown that the type of oor the DNA is being deposited on matters substantially. The extra abrasive the oor, the higher the threat extra skin cells may be sloughed off in the course of touch. The terms like direct and indirect transfer pertains to the routes by way of which DNA can be passed on. Some other terms such as primary, secondary, and tertiary are also used while referring to the transfer of DNA. DNA can be passed on multiple time and can be called as multistep transfer pathway; one needs to be clear on what is meant with the aid of secondary switch within the context of the state of affairs at hand. For a few, secondary switch method is any switch event after the number one switch; for others it simply refers back to the singular transfer step after the preliminary deposit. In addition, while relating to a selected touch event inside a long collection of a couple of contacts, one may also talk over with the number one and secondary substrates worried in a specific contact even though they will now not be the primary or second substrates inside the collection of contacts. As in most case situations, when taking into account the possibility of direct versus indirect transfer, the range of oblique steps are unknown, consequently forensic expert decides on the use of the term “indirect transfer” in place of “secondary switch,” until the situations put forward by means of prosecution or defence, or known facts within the case set up that the indirect transfer is primarily based on best a single step after preliminary deposit. However, on account of a rape by an odder, finding the suspect’s DNA anywhere on the victim’s dress might be of evidentiary worth. In these circumstances it is critical to assemble however much data from the victim as could be expected (if living), or to endeavor to reproduce the occasions if the victim is expired. For example, in the event that the victim’s pants were pulled somewhere around the culprit, at that point the examiner and criminological researcher ought to consider testing regions for touch DNA where one would imagine that the suspect would have snatched during the attack. Finding the suspect’s DNA on the victim’s dress, and in specific regions of the apparel, may help validate the victim’s rendition of occasions

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and help address the charges being referred to. It is additionally significant that the examiner endeavors to gather the apparel of expired people or gather tests from the garments before the deceased being taken out from the scene. Assortment of the apparel at the scene and ideal safeguarding considers the chance of acquiring touch DNA sometime in the future, regardless of whether it is not at first shown to be available at the crime scene. It is important that the investigator furnish the forensic scientist with some case foundation data to get the best guidance on the possible estimation of DNA evidence, just as proposals for testing. The arrangement of crime scene photographs can regularly be very valuable. Thus, it is similarly significant that the forensic scientist is outfitted with appropriate inquiries for the investigator to reply or consider (Oldoni et al. 2017).

Limitation Sampling techniques of touch DNA and the procedures of processing the examination for the DNA are very sensitive. So, it has a huge chance of getting contaminated by the investigating officers even while they are covered in a PPE kit. It very well might be important to acquire end tests from key individuals in the situation where unfamiliar DNA profiles are acquired that cannot be credited to a suspect or the victim. There is a huge chance of encountering a mixed DNA profile as it might be possible that a few individuals have come in contact with the victim or the surface of evidence; this mixed DNA might contain the DNA of victim, suspect, or any other individual who has been in the crime scene at any time. The investigator may likely to be confronted with the examination of what it implies if unexplainable DNA is encountered (Nunn 2013). For instance, an unfamiliar male profile from a touch DNA test might be acquired from proof relating to a female victim. On the off chance that the male DNA profile does not coordinate the suspect being referred to the specialist needs to think about its importance to the case. The unfamiliar profile could be from the genuine culprit and the first suspect could be blameless. Or on the other hand maybe the DNA profile is from unusual exchange from crime scene staff, specialists on call, lab experts, or crime scene equipment, for example, brushes for fingerprint collection. These are largely conceivable outcomes that law requirement may need to assess and deliver to push ahead with the examination (Tobias et al. 2017). Some evidence things are likewise not suggested for the examination of touch DNA tests. Such things incorporate those that are seriously debased (for example, rotten apparel) have been presented to extraordinary ecological conditions, (for example, weapons left outside for quite a long time or years), have been washed, or are vigorously absorbed the victim’s body uids. Additionally, things that are probably going to have been touched by numerous individuals, for example, a public compensation telephone or store counter are generally bad hotspots for probative or interpretable touch DNA profiles. Most forensic scientists will not agree with the proposal of the inspection of these things (Hanson and Ballantyne 2013).

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Case Study In Missouri, the Kansas City Police Department’s (KCPD’s) cold case crew and crime research facility, and the Jackson County Prosecutor’s Office structure a triumphant group. The sum of what three have been granted National Institute of Justice (NIJ) DNA awards. Since January 2008 (the first of the 3 years that the KCPD Sex Crimes Cold Case Squad got NIJ Solving Cold Cases with DNA award subsidizing), the group has gotten more than 150 CODIS hits, outstandingly cleared in excess of 100 cases and gave in excess of 50 charges. Capt. Imprint Folsom, authority of the KCPD Special Victims Unit, says the Sex Crimes Cold Case Squad, which was rebuilt under the Violent Crimes Cold Case Squad in March, has a commendable working relationship with the crime research center and the Jackson County Prosecutor’s Office. Returning to 2000, around 115 virus cases, including manslaughters and rapes, have since been indicted to conviction and speak to a 96% conviction rate, says Ted Hunt, boss preliminary lawyer in the Jackson County Prosecutor’s Office.

A Valid Example: Charging the Waldo Attacker In May 2010, collaboration and DNA evidence helped Jackson County accuse Bernard Jackson of 15 lawful offenses from four assaults in 1983 and 1984, in the Waldo and Armor Hills zones of Kansas City. At that point, KCPD was researching Jackson as an individual of interest in five comparable rape cases occurring in the Waldo zone in 2009 and 2010. Primer reports from the 1980s were pulled, alongside evidence from the property room and the lab’s drawn out capacity cooler. In the stretch of time of a Friday evening to Monday morning, criminalists found a coordinating profile. “That sort of correspondence and fast activity, and the eagerness of the lab to drop everything and work overtime to settle the case upgraded our capacity to charge the suspect rapidly and get him off the road,” Hunt says. The supposed chronic attacker had been captured and indicted after a 1984 robbery and assault, invested energy in jail, was delivered in 2008, and purportedly again began an example of crime. Folsom, who drove the Waldo attacker examination team, says the crime research center group was a gigantic assistance, especially those working in DNA and trace evidence who needed to organize which of thousands of things would be broke down first. In February 2011, the Jackson County Prosecutor’s Office accused Jackson of 22 lawful offenses for two of the five cases from 2009 to 2010. In one case, a girl become sexually assaulted and strangled to demise. She has been certain with a couple of ligatures, which include leather straps. Initial checking out found out that DNA from a semen stain at the victim’s nightgown matched a convicted perpetrator. However, this became not sufficient evidence for a conviction due to the fact the suspect had recognized the victim and claimed they had had a consensual relation. The scraping method become performed on the ligatures to gather possible skin cells and the suspect’s DNA turned into recognized on one of

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the leather-based straps, offering compelling proof that the sexual come across changed into now not consensual. The suspect pled guilty following the presentation of the DNA proof at trial and is presently serving a life sentence.

Conclusion The objective of forensic science is to boost the estimation of evidence. Distributed information and reports from operational scientists have demonstrated that up to 90% of the DNA in an example can be lost during assortment and examination (Cavanaugh and Bathrick 2018), anyway different investigations normal this misfortune at around 39% (Kemp et al. 2015). An expansion in comprehension of the difficulties in effectiveness of each progression in the DNA test work process has significant repercussions regarding likely improvement for all organic example types, including semen, blood, and salivation. Enhancements in proficiency in acquiring tests from substrates and ensuing extraction will expand the quantity of tests that will yield effective profiles, especially when the beginning amounts are low or ecologically tested, like the case with touch tests. With better comprehension of DNA yields and the components of misfortune, directed cycle enhancements will bring touch DNA tests into significantly more normal use with normalized improved strategies. Contingent upon substrate type and porosity, it is a test to acquire all the accessible DNA.

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Kemp BM et al (2015) How much DNA is lost? Measuring DNA loss of short-tandem-repeat length fragments targeted by the powerplex 16 ® system using the Qiagen Minelute purification kit. Hum Biol 86(4):313–329. https://doi.org/10.13110/humanbiology.86.4.0313 Kirgiz IA, Calloway C (2017) Increased recovery of touch DNA evidence using FTA paper compared to conventional collection methods. J Forensic Legal Med 47:9–15. https://doi.org/ 10.1016/j.j m.2017.01.007 Mishra A, Sathyan S, Shukla SK (2015) Application of DNA Fingerprinting in an Alleged Case of Paternity. Biochem Anal Biochem 4:165. https://doi.org/10.4172/2161-1009.1000165 Mishra IK, Singh B, Mishra A, Mohapatra BK, Kaushik R, Behera C (2020) Touch DNA as forensic aid: a review. Indian J Forensic Med Toxicol 14(2) Nunn S (2013) Touch DNA collection versus firearm fingerprinting: comparing evidence production and identification outcomes. J Forensic Sci 58(3):601–608. https://doi.org/10.1111/15564029.12119 Oldoni F et al (2017) Sensitive DIP-STR markers for the analysis of unbalanced mixtures from “touch” DNA samples. Forensic Sci Int Genet 28:111–117. https://doi.org/10.1016/j.fsigen. 2017.02.004 Sessa F et al (2019) Touch DNA: impact of handling time on touch deposit and evaluation of different recovery techniques: an experimental study. Sci Rep 9(1):1–9. https://doi.org/10.1038/ s41598-019-46051-9 Singh Sankhla M, Kumar R (2017) Identification of criminal by using touch DNA: a new tool for investigation in forensic science. Imp J Interdiscip Res 3(5):2454–1362. Available at: http:// www.onlinejournal.in Templeton J et al (2013) Genetic profiling from challenging samples: direct PCR of touch DNA. Forensic Sci Int Genet Suppl Series 4(1):224–225. https://doi.org/10.1016/j.fsigss.2013.10.115 Tobias SHA et al (2017) The effect of pressure on DNA deposition by touch. Forensic Sci Int Genet Suppl Series 6(September):e12–e14. https://doi.org/10.1016/j.fsigss.2017.09.020 Verdon TJ, Mitchell RJ, Van Oorschot RAH (2014) Evaluation of tapelifting as a collection method for touch DNA. Forensic Sci Int Genet 8(1):179–186. https://doi.org/10.1016/j.fsigen.2013.09.005

The Use of Rapid DNA Technology in Forensic Science

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Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . History of Forensic Rapid DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Instrumentation and Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ease of Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Portability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Uses and Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Booking Station . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Crime Scene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Satellite DNA Laboratories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Disaster Victim Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Match Confirmation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Removing Bias in Mixture Interpretation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Retesting of Swabs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Comparison to Traditional DNA Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sample Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PCR Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Data Interpretation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Areas for Improvement and Future Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cost . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Flexible Throughput . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Speed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Expanded Testing Capabilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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R. O’Brien (*) National Forensic Science Technology Center, Largo, FL, USA e-mail: robrien@fiu.edu © Springer Nature Singapore Pte Ltd. 2022 H. R. Dash et al. (eds.), Handbook of DNA Profiling, https://doi.org/10.1007/978-981-16-4318-7_46

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Abstract

Forensic rapid DNA is a technology that develops a DNA profile from a sample in under 2 h, without human intervention. The entire process is performed on a single automated instrument and is much faster than conventional DNA methods. In addition to its speed benefits, rapid DNA supports processing outside of the laboratory and can be used at booking stations, crime scenes, border checkpoints, and disaster sites to aid in victim identification. This chapter brie y examines the history of rapid DNA, as well as currently available rapid DNA instrumentation and the characteristics of rapid DNA technology. It compares rapid DNA to traditional DNA methods, including the process, data interpretation, troubleshooting, and ability to perform swab retesting. It also explores the possible uses of rapid DNA in various applications and environments. Finally, it examines areas for improvement and potential future development. Keywords

Rapid DNA · Forensic DNA · Disaster Victim Identification · DVI · mobile DNA

Introduction Forensic DNA typing was first used as an investigative tool in 1986. There have been significant improvements to the speed and sensitivity of forensic DNA testing, and it has grown to become the gold standard in forensic testing (Butler 2011). Decades ago, forensic DNA processing took weeks. With rapid DNA technology, testing can now be completed in under 2 h. Rapid DNA analysis is commonly defined as the fully automated process that develops a forensic DNA profile from a sample in under 2 h, without human intervention (U.S. Federal Bureau of Investigation “Rapid DNA” 2021). While the improved processing time is critical for certain use cases of this technology, the implications of rapid DNA are much more significant. By reducing or eliminating the need for forensic analyst review, the technology expands the possibilities of who can process DNA samples. This technology has demonstrated the potential to move DNA processing from specialized laboratories into booking stations, crime scenes, and even border checkpoints and disaster scenes. A powerful and versatile new tool in human identification, rapid DNA is poised to revolutionize the field of forensic DNA testing. This chapter will explore the world of rapid DNA testing, including its history, its technology, its current uses, and what the future might hold for this branch of forensic DNA testing. The information outlined in this chapter is intended to serve as an introductory overview based on current developments. Additional updates will surely be required with time as rapid DNA testing becomes as commonplace in the forensic field as traditional DNA testing is today.

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History of Forensic Rapid DNA Traditional forensic short tandem repeat (STR) testing methods require multiple work ow steps, including DNA extraction, quantification, PCR amplification, STR fragment analysis via capillary electrophoresis, data review, and interpretation. The process also requires specialized equipment located in a carefully controlled laboratory environment, run by trained forensic DNA analysts. Given this complexity and other factors, including case volume relative to capacity and available resources, it should come as no surprise that it can require multiple days or even sometimes months to obtain reported results. Rapid DNA testing technology required significant advances in both amplification chemistry and instrumentation to fully integrate and accelerate processing time and enable analysis outside of the laboratory environment. The first step toward rapid DNA technology was taken in the late 2000s, when researchers developed a faster polymerase chain reaction (PCR) amplification method (Vallone et al. 2008; Giese et al. 2009). The total time required for PCR amplification decreased to about 30 min. There were also significant advancements at that time in micro uidic efforts for PCR, separation, and detection (Horsman et al. 2007). The initial development of the current rapid DNA systems was spurred in the late 2000s by funding released by a consortium of US federal agencies, including the Federal Bureau of Investigation (FBI), Department of Defense (DOD), Department of Homeland Security (DHS), and Department of Justice (DOJ). These agencies recognized the need for automated, integrated DNA analysis platforms with a simplified “swab in – profile out” process. Ideally, such platforms could be used for identification at locations ranging from local booking stations to mass disaster sites and operated by nontechnical personnel. By 2012, these funded development efforts had led to the release of two rapid DNA systems: the NetBio ANDE ®/ DNAscan™ and the IntegenX RapidHIT™ 200. With the advent of rapid DNA systems capable of producing DNA profiles in less than 2 h, law enforcement agencies began to adopt the technology. A commercially available rapid DNA system was installed at the Palm Bay Police Department in 2012, leading to reports of successful use in active investigations not long after ("Rapid DNA Used by Prosecution,” 2014). In 2014, rapid DNA programs were launched by the Arizona Department of Public Safety and Orange County District Attorney’s Office in California. As of May 2020, the Orange County District Attorney’s Office reported they had processed 427 cases using rapid DNA technology resulting in 138 investigative leads (Dadhania et al. 2020). In June 2020, the Arizona Department of Public Safety reported that rapid DNA had been used in over 530 cases and generated over 170 investigative leads (Arizona DPS SAB 2021). A major development occurred in the USA with the passage of the Rapid DNA Act in 2017, which amended the DNA Identification Act of 1994 in order to permit upload to the National DNA Index System (NDIS) of DNA profiles generated by criminal justice agencies using rapid DNA instruments approved by the FBI Director

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in compliance with FBI issued standards and procedures (U.S. Congress 2017). This enabled the FBI to work with the Criminal Justice Information Services (CJIS) Division and the CJIS Advisory Policy Board (CJIS APB) Rapid DNA Task Force to plan the effective integration of rapid DNA into the booking station process (U.S. Federal Bureau of Investigation “Rapid DNA” 2021). September 1, 2020, marked the FBI release of the Standards for the Operation of Rapid DNA Booking Systems by Law Enforcement Booking Agencies (U.S. Federal Bureau of Investigation “Standards” 2020b) and the National Rapid DNA Booking Operational Procedures Manual (U.S. Federal Bureau of Investigation “National” 2020a), thus creating the operational framework for mainstream deployment of rapid DNA systems in booking station environments. A number of law enforcement agencies outside the USA are also implementing or evaluating rapid DNA for custody suites/booking stations, crime scenes, and other scenarios. For example, the Italian Carabinieri Scientific Investigation Group (Raggruppamento Carabinieri Investigazioni Scientifiche (RACIS)) received ISO/IEC 17025 accreditation for rapid DNA in 2019 and thereafter started utilizing the technology to successfully resolve multiple forensic cases, including a homicide, and upload profiles to the nation’s genetic database, the Banca Dati Nazionale del DNA (Thermo Fisher Scientific “Italian” 2020b). In Singapore, a comparison study was conducted on the performance of rapid DNA work ow and conventional forensic DNA work ow (Thong et. al. November 2015a). The study supported the use of rapid DNA for providing quick intelligence through the concomitant use of offender database searches. The authors subsequently published another article that reviewed several actual crime cases, which were resolved using rapid DNA technology (Thong et. al. December 2015b). Due to international differences in legislation and approaches to forensic DNA in general, implementation and potential final solutions using rapid DNA could vary. However, the growing global acceptance and support for DNA databases as an effective means to reduce and deter crime (Anker et al. 2021) provides a strong incentive for many jurisdictions (DNA Resource) to adopt rapid DNA in order to accelerate and increase the scalability of these benefits.

Instrumentation and Technology All current rapid DNA instruments perform the complete forensic DNA work ow using disposable consumables which include all reagents, materials, and waste containment required for STR analysis within a single instrument. This compact size allows users to deploy the instruments at a variety of locations, including laboratories, police booking areas, disaster sites, and border crossing stations. In addition, the most recently introduced rapid DNA instruments have been further optimized for use outside the laboratory in decentralized environments to enable operation by nonscientists (Salceda et al. 2017). In 2018, the three current rapid DNA instruments were analyzed as part of a multiagency technology maturity assessment (Romsos et al. 2020). This included the

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ANDE 6C System, developed by ANDE (Longmont, CO), and the RapidHIT 200 and RapidHIT ID instruments, developed IntegenX (Pleasanton, CA), which was acquired by Thermo Fisher Scientific (Waltham, MA) in 2018. The assessment was funded by the FBI Laboratory and the Biometrics Center of Excellence, and results were analyzed by the National Institute of Standards and Technology. Rapid DNA analysis for the systems tested resulted in an overall 85% success rate for generating 20 CODIS core loci profiles with automated analysis and an overall success rate of 90% (with 240 tested swabs) in generating data. In addition to the technology maturity assessment, numerous studies have evaluated and/or validated the performance of individual instruments to ensure consistent, balanced, and precise results. The RapidHIT 200 instrument was evaluated in several studies (Hennessy et al. 2014, Jovanovich et al. 2015). The original DNAscan instrument was evaluated in 2016 (Della Manna et al. 2016; Moreno et al. 2017). The RapidHIT ID instrument was also evaluated (Salceda et al. 2017; Wiley et al. 2017), as was the ANDE 6C (Carney et al. 2019; Ragazzo et al. 2020; Turingan et al. 2020).

Ease of Use The traditional DNA analysis methods require specialized education and training. Ease of use is a major factor in the adoption of rapid DNA technology, which is intended to be a fully automated (hands free) process requiring minimal operator training. First responders and individuals trained to deal with hazardous situations can be easily trained to set up and operate these instruments. Standard instrument operation could be taught in less than an hour. The instrument guides the user through sample processing with on-screen prompts. This simplicity makes it possible for the instruments to be readily and successfully deployed across a variety of agencies with users of many different skill levels. This expands the use of rapid DNA to include: • Police officers at booking stations: DNA is collected at arrest and processed and searched before the individual is released. • Crime scene personnel at crime scenes: DNA profiles can be developed from biological evidence and known reference samples to more quickly include or exclude potential suspects or develop investigative leads. • Medical examiners and coroners: to confirm identity of victims of accidents or mass disasters. • Military personnel: to identify human remains and enemy combatants in theater. • Border patrol and immigration personnel at border crossings: to verify familial relationships and prevent human trafficking. The straightforward operation of these instruments is as important to the rapid DNA process as the technology contained within. Enabling nontechnical personnel to generate DNA data can allow forensic scientists to focus on more complex

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analyses while also preventing them from being deployed into potentially hazardous conditions. Data can be transmitted to a scientist located miles away for review before it is acted upon. The “sample in, answer out” means there is very little preprocessing of samples and no manipulation of the sample once the DNA process is started. Most samples can be collected by simply swabbing and can go directly into the instrument without any further manipulation. However, this also means that any problems with the samples or the process cannot be detected or remedied during testing. If there is a problem, it is only detected at the end of the process based on the quality of the profile developed. Therefore, additional training is recommended to educate the user on what sample types are most likely to yield usable results (i.e., single-source buccal, blood, and saliva as opposed to touch/trace samples with potential mixtures) and the optimal methods for collection and loading into the rapid DNA instrument.

Portability Traditional DNA laboratories require sufficient space to house the instrumentation, reagents, and consumables and a staff of trained forensic scientists. Certain processes also require a physical barrier between workspaces. Rooms must be routinely cleaned to avoid introducing contamination from the environment. Certain instruments are also susceptible to environmental factors, so controls are required for instruments to perform optimally. Given these limitations, it is not a simple process to quickly establish an impromptu DNA laboratory at a mass disaster or a crime scene. Shipping samples to an established laboratory for analysis delays processing. Samples are generally shipped in batches, introducing a delay while the items are gathered. Transportation time can be a significant factor, especially to distant laboratories. The portability of rapid DNA instruments brings the power of DNA processing to the front lines. The instruments are more rugged than traditional instrumentation used in DNA laboratories, allowing them to be moved between locations. The size of these instruments allows for easy shipping in a protective case. The instrument setup is as easy as plugging it in and turning it on. In less than an hour, a location can have DNA processing capability. Unlike traditional instruments, rapid DNA instruments do not require calibrations after shipping, a controlled laboratory environment, or highly trained scientists for operation. Instrument portability means DNA samples can be processed on site. Only data files would need to be transmitted, rather than DNA samples. This means within 2 h a sample can be processed, the data sent to a laboratory for comparison, and a result reported back to personnel on location for immediate action. A process that historically takes weeks or even months can now be accomplished in hours. This is the true power of rapid DNA.

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Uses and Applications Booking Station Booking is the procedure of collecting and recording information about an arrested person and the charge against that person into police records. In many jurisdictions, arrestee DNA is collected for certain crimes, i.e., felonies or violent offenses. The rationale behind this collection is to test the arrestee DNA sample and compare it to a local, state, or national DNA database, to determine if the individual, currently in custody, matches or “hits” to any DNA left behind at unsolved crimes. For many agencies, it can take months to process arrestee samples. Once collected, the samples must travel to a forensic laboratory for DNA testing and are uploaded and searched against the relevant database. If a match occurs, this investigative lead is provided back to the law enforcement agency to pursue. This often requires locating and rearresting the person of interest who was previously in custody, which consumes valuable time of law enforcement officers and potentially exposes them to danger when reapprehending violent offenders. In addition, if a suspect was released from custody in the time it takes to collect, process, upload, and search the profile, that person could potentially ee the jurisdiction or commit further crimes. If booking stations implement rapid DNA processing and standard operating procedures and DNA networks are modified to accommodate rapid DNA booking searches, crimes can be solved more expeditiously and suspects can be identified while still in custody, reducing the need to rearrest them. This also reduces the likelihood of improperly released individuals committing more crimes and impacting more victims.

Crime Scene The typical procedure when dealing with biological evidence at a crime scene focuses on identifying the materials and then collecting, preserving, and preparing them for shipment to the laboratory. After the entire scene is processed, which could take hours to days depending on the type of scene, then those items collected are transported back to the agency. At this point, the agency then must log these items and perform the necessary paperwork to get the case entered in their system. Following this, if the agency that did the collection has a DNA laboratory, the items need to be put into the evidence vault and wait for the case to be assigned, before DNA testing can start. If the agency does not have its own DNA laboratory, then the items collected must then be transported to the laboratory that offers services to that agency. That process could take days, and once it arrives at the new laboratory, it may once again sit waiting for the case to be assigned.

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Once the case is assigned, the analyst often has limited information on what pieces of evidence would give the best result. If multiple samples were collected from the same area, an analyst should ask themselves: Should all of those be processed, or only one or several? Which are the best ones? He or she must rely on whatever information is relayed in the case file, along with their training and experience. If case file information is limited, the analyst must contact the investigator for clarification. The next potential issue could be finding the people who collected the reference samples. It is no wonder cases can sometimes take weeks to months or even years to complete! How can rapid DNA help to improve this situation? First, known reference samples from individuals at the crime scene can have their DNA processed at the scene. These profiles can be electronically sent to the DNA laboratory, rather than consuming valuable processing time for those typically routine samples. In that way, the laboratory can concentrate on processing the more complex or challenging samples that are more likely to yield reportable data with traditional methods. With actual crime scene samples (i.e., biological evidence), care must be taken to triage what will be processed using rapid DNA and what will be sent back to the laboratory. However, if there are many samples present, they can be run at the scene and, using the instrument software, matched to any known reference samples. Rapid DNA could be used to determine which samples contain DNA foreign to the victim and those can be prioritized for processing at the laboratory. Could this go one step further and lead to an arrest based on DNA results processed at the scene with rapid DNA technology? This has already occurred in Miami, Florida, on more than one occasion (Plasencia 2017). In one such instance, with a fatal “hit-and-run,” DNA from the gear shift in the automobile was matched to a suspect in a crime where he was detained (Plasencia 2017). Samples from the vehicles were taken both for rapid DNA testing and for testing in a traditional laboratory. However, once the match was made to the individual, detaining officers had definitive proof to arrest the individual. The other samples sent to the laboratory would only need to be analyzed if the case went to court. It could be argued that the result may have eventually been the same if rapid DNA was not involved. However, factoring the time it would have taken to get these samples processed, barring a confession from the individual, the suspect would most likely have been released while waiting for these results from the laboratory. If the individual chose to ee the area, this case could possibly take months to be resolved, as opposed to the resolution in only several hours enabled by rapid DNA. This case and others like it show demonstrate the utility of rapid DNA technology.

Satellite DNA Laboratories In the city of Boca Raton, Florida, a small ancillary laboratory was brought online to screen items of evidence. The items that are positive for pertinent biological material are then submitted to the Palm Beach County Crime Laboratory for DNA testing. Screening of evidence can be very time-consuming and does not lend itself to

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automation. Having a smaller screening laboratory with well-trained personnel takes that burden from the main DNA laboratory. These screening laboratories can be smaller in size and can operate with less staff than a traditional DNA laboratory. They can screen a high volume of cases because that is their primary focus. The main DNA laboratory benefits because they no longer receive large items for testing. They will instead receive a cutting from an item, which contains the biological material needed for DNA processing. Rapid DNA can give a screening laboratory additional capability. It can be used to help triage the evidentiary items and run reference samples, allowing the main DNA laboratory to receive items ready for DNA processing along with known reference sample profiles for comparison. In remote locations, such as islands or rural areas, rapid DNA can also be used in satellite locations to generate profiles for analysis on-site or via secure electronic data exchange with a centralized laboratory, thus providing efficient DNA analysis capabilities for communities that are typically underserved by forensic services.

Disaster Victim Identification Disaster victim identification (DVI) is typically performed under difficult conditions and, at times, in remote locations. Typical scenarios not only include natural disasters, such as hurricanes, tsunamis, wildfires, and earthquakes, but also include aircraft crashes, capsized ships, mass graves, and military incidents. Any mass fatality situation can quickly overcome local resources. Given the additional potential compounding infrastructure factors such as power and water supply disruption, setting up another standard laboratory work ow can be a prohibitive challenge. The International Criminal Police Organization (Interpol) is a well-known intergovernmental organization that investigates many DVI incidents, since not all countries have a fully resourced and trained DVI response team. Interpol methodologies recommend that victims are first identified using dental and fingerprint records. However, depending on the situation and the condition of the recovered body, DNA analysis may be required to identify victims. Disasters can occur anywhere and include victims from many different origins. Multinational cooperation is sometimes necessary when providing support but also when repatriating human remains. Having a DNA solution that can be deployed and provide answers quickly, without the need for a complex setup, can make a significant difference in these efforts. As part of the postmortem phase of a mass disaster, rapid DNA provides a portable solution that can be taken to these scenarios and set up quickly with minimal requirements for additional instruments and reagents (Murakimi et al. 2020). In some situations, body preservation may also be difficult, and a quick response is necessary to ensure a DNA profile can be retrieved. Extraction of DNA from bone using traditional laboratory methods is typically a laborious process involving physical and chemical techniques that can, depending on the method used, take over a day of hands-on time. Rapid DNA protocols have

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been developed that streamline this process and allow investigators to obtain interpretable data from medium- to high-quality bone samples in significantly less time (Thermo Fisher Scientific “Bone Sample” 2020a). Rapid DNA can also be used to identify and match the relatives of the victims of DVI incidents in the ante mortem phase, where other means of identification have failed or are not available. This can be an extremely time-consuming process, especially when families are not aware that their relative is a victim in the disaster. A mobile rapid DNA solution can be used to transport the instrument to the suspected families of the victims, providing a quick conclusion to an open case. Rapid transport vehicles and transport boxes have been developed to support the mobility of the instrument. Some transport solutions include battery power, enabling instruments to be used in extreme locations where traditional work ows would otherwise be impossible. Given the mobility of the instrumentation, decisions in DVI situations can be made in the field. The goal of DVI is to identify and provide closure for families of victims as quickly as possible, and rapid DNA can be a valuable part of that solution.

Match Confirmation Offender DNA database systems such as the CODIS (Combined DNA Index System) are a very powerful tool. They are especially powerful in cases where no suspect is named in the case, but DNA has been found that could potentially identify the perpetrator. The DNA profile is entered and searched against the database, and any possible database “hit” must be confirmed. Under normal procedures in the USA, the hit confirmation must be done with a separate sample collected following the rules of evidence collection and preservation. Evidence chain of custody must also be maintained to be used for court purposes. In court, the analyst will not be allowed to say the profile generated from the crime sample matched against a profile in CODIS. This would potentially raise questions as to why this person’s sample was in CODIS, which could introduce evidence of prior bad acts, which could in turn cause the jury to be biased. Therefore, the analyst must only testify to the work conducted to compare the DNA profile developed from the evidence against the DNA profile developed from a fresh buccal swab taken from the person of interest. Often, when these new reference samples are submitted for CODIS confirmation, the analyst has already moved on to another case. They must then stop what they are doing to work on this single buccal swab. This causes a disruption in the case they are currently working on, and if they do not process the reference sample right away, this can delay the confirmation of the CODIS hit. It is important to realize that the confirmation of the CODIS hit requires putting this one reference sample through the entire DNA process from extraction to capillary electrophoresis and data analysis. This consumes time and can seriously impact the work ow of the laboratory. It would be ideal to batch confirmations, but that would mean waiting and delaying the closing of the cases to which they are related.

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Rapid DNA can play a major role in these CODIS hit confirmations. Rapid DNA instruments can be run on-demand when the buccal swab is submitted to the laboratory, especially the instruments that can process one sample at a time. No waiting or batching is required. Since the instruments are so easy to use, the actual running of the sample can be performed by a less trained technician, rather than an analyst, and the resulting DNA profile sent to the analyst for comparison to the profile developed from the evidence in the case. This saves time on two fronts. Firstly, the CODIS confirmation sample is run as soon as it is submitted to the laboratory, accelerating closure of the case; and secondly, ongoing casework is not delayed when this sample must be run through the entire DNA process.

Removing Bias in Mixture Interpretation Mixture interpretation guidelines from the Scientific Working Group on DNA Analysis Methods (SWGDAM) indicate that determinations concerning the possible contributors to a mixture should be made before performing comparisons to reference samples (SWGDAM 2017). Utilizing rapid DNA for known reference sample processing, conducted in a separate work ow or facility by different personnel than the forensic DNA analysts who analyze evidentiary samples, can help remove bias in mixture interpretation. After the forensic analyst has performed deconvolution of the mixed profile in questions, developing profiles for possible contributors and documenting these results, only then would they formally request the profiles developed from the reference samples for comparison purposes. In court, the analyst could potentially testify that they never had custody of the reference samples until such time as it was necessary.

Retesting of Swabs One of the primary concerns with rapid DNA testing has been sample consumption. Sample consumption often requires permission from attorneys in order to prevent court challenges by defense. As a result, it is important that whatever sample is used to generate an investigative lead from a DNA sample does not consume all of the sample. Rapid DNA can be used to generate important investigative leads in a short period of time. However, if the DNA is consumed by the rapid DNA instrument, it prohibits additional DNA testing using conventional forensic laboratory methods. This is a major reason why the Non-CODIS Rapid DNA Considerations and Best Practices for Law Enforcement Use from the FBI (U.S. Federal Bureau of Investigation “Non-CODIS” 2019) advises that two swabs should be collected when using rapid DNA instrumentation – ensuring that a swab can be sent to the laboratory for traditional DNA testing and court purposes once an investigative lead is created on a rapid platform. These two swabs can be either collected at the same time using a “bouquet method” or one swab at a time (“A-swab, B-swab”), providing one swab for laboratory testing followed by one swab for rapid DNA testing.

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This is a good approach; however, one can still envision scenarios, where it would be beneficial to retest the swab run on the rapid instrument. To date, only one rapid DNA system has been shown to enable sample retesting using either rapid or traditional DNA methods as reported below (O’Brien and Barnhart 2021). The RapidHIT ID System (Thermo Fisher Scientific) allows a processed swab to be removed after testing. The extraction method used on the RapidHIT ID is not as rigorous as conventional DNA methods, potentially leaving sufficient DNA on a swab for reanalysis. If a sample contains enough DNA, it is possible to reanalyze the swab using conventional DNA methods after it has already provided a profile via the RapidHIT ID. If so, a single swab may provide both quick investigative information and a confirmatory result. Studies were performed to determine if the same swab run on the rapid system can be removed and used again with conventional DNA techniques to achieve similar quality profiles. After the rapid DNA instrument finished running, the swabs were removed and placed into a 1.5 ml test tube to air-dry. Some swabs were air-dried for a week, while others air-dried for a month or more. The swabs were then put through the entire traditional DNA testing process from extraction through capillary electrophoresis. The DNA profiles developed from these swabs were compared to DNA profiles developed from the rapid DNA instrument to check for the accuracy and overall quality of the profile. In all cases, if a high-quality profile was developed by the rapid DNA instrument, the swab was also able to generate a high-quality profile upon reprocessing, using traditional DNA testing methods. Furthermore, 100% concordance was obtained between the duplicate samples and those samples run with the RapidHIT ID and reanalyzed using conventional analysis. Similarly, with crime scene-type samples, like cigarette butts and drinking containers, enough DNA was present after running the swab through the RapidHIT ID to produce, in most cases, a full profile using conventional DNA analysis methods (O’Brien and Barnhart 2021). These data exhibited that rapid DNA processing does not consume the entire sample on certain rapid DNA instruments. Swabs that are removed properly, taking the necessary precautions to avoid contamination, can be reprocessed with traditional DNA methods to generate a profile consistent with rapid DNA results. Although retesting swabs would not be a first choice, if an unexpected problem occurs with the swab sent off to the laboratory, then it is important to note that the swab used for rapid DNA testing can be retested. For this to work, however, the swabs used on the rapid DNA instrument must be preserved until the laboratory has successfully processed the evidence sent to them. The frontline operators of the rapid DNA instrument will have to develop a process for preserving and maintaining the chain of custody for swabs after they are processed and communicating with the laboratory to track their progress. This process will require coordination between agencies as the time between samples run on rapid DNA instruments and in the laboratory can be months apart.

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Comparison to Traditional DNA Methods Rapid DNA systems are smaller, faster, and less complicated to operate than traditional DNA testing methods. Therefore, why hasn’t traditional DNA testing been replaced by rapid DNA thus far? There are still limitations on what rapid DNA instrumentation can do when compared to traditional DNA testing methods. Traditional DNA testing methods still tend to be more affordable and sensitive than rapid DNA and able to handle a wider range of sample types. The sections below provide a brief comparison of the traditional forensic DNA work ow with rapid DNA, to help elucidate differences and future areas for improvement.

Sample Collection Both traditional and rapid DNA methods require collection of the samples so that they can be run through the extraction process. While some rapid systems use conventional swabs commonly used in traditional DNA processing, a study indicates that one rapid DNA instrument performs demonstrably better when using specialized swabs that are designed for use with that system (Manzella and Moreno 2020). These particular swabs use RFID-tagged caps that lock inside the biochip, meant to avoid the potential for switching samples during a run. While these RFID swabs provide an advantage to the rapid DNA instrument user, the need to use such swabs increases cost and reduces the exibility to submit samples to a traditional or rapid DNA process as needed.

Extraction In both traditional and rapid DNA methods, the first step is lysis, which breaks open cells, releasing DNA to be transferred for further processing. During most traditional DNA extraction methods, lysis is just the first step of a longer process called extraction. The lysis buffer is often coupled with mechanical processes like heating and agitation for a period which can be adjusted based on the sample type. High content DNA samples can be exposed to these conditions for a shorter period, whereas low content DNA samples can be incubated in these conditions for up to 24 h, if necessary. The exibility to add these extra mechanical processes and increased incubation times make the lysis step in traditional DNA processing more effective at breaking open most, if not all, of the cells contained within the sample. In traditional DNA extraction, lysis is followed by purification. The purification process removes cellular components that are released from the cell during lysis. In the rapid DNA systems, there is currently no mechanism to perform a thorough purification process. This means that cellular components other than DNA may pass

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through the system after the lysis step. As a result of these differences in the lysis and purification processes, more DNA may be obtained using traditional extraction methods compared to lysis on the rapid DNA instrument.

Quantification Traditional DNA testing uses a quantification step to estimate the amount of DNA present in samples prior to PCR amplification. Using the quantification results, the scientist can adjust the sample concentration to optimize the DNA input that works best for the amplification kit used. The current rapid DNA instruments do not perform quantification of DNA. Eliminating this step saves time, because the sample is transferred directly to the amplification step following lysis. Because there is no quantification step, the DNA input for amplification cannot be adjusted, which may affect the results. To optimize DNA recovery, rapid DNA instruments currently employ two different sample cartridges: one designed for high-quantity samples and one for lower-quantity samples. The protocol for each cartridge has been optimized to maximize DNA recovery and interpretable results. If too little sample is placed into the cartridge, partial or incomplete profiles may be generated.

PCR Amplification Traditional DNA and rapid DNA methods use similar or the same PCR amplification chemistry, depending on the platform, with a common set of standardized loci. The amplification kits in use today target the 20 CODIS core loci established by the FBI as well as additional, kit-specific loci. Therefore, profiles resulting from either rapid or traditional DNA processing can be compared. The profiles can be uploaded into local or national databases and compared to other profiles generated worldwide. The amplification chemistries, used in both traditional and rapid DNA instruments, have received FBI NDIS approval in the USA, so reference samples generated using these chemistries can be uploaded into the national level of the CODIS database, so long as all FBI standards and procedures are followed (U.S. Federal Bureau of Investigation “Rapid DNA” 2021).

Electrophoresis Electrophoretic separation is the process used to separate and detect DNA in both rapid DNA and traditional DNA methods. There are differences between the electrophoretic separation processes on different rapid DNA instruments. The RapidHIT ID instrument is very similar to traditional DNA methods in that it employs capillary electrophoresis. The cartridge on this instrument contains one capillary that can run at least 100 runs and as many as 150 runs with consistent use.

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The instrument will alert the user when the cartridge containing the capillary needs to be replaced. Each new capillary placed on the instrument can be tested by running a ladder and positive and negative controls to ensure that the capillary and reagents are functioning properly and are not contaminated. The RapidHIT 200 instrument utilizes an eight-capillary system that is embedded inside the instrument, which is guaranteed for 100 runs. A single run includes a native ladder and up to seven samples utilizing two sample cartridges. Positive and negative controls can be run at the user’s discretion. The ANDE 6C system utilizes single-use electrophoretic separation channels that are injection molded into the cassette. A new capillary is used for every run. The operator will rely on the manufacturer’s quality control measures as there is no way to verify the quality of the capillaries before use. This system requires very tight quality control employed at the production level to ensure every capillary in each cartridge functions to the same specifications. Any differences can impact the result.

Data Interpretation In 2004, the FBI incorporated guidelines and acceptance standards, enabling laboratories to utilize expert system software tools to automatically evaluate the quality of a single-source DNA profile. This allows for the upload of profiles to a DNA database without requiring manual review. Rapid DNA systems employ expert system software to perform data interpretation according to defined rules, like those used in traditional DNA analysis methods. Expert system software automates data analysis and allows the rapid DNA system to be operated by nonscientists who are not trained in advanced data interpretation. Manufacturers set static interpretation thresholds based on the type of cartridge used (high- or low-level DNA). Interpretation thresholds and quality ags are used to identify profiles requiring review, such as mixture profiles or profiles resulting from poor-quality samples. If the sample passes the interpretation thresholds, the DNA profile is considered acceptable and manual review is not required. The difference between the data interpretation systems becomes more evident with low-level samples or samples involving a mixture of DNA contributors (Hares et al. 2020). For these types of samples, trained scientists must perform the interpretation. Scientists use previously validated thresholds and interpretation protocols to evaluate these challenging profiles. Additional software tools can perform mixture deconvolution; however, extensive DNA analysis training is required to correctly identify and enter profiles into these tools. Typically, mixture interpretation should not be performed on profiles generated from rapid DNA systems. The reduced sensitivity and peak height ratio balance of rapid DNA systems can make detection of minor contributors more difficult than traditional methods. Rapid DNA samples that yield complex profiles will require interpretation and will be agged by the quality system. If a more in-depth look at the data generated from a rapid DNA system is desired, a scientist trained in such interpretation methods should be consulted.

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Troubleshooting Traditional DNA processing is a step-by-step process that allows for detailed troubleshooting to identify if the problem is sample specific or can be attributed to a specific process, instrument, or reagent used in the work ow. Rapid DNA, in contrast, is a fully automated process from extraction through to capillary electrophoresis and data analysis, which can make it more difficult to target the problematic step. There are only three main areas to troubleshoot: the instrument, consumables, and the sample. Issues are often identified by the instrument itself or by the outcome after a sample is processed. Certain instruments have internal diagnostics that alert the user to potential errors. When a specific code is displayed, the manufacturer should be consulted to assist with specific troubleshooting steps. If there is no sample profile at the end of a run, the presence of a size standard in the resulting data would lead the user to believe that the run successfully completed, and the instrument is still likely to be functioning properly. In this case, it is possible the sample was at too low level (contained too little DNA) to produce a result. If the sample was expected to contain a high level of DNA, like a reference buccal sample or blood sample, then the problem could have occurred either in the transfer of sample through the cartridge or the instrument, or during a part of the process such as lysis or amplification. Depending on where these processes occur, i.e., whether it is on the cartridge or instrument, then the problem can be corrected by using a new cartridge or it may require an instrument repair. Typically, troubleshooting problems that occur on rapid DNA instruments require the user to send log or data files to the manufacturer. Based on the review of log and data files, the manufacturer can potentially determine the problem and advise the user on the next steps.

Areas for Improvement and Future Development The current rapid DNA systems have already made a significant impact on both the forensic and law enforcement communities and are poised to make an even greater impact in the future. However, there is still a need for further development to address the remaining performance gaps and barriers to adoption. In July 2020, a letter to the editor of Forensic Science International: Genetics was published entitled “Rapid DNA for crime scene use: Enhancements and data needed to consider use on forensic evidence for State and National DNA Databasing – An agreed position statement by ENFSI, SWGDAM and the Rapid DNA Crime Scene Technology Advancement Task Group” (Hares et al. 2020). This document describes five major areas that should be addressed by manufacturers to improve rapid DNA analysis of forensic evidence samples: 1. An integrated method of human specific internal positive controls must be incorporated to identify low quantity, DNA degradation, and inhibition. This is

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intended to address the human quantification requirements shared by SWGDAM and many of the member countries of the European Network of Forensic Science Institutes (ENFSI). It must be possible to export analyzable raw (optical preprocessed) data. This ensures the availability of data for analysis or reanalysis by qualified forensic DNA scientists if needed for mixture interpretation and court purposes. An onboard fully automated expert system must be incorporated and programmed with rules to accurately ag allele calls in both single-source and mixture data that require analyst evaluation. Improved peak height ratio balance (per locus and across loci) for low-quantity and mixture samples (high and low quantity) must be achieved to facilitate interpretation of DNA mixtures and low-level samples by qualified forensic DNA scientists. Rapid DNA manufacturers must perform a well-defined publicly available developmental validation on a wide variety of forensic evidence-type samples commonly encountered in the forensic DNA laboratory.

It should be noted that several of these areas have already been addressed. For example, it is already possible to export raw data files (#2) for several on-market rapid DNA systems, and extensive developmental validations have been published by manufacturers (#5). In addition, expert system software (#3) is already incorporated into rapid DNA systems as described earlier in this chapter; however, it is recognized that further optimization of these software systems could increase automation in order to significantly reduce the amount of manual data review required by trained forensic scientists. Additional development is clearly required to address area #1 (internal positive controls) and #4 (improved peak height ratio balance). It is interesting to note that while there is wide recognition of the need for increased sensitivity with rapid DNA systems in order to facilitate improved detection of low-level DNA samples, the authors of this article specifically focused on improved peak height ratio balance. Thus, a preference for enabling enhanced mixture interpretation as opposed to solely focusing on increased sensitivity can be inferred. Once the areas above have been developed and validated, rapid DNA systems will in many respects possess performance capabilities that are on par with the current traditional laboratory methods. Still, there are several additional areas for potential improvement, as outlined below.

Cost Presently, it is more expensive to process a sample using a rapid DNA system versus traditional bench processing, when only considering the cost of reagents. For some applications, this is not prohibitive, as rapid DNA processing presents cost savings when considering all factors. For example, the Kauai Police Department reported that implementation of rapid DNA technology saved the agency significant time and

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several thousand dollars in one case, projecting that it will save over ninety thousand dollars annually (Thermo Fisher Scientific “Kaua’i” 2020c). For broader adoption, however, especially within the laboratory environment, rapid DNA processing is costly for routine STR analysis. Decreasing the cost of instrumentation and the cost per sample will likely be an important factor in widescale adoption.

Flexible Throughput The current rapid DNA instruments can run between one and seven samples at a time. For the instruments that enable multi-sample runs, the user may feel obliged to wait until they have gathered sufficient samples to run the maximum, or risk wasting costly consumables. This can defeat the purpose of possessing “rapid” analysis capabilities and is in stark contrast to traditional DNA laboratories, which can batch samples to increase throughput and reduce costs simultaneously. Future rapid DNA systems will need to evolve to enable more exible throughput capabilities.

Size While current systems are portable as discussed above, they range from about 50 pounds to over 100 pounds, with the larger systems requiring more than one person to move. If the devices were smaller, lighter, and internally battery-powered, this would further enhance portability and opportunities for field deployment.

Speed Although generating an STR profile in under 2 h is fast, producing results even faster does present advantages. Systems currently process between one and seven samples per run and decreasing the time to generate a result would increase the potential throughput per instrument. Increasing the number of samples per run would also accomplish this, but the advantage gained may be mitigated by decreased portability and increased cost, as discussed above.

Expanded Testing Capabilities To date, rapid DNA instruments have focused on producing autosomal STR profiles that contain the same genetic loci that are used in CODIS and similar databases, to facilitate upload and searching. There is, however, benefit in expanding the scope of markers tested with rapid DNA.

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For example, Y-STR profiles can be used to identify the presence of male DNA even when there is an overwhelming amount of female DNA present. Many laboratories use Y-chromosome-based screening on sexual assault samples, as it is a quicker way to determine the presence of male DNA than chemical identification and differential extraction. If no male DNA is detected with a “Y-screen” method, the case is deemed negative, and no further testing is performed. If rapid DNA technology could also generate Y-STR profiles, this could greatly aid in the screening of sexual assault kits. Instead of sending sexual assault kits directly to the laboratory, they could be screened by the submitting agency or even at the hospital where the sexual assault kit was collected, thus increasing the efficiency of downstream testing performed by DNA laboratories. However, an increase in sensitivity would also be necessary for rapid DNA systems to utilize this method and enable the operator to identify true negatives confidently.

Conclusion Rapid DNA testing is still in the early stages of adoption, yet it has already made significant impacts in the identification of disaster victims and proven the ability to solve crimes in hours rather than days, weeks, months, or even years. As rapid DNA testing becomes more widespread, it is destined to become a dominant force in forensic science. Some are hesitant to put this power into the hands of nonscientists, and others are concerned that rapid DNA testing may replace traditional DNA testing methods. However, the most likely near-term progression will be that an increasing percentage of simpler cases, as well as less violent offenses such as property crimes, will be solved quickly using this technology, while more complex cases will continue to be the focus of forensic scientists using conventional methods. Advancements in rapid DNA technology will continue to contribute to the field of forensic science, enhancing the power of forensic DNA testing and databases. Looking further ahead, it may one day replace the current laboratory methods as the primary routine work ow.

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Bone-sample-processing-RapidHIT-ID-system-RapidINTEL-cartridges-application-note.pdf. Accessed 15 Mar 2021 Thermo Fisher Scientific (2020b) The Italian Carabinieri achieves success using rapid DNA technology. http://assets.thermofisher.com/TFS-Assets/GSD/Reference-Materials/italian-carabi nieri-rapid-dna-customer-profile.pdf. Accessed 15 Mar 2021 Thermo Fisher Scientific (2020c) The Kaua‘i Police Department uses rapid DNA technology to save time and cost in disaster victim identification. http://assets.thermofisher.com/TFS-Assets/ GSD/Reference-Materials/Kauai-police-dna-technology-disaster-victim-identification.pdf. Accessed 10 Mar 2021 Thong Z et al (2015a) Evaluation of the RapidHIT™ 200 System: A comparative study of its performance with Maxwell ® DNA IQ™/Identifiler® Plus/ABI 3500xL work ow. Forensic Sci Int Genet 19:22–27. https://doi.org/10.1016/j.fsigen.2015.05.006 Thong Z et al (2015b) Investigative leads from DNA: Casework experience from the IntegenX RapidHIT™ 200 System. Forensic Sci Int Genet Suppl Ser 5:e69–e70. https://doi.org/10.1016/j. fsigss.2015.09.028 Turingan RS et al (2020) Developmental validation of the ANDE 6C system for rapid DNA analysis of forensic casework and DVI samples. J Forensic Sci 65(2020):1056–1071. https://doi.org/10. 1111/1556-4029.14286 U.S. Congress (2017) H.R.510 – Rapid DNA Act of 2017. https://www.congress.gov/bill/115thcongress/house-bill/510/all-info. Accessed 9 Mar 2021 U.S. Federal Bureau of Investigation (2019) Non-CODIS rapid DNA Considerations and Best Practices for Law Enforcement Use. https://www.fbi.gov/file-repository/non-codis-rapid-dnabest-practices-092419.pdf/view. Accessed 15 Mar 2021 U.S. Federal Bureau of Investigation (2020a) National Rapid DNA Booking Operational Procedures Manual. https://www.fbi.gov/file-repository/national-rapid-dna-booking-operationalprocedures-manual-eff-090120.pdf/view. Accessed 15 Mar 2021 U.S. Federal Bureau of Investigation (2020b) Standards for the operation of rapid DNA booking systems by Law Enforcement Booking Agencies. https://www.fbi.gov/file-repository/standardsfor-operation-of-rapid-dna-booking-systems-by-law-enforcement-booking-agencies-eff090120.pdf/view. Accessed 15 Mar 2021 U.S. Federal Bureau of Investigation (2021) Rapid DNA. https://www.fbi.gov/services/laboratory/ biometric-analysis/codis/rapid-dna. Accessed 9 Mar 2021 Vallone PM, Hill CR, Butler JM (2008) Demonstration of rapid multiplex PCR amplification involving 16 genetic loci. FSI Genetics 3(1):42–45 Wiley R et al (2017) Internal validation of the RapidHIT ® ID system. Forensic Sci Int Genet 31: 180–188. https://doi.org/10.1016/j.fsigen.2017.09.011

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The Interpretation of Mixed DNA Samples Historical Perspective and Current Developments Francesco Sessa, Monica Salerno, and Cristoforo Pomara

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mixture and Complex DNA Profiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Issues Surrounding the LCN Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Probabilistic Genotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Application of Probabilistic Genotyping Methods During the Process . . . . . . . . . . . . . . . . . . MPS Approaches to Forensic Genetics for DNA Mixture Deconvolution . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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The interpretation of mixed DNA samples represents a very compelling research field for the forensic community. Modern short tandem repeat (STR)-based kits can sequence several markers with a high degree of diversity; in the same way, the analysis of STR sequence variation in sample population groups highlighted that several current STR loci lack the necessary sequence diversity. Moreover, in the past few years, new forensic DNA profiling methodologies have been developed based on the use of massively parallel sequencing (MPS), also referred to as nextgeneration sequencing (NGS). The current human STR kits use markers with different heterozygosities, showing some highly heterozygous loci, while others are only marginally heterozygous. New methods offer the opportunity to enhance human identification for forensic purposes in DNA mixtures: using new multiplex kits with highly F. Sessa (*) Department of Clinical and Experimental Medicine, University of Foggia, Foggia, Italy e-mail: [email protected] M. Salerno · C. Pomara (*) Department of Medical, Surgical and Advanced Technologies “G.F. Ingrassia”, University of Catania, Catania, Italy e-mail: [email protected]; [email protected] © Springer Nature Singapore Pte Ltd. 2022 H. R. Dash et al. (eds.), Handbook of DNA Profiling, https://doi.org/10.1007/978-981-16-4318-7_47

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heterozygous STRs, it is possible to solve more effectively the interpretation of many complex mixture samples. Regarding these concerns, MPS methods have improved the discrimination power by investigating genetic diversity through the analysis of both STRs and single-nucleotide polymorphisms (SNPs) located near the anking region. Nevertheless, to date, the mixtures generated by more than two contributors (three or more persons) remain frequently unsolved because of the difficulties in the evaluation of stochastic effects and/or other technical problems, such as the differences between true alleles and stutter products. For these reasons, future studies are needed in order to overcome both these technical and interpretation difficulties. Finally, it is important to remark on the importance of statistical methods to establish the “weight of the evidence,” particularly for the interpretation of MPS multiplex kits for forensic DNA analysis. The interpretation of MPS results remains another interesting research field for the forensic community, particularly in the application of statistical methodologies. Keywords

Mixed DNA samples · Short tandem repeats · Mixture and complex profile · Probabilistic genotyping · Massive parallel sequencing · Next-generation sequencing

Introduction In the first half of the 1980s, Jeffreys used restriction enzymes that cleaved DNA while investigating hereditary diseases, pointing out the possibility of fragmenting DNA samples. Applying this experimental technique, small and large DNA sections were obtained. Using a radioactive probe at low stringency combined with electrophoresis, exposing a film directly on the gel, an image similar to a barcode was obtained (Jeffreys et al. 1985). Based on this evidence, Jeffreys understood the potential use of identifying a subject responsible for a crime: “a single barcode for a single individual.” A few years later, this profiling method was applied for the first time to solve two homicide cases in the forensic field; particularly, it was used to exonerate an innocent person and, only after a mass screening, to identify the guilty subject (Hecht 1989). This case may be considered the start of the forensic application of DNA analysis. A few months later, this technique was purposed for forensic applications. Demonstrating somatic and germline stability combined with individual specificity, the DNA fingerprints produced by Southern blot hybridization have suggested the use of this application as a key element to revolutionize forensic biology, particularly with regard to the identification of suspects in different crimes, such as rape, robbery, and homicide (Gill et al. 1985). DNA fingerprinting, at that time known as the MultiLocus Probe (MLP) technique, demonstrated its great power to both implicate and exonerate individuals as offenders in criminal cases; MLPs

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were compared visually, rather than relying on any agreed statistical methods. Matching patterns were compelling, and if sufficient numbers of bands were present, most practitioners reported that the evidence conclusively identified the samples as coming from the same individual (Aronson 2007). In the early 1990s, several criticisms and disagreements were moved to this technique by the forensic community, generating the so-called DNA wars. For example, problems were found with cases where only some of the MLP bands matched (such as cases involving relatives) and the application of the method to mixed samples or paternity work. There was also the issue of whether individual MLP bands were present in the profile or not. Another disadvantage of the MLP technique was that relatively large biological samples were required to give a reliable result. Subsequently, the single locus probe (SLP) technique was introduced in the UK in around 1988. In forensic science, DNA analysis of restriction fragment length polymorphisms (RFLP) has become the most powerful method. Almost all laboratories working in the field of stain analysis prefer SLPs, because these probes have a better sensitivity than MLPs and offer the possibility to build an allele frequency database. By using specific probes to target different regions of DNA, different chromosomes were typed. Each probe produced one or two bands, called homozygous and heterozygous, respectively, depending on whether the versions of the DNA sequence that an individual inherited from his two parents. Based on this theory, different methods were described (Gill and Werrett 1990; Budowle et al. 1991). In the second phase of the analysis, a database was required to estimate the relative frequency in the population of each of the corresponding SLP bands. Based on the assumption that the population was approximately in a Hardy-Weinberg equilibrium (HWE), which means that the bands are inherited independently within the loci, and using about 4 different SLP probes, the method was able to produce very strong evidence with matching probabilities in the range of 1 in several millions. The probability of matching is an estimate that non-correlated members of the population would have the same profile as an SLP. Although different statistical tests were described (Evett and Gill 1991; Evett and Pinchin 1991), several critical issues were raised: the dimension and representativity of the databases, predetermined band frequency, independent data in the different loci, measurement and laboratory errors, and management of relatives and mixed DNA samples from more than one person. Berry et al. (1992) and Evett et al. (1993) described a more impartial and continuous approach to the evaluation of SLP evidence. This avoided the necessity of a two-step comparison; however, the evidence was reported as a likelihood ratio (LR) rather than a random match probability (RPM). The appropriateness of the likelihood of concordance and LR values resulting from the above methods has been proven by a large-scale experiment performed by Risch and Devlin (1992) and Lambert et al. (1995), involving comparisons among millions of people. In the 1990s, the importance of statistical analysis was stressed, describing different methods on which to base the interpretation of DNA profiles (Morton 1997; Thompson 1997).

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Technological changes since those times have been considerable. DNA profiling systems were further improved by the development of the short tandem repeat (STR) method in 1993. This followed the pioneering work of Mullis who reported a new technique (called PCR) that uses small amounts of DNA to be typed. STR regions typically involve a special sequence of nucleotides that are repeated about 5–30 times. There are many hundreds of these STR regions in the human genome, but several sequences are sufficiently efficient for providing very high levels of human discrimination. The loci targeted for forensic analysis are referred to as short tandem repeats, or STRs. STRs are discrete sequences of DNA that are repeated end to end. They can be repeated in tandem up to 100 times within the genome. The sequence of an STR consists of two to five nucleotides, and these are termed di-, tri-, tetra-, and pentanucleotide repeats. Tetranucleotide repeats are the most common type used in forensic DNA profiling (Fig. 1). The obtained STR profiles look like a series of peaks on a graph; each peak is labeled with a special number according to its position on the graph. This number represents the number of times that the core sequence is repeated. These numerical markers are known as alleles. The STR-PCR technique has provided some distinct advantages over the SLP system: the size of STR peaks can be accurately and unequivocally determined. Therefore, the STR peaks are simply shown as a number series (“alleles”), two for every locus inherited from each parent. This facilitated the creation of a complete intelligence database containing the profiles of all typed subjects; moreover, it amplified several STR regions, analyzing smaller DNA samples compared to SLPs; finally, in the human genome there is a large number of STR regions; therefore, there is no theoretical limitation to the power of the technique.

Fig. 1 Summary of the main STR characteristics

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The first STR kit was developed using 4 STRs, followed by a growing system that could analyze up to 26 STRs (Gill and Evett 1995; Vraneš et al. 2019). Modern techniques have also greatly improved the speed at which a DNA profiling result can be obtained. High-throughput laboratories now have the capacity to generate a plethora of profiling results in just a few hours. More data have meant increased pressure for efficient and effective interpretation methods. Multiplexing is a rapid and convenient way to type many STRs and generate a DNA profile, also maximizing the amount of information obtained from forensic DNA samples, which can be limited in quantity and/or compromised in quality. The STR technique has not led to new statistical questions in forensic cases. However, the question persisted as to which databases were used to estimate the proportions of the alleles. Different authors argued about the bias involved in these methods. Basically, the process is awed because it calls for the idea of proving a null hypothesis of perfect independence (both within and between loci) that cannot be true in a real population (Buckleton et al. 2001). Therefore, this is obtained via a Bayesian estimation of allele proportions using a binomial or trinomial probability and uniform priors (Foreman et al. 1997). The rapid and progressive evolution of analytical techniques and the advent of next-generation sequencing (NGS) have completely revolutionized the DNA sequencing approach. Other markers were suggested as an alternative to STRs, such as single-nucleotide polymorphisms (SNPs) and insertion/elimination polymorphisms, for the identification of individuals. These types of markers have some advantages compared to STRs, such as their smaller size, high discriminatory power, and the fact that they are efficient for typing degraded samples (Oldoni and Podini 2019). Even though this method is widely used in different diagnostic fields, to date, there is no general agreement on the interpretation rules of forensic data produced via NGS technology. Moreover, they are not commonly used by forensic labs, which rely on historical databases of STR profiles, often containing thousands of known individuals to identify criminals. While the gold standard of capillary electrophoresis (CE) in forensic DNA typing is robust and reliable, the ability to determine the number and appropriation of contributors in DNA mixture profiles remains a challenge.

Mixture and Complex DNA Profiles In the 1990s and the first decades of the 2000s, new techniques revolutionized the progress made in the fields of processing and interpreting of data. More sensitive DNA profiling methods generate a profile even if the quality and quantity of DNA are poor. STR typing relies primarily on polymerase chain reaction (PCR) coupled with capillary electrophoresis (CE), where STRs are amplified using extensively validated primer-based multiplexes and separated by size, resulting in precise allele designations on the basis of DNA fragment length (Lohmueller et al. 2014; Ludeman et al. 2018). In forensic genetics, current STR genotyping kits are able to amplify starting from 9 to 27 forensic relevant STRs (Ludeman et al. 2018).

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To date, several STRs are currently used worldwide in forensic cases and are chosen, considering their characteristics and their human genome distribution (Moretti et al. 2016). The current CODIS core loci are composed of 20 STRs: D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, CSF1PO, FGA, TH01, TPOX, vWA, D1S1656, D2S441, D2S1338, D10S1248, D12S391, D19S433, and D22S1045. Moreover, the STRs are chosen considering their inherited characteristics: specifically, they should be independent. It is possible to obtain a profile starting from different sample types, increasing both DNA detection from multiple contributors (called mixed DNA profiles) and the determination of incomplete (partial) and degraded profiles (Fig. 2). For all of these reasons, profile interpretation became very complex and challenging. As shown in Fig. 3, more than two allelic peaks are found for each locus representing a mixed profile. In general, a mixed profile appears to be degraded with the height of the peak decreasing through the profile as molecular weight increases. Moreover, as shown in Fig. 3, one allele is detected at the higher-molecularweight loci (such as D2S1338): no other unlabeled peaks are visible, even though this could be related to the quantity and quality of the DNA. Furthermore, in the D16S539 locus, it may be difficult to distinguish peaks originating solely from stutter from composite peaks, with both allelic and stutter contributions, or from

Fig. 2 The interpretation of incomplete (partial) profiles is challenging for the forensic examiner

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Fig. 3 Mixed profile: considering that no more than four alleles were found for each locus, it could be thought that this is composed of two contributors

allelic peaks originating from a minor contributor. As previously described, at the high molecular weight, it is possible that there is the “dropout” phenomenon. A further difficulty that can occur, especially when using increased sensitivity techniques, is the presence of peaks that are extraneous to the profile. These usually occur in the profile as single peaks (or rarely as more than one peak), characterized by low peak heights, which are referred to as drop-ins. A drop-in is believed to originate when small fragments of human DNA ubiquitously found in the environment are introduced into the DNA sample during PCR setup. Drop-in is not replicable (except by accident), and it is impossible to establish its sources. On the contrary, the contamination phenomenon is typically manifested as reproducible foreign DNA within the worked profile and can often be attributed to a particular source (i.e., the analyst who processed the sample in the laboratory). When a biological trace is recovered from a crime scene, one of three general scenarios can be reconstructed: the comparison is inconclusive; the person of interest (POI) can be excluded as a contributor to the DNA profile obtained from the crime scene evidence; the POI cannot be excluded as a possible contributor to the complex profile obtained from the evidence. This last scenario is very interesting and it appears necessary to supply the weight of this evidence. For this purpose, statistical analysis should be performed: the weight of the evidence is strictly related to the number of detected and included loci in the evidence profile and to the rarity of the observed alleles. Several important organizations, such as the Scientific Working Group on DNA Analysis Methods (SWGDAM), remark in their guidelines on the importance to perform statistical analysis to support inclusion evidence. Moreover, another important aspect is related to the procedures that should be followed for qualifying the significance of associations in the report, whether by a statistic or a qualitative statement (SWGDAM 2010; Moretti et al. 2017). The most important methods used to evaluate the weight of evidence are the combined probability of inclusion (CPI) and the random match probability (RMP). CPI represents the probability that a person could be included as a DNA profile contributor; the RMP is the probability that a random person could have the same genotypes found in the DNA profile. Obviously, these tests are perceived as simple and easily explained during a juridical process. In cases of mixture samples generated by multiple contributors, the DNA profiles may contain several alleles in each locus and in turn the DNA profiles are made more complex. As a result, the combinatory power of the mixed DNA profile using the product rule becomes

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compromised as the complexity of the mixed DNA profile increases. In particular, RMP works best for single source DNA profiles and DNA mixtures that can be resolved into unique components (i.e., mixed DNA profiles with clear major and minor contributors that can be interpreted independently) (Bille et al. 2013). Another commonly used method is the likelihood ratio (LR): this ascertains the probability of two mutually exclusive propositions; in particular, it represents the ratio between the prosecution hypothesis (Hp) and the defense hypothesis (Hd): this method is frequently applied in real forensic cases and it is well-supported by the forensic community. As previously described, considering that recent forensic kits have a high limit of detection, the interpretation of a DNA profile could be very complex. Particularly, several forensic laboratories continued to use inadequate methods, both procedurally and statistically, and these situations generated several failures at important trial procedures. Several significant papers were published to describe inter- and intra-laboratory inconsistency, remarking on the importance to follow well-established guidelines in order to avoid or reduce procedural bias. In 2011, Dror and Hampikian reported an exercise studying the variability in DNA profile interpretation (Dror and Hampikian 2011). In their study, the authors underlined intra-laboratory variability: this conclusion pointed out the so-called “fallibility of DNA evidence,” which could in uence the decision about suspected people (Geddes 2010). In a recent publication, interlaboratory DNA methods were also discussed (Butler et al. 2018). Even though the study presented several criticisms, it could be considered an important example to highlight the variability in DNA interpretation. Due to subjectivity in the interpretation of the guidelines, several different interpretations were made by different scientists involved in the evaluation of the evidence (Butler et al. 2018). In order to better define the weight of forensic evidence, the forensic scientist should apply a probabilistic expert system particularly in the case of DNA mixtures originating from more than one person. DNA mixtures are currently the most common challenge in forensic genetics casework. However, mixed DNA profiles involving more than two contributors are more complex than two-person mixtures. Figure 4 shows that the presence of more than two alleles in one or more loci represents the so-called mixed profile. When using the STR technique, it is very important to evaluate several characteristics of the peak, such as the peak areas: for example, when the mixture is composed of two contributors, it could be possible to distinguish the two alleles of one contributor from the peak height, expecting that they are similar in both height and area; in contrast, excessive differences could show different contributors. On the contrary, when the mixture is composed of more than two contributors, the probability of solving the mystery behind the profile decreases. Obviously, several parameters are more important in order to solve the mixture: first, the quality and the quantity of DNA and, second, the choice of the DNA typing kit and the instrument for the capillary electrophoresis with the relative parameters are both of utmost relevance (Gill et al. 2015; Butler et al. 2018). These kinds of profiles show multiple alleles for each locus, and in several cases, it may be possible to find the profile of an unknown person who has only handled the evidence before or after the crime (Fig. 5).

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Fig. 4 Simulation of a two-person resolvable mixture: the peaks might may be useful to distinguish major and minor contributors

Fig. 5 When the contributors to the mixture are more than two persons, it is hard to distinguish the real profile. Moreover, it is very difficult to understand if several peaks are realistic or artifacts such as stutter (e.g., in locus B, allele 8)

These factors in tandem may generate profiles hard to be deconvoluted in the different contributors’ genotype, reducing the weight of DNA typing and leaving cases unsolved.

Issues Surrounding the LCN Technique As previously stated, the current forensic techniques are able to detect a complete profile working with a low copy number (LCN) DNA. LCN typing, particularly for current short tandem repeat (STR) typing, refers to the analysis of any sample that contains less than 200 pg of template DNA (Budowle et al. 2009). These techniques are extremely sensitive and enable profiles to be obtained from only a few cells of starting DNA. In this context, it is important to highlight the possibility to transfer skin cells with the relative profile under different circumstances (Phipps and Petricevic 2007; Sessa et al. 2019); the adequate interpretation of LCN profiles should take into account modeling contamination, the possibility to obtain the so-called drop-in of spurious alleles, and also the reproducibility of DNA profiles. Concerning this last issue, LCN

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analysis is by nature not reproducible: for this reason, it cannot be considered as robust as that associated with conventional DNA typing. Nevertheless, LCN typing is commonly used for the identification of missing persons and human remains and for developing investigative leads: in this scenario, caution should be taken with its use in other endeavors until developments are made that overcome the vagaries of LCN typing. The most important limitation in the case of LCN analysis is related to a great degradation of DNA, both because of the limited number of cells and because of the storage conditions of the source. Therefore, the obtained profile can be characterized by an incomplete representation of alleles (often called “allelic drop-out”). Indeed, in different loci, a single allele could be observed, but it is not possible to determine if the subject is homozygote or if it is a dropout phenomenon. Moreover, to allow for a complete interpretation, several parameters are frequently modified, such as the baseline value on the graph; contrariwise, this choice generates imbalances in the interpretation of the profile, particularly in heterozygote alleles. Working with an LCN profile, the more complex biases are related to the undetected loci (no peaks) or when the peak is very difficult to assess. Moreover, the possibility of a stochastic contamination event should be considered: inserting a few exogenous cells into the reaction can generate the presence of other additional alleles. This contamination could be traced back to degraded human epithelial cells (i.e., dust particles), referred to as “Touch DNA,” or it may itself be the product of an earlier PCR amplification. Genetic profiles generated from fingermarks were first described in 1997 (van Oorschot and Jones 1997). As regard the so-called Touch DNA, there is a growing interest in it by the forensic community. Several important aspects of “Touch DNA” have been studied such as the “handling time,” analyzing the contact time needed to deposit a sufficient amount of DNA on a garment to produce an interpretable profile, or the technique that guarantees the best recovery (Sessa et al. 2019). Moreover, this kind of DNA has been investigated under different scenarios in order to better clarify several aspects linked to the deposition or transferring conditions (Gosch et al. 2020; Neckovic et al. 2020; Sakurada et al. 2020). To reduce all of these biases, a consensus profile approach was suggested (Whitaker et al. 2001): the reproducibility of the allele is taken to be present in the profile. Obviously, the consensus profile restricts the designation of the artifacts and the potential dropout or drop-in of alleles, improving the statistical analysis of the obtained mixed profiles.

Probabilistic Genotyping Statistical evaluation is very important in order to provide an estimate of the number of people who would match the crime profile in a specified population. In fact, the improvement of the STR methods poses a number of relevant questions to issues that may be critical for the defense and can include the following: how and when the DNA was deposited, its relevance to the alleged crime, establishing continuity of exhibits and integrity of the evidence, and, ultimately, laboratory error.

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One of the first methods used to evaluate the mixture profile is related to the calculation of the probability of exclusion (PE) (Devlin 1993). This case is represented by the event of the allele not being shared in the mixture profiles. Obviously, the main limitation of this proposed approach is the presence of the dropout event, particularly in the case of degraded DNA. To improve this approach, new methods were proposed that considered the mixture profile of a subject involved in the crime as a contributor (e.g., the victim’s profile): this profile could be very useful in order to ascertain the unknown profile (Devlin et al. 1990). As previously described, the use of modern forensic kits has improved the possibility to obtain LCN profiles, which, unfortunately, has increased the background noise in a profile. Several experimental models have been proposed in order to solve these difficulties, describing different scenarios and the relative software or approaches (Weir et al. 1997; Curran et al. 1999; Fung and Hu 2002; Fukshansky and Bär 2000). All of the LR approaches discussed above are based solely on which alleles or peaks are present in the mixture. Therefore, if we consider the case of a two-person mixture, where “a” denotes the vector of alleles observed at a locus, “G” denotes the genotypes of the two contributors, and “H” denotes the hypothesis, the LR is then composed of terms of the type: p ðajG, H ÞPr ðGjΗ Þ As previously described, to interpret an STR mixture profile, several parameters should be evaluated, such as the peak intensities or areas, because these data could supply the indication of the amount of DNA being contributed by the allele to the mixture. The areas are usually indicative as to the relative DNA contributions; to date, forensic scientists may use this information to assign the value of “0” or “1” to the possible combination of contributors. This information is summarized in several papers that describe this approach in the deconvolution of two-person mixtures (Clayton et al. 1998; Gill et al. 1998; Gill 2002). One of the first methods to analyze the mixture profile (two-person mixed profile) was provided by Evett et al. (1998). This method considered the peak area evidence more formally by modeling ratios of peak areas using normal probability distributions. A few years later, Balding (1999) indicated a new recursive formula to interpret the evidence considering the population substructure, although this approach can only be applied to the mixture of two contributors. In 2001 (Perlin and Szabady 2001), a new deterministic approach was applied to identify the contributor in mixture profiles using an intelligence database. This is reached by a process of optimization based on a least-squares-type metric following the removal of the random PCR processes that act on STR profiles. Although providing an adequate solution for intelligence purposes, this is wholly inappropriate once the evaluative stage is reached. Modern forensic technologies improved the DNA profile results; in the same way, the complexity of profile interpretation became very difficult and demanded more complex methods to correctly validate the results. Starting in 2010, the method required to validate the results have been the so-called “probabilistic genotyping (PG).” PG evaluates several different parameters

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such as peak height, and peak area, using specific software based on mathematical processes. This software increased both the objectivity and the weight of the DNA evidence, reducing the analyst’s subjective interpretation as well as improving the overall interpretation of the DNA profile. The PG software is based on the LR (1), meaning the ratio of two mutually exclusive hypotheses (two conditional probabilities).
 Pr OjΗ p , I LR ¼ Pr ðOjΗ d , I Þ

ð1Þ

In this formula, “O” indicates the “observation”; in other words, it represents the DNA findings, “Hp” is one proposition (in general, it represents the prosecution theory including the POI), “Hd” is the other proposition (in general, it represents the defense theory excluding the POI), while “I” represents the other information named “framework of circumstances.” To implement the PG, another form is used (2): ð


 Pr OjS j ¼ p OjS j , M dM




ð2Þ

M

In this equation, “Sj” is the possible genotype set that may be included in the profile, and “M” re ects the parameters used to obtain the DNA profile. These parameters are commonly defined as “weights,” and different approaches may be applied to improve the value of these terms. The most used approach is a “binary” interpretation approach, assigning the probabilities of “0” and “1” to these weights. Semicontinuous models were applied, establishing the presence or absence for each allele, calculating the probabilities for the “drop-in”/“dropout” phenomena. This approach is easier to understand than its counterparts. The main limits of this approach are the absence of the evaluation of peak height and the operator decision about the stutters. The semicontinuous models have good discrimination power in order to distinguish the true/false donors; nevertheless, these methods are unable to separate a mixture profile into individual profiles: for this reason, they cannot be used in DNA mixture interpretation when no reference profile is available for a POI. To mark the naissance parameters indicated with “M,” a set of approaches can be applied, using the maximum likelihood estimation (MLE) that does not lead to Eq. (2). To obtain functions proportional to the integral in Eq. (2), several applications can be applied, such as Markov chain Monte Carlo (MCMC). This model represents a continuous model, assigning a value between 0 and 1 to show the likelihood of a proposed genotype given the observed DNA profile. The very important characteristic of the continuous model is related to the possibility to assign a relative probability, whereas the previous analyses are based on the determination of a possible (1) or not possible (0) genotype. In this way, the MCMC models may deconvolute a mixed profile into separate profiles, generating different combinations as well as calculating match statistics for comparison to the POI.

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MCMC is applied in different fully continuous models, such as the PG software STRmix™. This software, developed by scientists with Forensic Science South Australia (FSSA) and Environmental Science and Research (ESR) in New Zealand, is one of the most used software packages for the interpretation of forensic DNA profiles today.

The Application of Probabilistic Genotyping Methods During the Process There is a growing interest in the identification of a new method to establish an LR. The degree of mistrust or belief of any particular PG method is strictly related to the individual profile and among all participants involved in the relative scenario. Another important question is related to how to present the statistical evaluation. At first, the presentation of the match probabilities, even in order of several billions or less, was considered insufficient by the judge. For this reason, it was necessary to translate the match probabilities into frequencies in order to clarify the correspondence between the profile obtained from the evidence and the suspect (Hoffrage et al. 2000). Similarly, insufficient knowledge of statistics in general and incorrect Bayesian reasoning in particular can result in false convictions or acquittals made by juries in the court, for example, when they have to evaluate evidence based on a fragmentary DNA sample. Another important question is related to the techniques applied in the profiling: current methods are able to obtain a complete profile starting from only few cells, referred to as LCN (Foreman and Evett 2001). This possibility should be evaluated in the two-way analysis. On the one hand, it is possible to obtain a profile in very difficult conditions; on the other hand, in other conditions, only a mixture profile or a partial profile may be obtained. This situation must be analyzed with the relative statistical tests in order to ascertain the weight of the evidence. In the definition of a repeatable method, it is very important to ascertain if the theory or the technique used for the statistical analysis has been tested, has been subjected to peer review and publication, and has been generally accepted by the scientific community. Moreover, it is very important to define if the potential error rate has been defined and if there is a standard to check the effectiveness of the methods. Obviously, one of the most important open questions concerns the standards that exist for PG and the applicability with the related software. Several different international organizations, such as the UK Forensic Science Regulator (UKFSR), the International Society for Forensic Genetics, and the US Scientific Working Group on DNA Analysis Methods, have published guidelines to validate PG software, focusing on the desired actions (SWGDAM 2015; Coble et al. 2016; Coble and Bright 2019; Leib 2019). During the processes, there are two opposite statements: these are the prosecution and the defense theories. In this scenario, Bayes’ theorem is very important in order to provide a logical evaluation of evidence “E” in the DNA analysis:

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 Pr H p jE, I Pr EjH p , I Pr H p jΙ ¼ Pr ðH d jE, I Þ Pr ðEjHd , I Þ PrðH d jΙ Þ

ð3Þ

in this formula (3), “Hp” indicates the prosecutors’ proposition, “Hd” the proposition of the defense, and “I” the circumstances (meaning the form of time, location, and witness statements describing the alleged actions). In this scenario, each value is strictly related to the reconstruction: if, during the process, the data changed, the new interpretation should be supplied. Moreover, the evaluation of LR is very important:
 Pr EjH p , I LR ¼ Pr ðEjH d , I Þ

ð4Þ

The evaluation of the LR represents a key point for evidence interpretation, impacting the content of the statements (Evett et al. 2000). Particularly, its value is very important in order to define the weight of the evidence and is strictly related to the framework of circumstances. Moreover, the forensic scientist plays a pivotal role in order to define the values of each proposition (Balding and Nichols 1994; Evett 1995). In light of this information, before any evaluation, it should be mandatory to elucidate the two alternative propositions for each criminal scenario. One of the most important concerns that several international courts have highlighted is relative to the closed source of software used in the processes, such as STRmix™. In this way, the rights of the defense could be prejudiced. The rights of the accused are exercised with the possibility to check the source code of the software. This opportunity is guaranteed with open-source software; on the other hand, closed source complicates this opportunity, and the cost, inconvenience, and the possibility to use Internet access should be evaluated. Moreover, the source should be consultable everywhere by everyone. In addition, PG software, in several cases, conceals the algorithm applied, becoming a “black box.” This situation is overcome when the mathematical processes of the software are published in a peer-reviewed journal: this is the case of STRmix™. For example, in this case, numerous training workshops have been organized, giving practical training to the participants, thus allowing them to understand all of the possibilities of the software. In this way, forensic scientists can be trained, improve their knowledge, and avoid the use of unsophisticated yet obsolete methods. Moreover, in this regard, the International Society for Forensic Genetics (ISFG) has created a specific section on its website with the indication of forensic software resources (available on https://www.isfg.org/Software). Particularly, by supporting open-source software projects in forensic statistics, the ISFG has created a list of software applications as a service to the forensic genetics community, even though it is clearly written that the ISFG is not endorsing any specific software. Moreover, it is evident that the responsibility to validate and check if the selected program meets any applicable casework standards is of the end-forensic-user.

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MPS Approaches to Forensic Genetics for DNA Mixture Deconvolution As previously described, the statistical evaluation of DNA evidence weight is a very old question, particularly for mixture profiles. Indeed, for several years the procedures in the assignment of the number of contributors in a crime scene profile were made in subjective way (Cook et al. 1998a, b). Moreover, considering the sensitivity of modern forensic kits, the question about how DNA may have been transferred remains an important issue in the evaluation of evidence weight (Kokshoorn et al. 2017; Taylor et al. 2018b). Each piece of DNA evidence should be supported through the analysis of several concerns, such as transfer, persistence, and recovery of the detected profile. For these reasons, this research field should be supported by forensic scientists. In the last decade, several different techniques have been combined with the standard STR methods to supply the PG evaluation, such as “Y” or “X” chromosome profiling (Asmundo et al. 2006; Presciuttini et al. 2011; Ventura Spagnolo et al. 2017). In this way, the weight of the evidence is improved (Taylor et al. 2018a, b; Andersen and Balding 2019). Moreover, the traditional methods based on STR typing (including Y-chromosome STRs and X-chromosome STRs) require about 500 picograms of DNA, and in several forensic cases, the collected evidence is enough for a single DNA analysis (Ludeman et al. 2018). Another important question related to the mixture profile is the use of the new methods in forensic DNA profiling that have been developed in the past few years, using MPS, also referred to as NGS. These methods are able to detect STRs, SNPs, and in/del (insertions and deletions) simultaneously. MPS is related to a variety of high-speed sequencing platforms (sometimes called “second generation” or “next generation”) that use a common technical approach to sequence a large number of fragments in parallel using spatially separated and clonally amplified DNA templates (Børsting and Morling 2015). Several phases are included in these techniques, such as the creation of a library, bridge amplification or emulsion PCR, and enrichment (Churchill et al. 2016). In particular, STRs may be amplified and subsequently sequenced: with this approach, it is possible to analyze the nucleotide differences observed in the selected STR repeat motifs and their anking regions. Each fragment is uniformly and accurately sequenced in millions of parallel reactions. It is important to note that these new techniques generate data that are compatible with those previously archived. In particular, moving forward with MPS will give the forensic community an opportunity to expand its current use of forensic DNA typing with increased STR and SNP genetic diversity without the loss of the use of the millions of DNA profiles currently held in DNA databases (Churchill et al. 2016; Novroski et al. 2016). To date, the commercial test used for forensic purposes can type hundreds of markers: in this way, adding the sequence information, the discriminatory power is improved (Fig. 6).

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Fig. 6 Summary of the result of a single locus in a mixture profile obtained through NGS. In this case, the two people shared both alleles (11,12). The signal detected in allele 10 is a stutter from allele 11. Allele 11 is indistinguishable because no SNP difference is reported. Contrariwise, allele 12 distinguished the major and minor contributors. Using CE methods, it is impossible to obtain this result

These techniques are able to generate a large amount of DNA data using minimal starting material: NGS overcomes a major limitation of CE typing that often necessitates multiple rounds of testing, which can be impossible on limited or poor-quality material, or may not yield sufficient information for a conclusive result. Indeed, considering that in several forensic cases the initial biological sample recovered at the crime scene can be very poor, these methods minimize the overall number of samples used. For example, the ForenSeq™ DNA Signature Prep Kit (Verogen, San Diego, California, USA) contains 58 STR loci (27 autosomal, 7 X-chromosome, and 24 Y-chromosome STRs) whose amplicon sizes generally fall within the 350 base pair read length. In the same way, the Precision ID GlobalFiler™ NGS STR Panel v2 (Thermo Fisher Scientific, Waltham, MA, USA) analyzes more markers (35), including 20 CODIS STRs, 9 multiallelic STRs, 4 markers for the determination of sex, and 2 penta-STR markers with high informativity. Moreover, MPS kits are able to supply further information such as physical characteristics, including eye color or ethnicity of the investigated subject (Wang et al. 2015). In contrast, even though MPS may offer the greatest opportunity to resolve DNA mixtures, to date it is necessary to improve the statistical methods used to interpret these data (Coble and Bright 2019; Hwa et al. 2019). One of the most important problems is related to the use of a database to evaluate the weight of evidence statistically. Several papers worldwide have published datasets, contributing to the growth of STR sequence diversity, which is now compiled in the STRSeq database (Novroski et al. 2016; Wendt et al. 2016; Phillips 2017). Nevertheless, the difficulty of interpreting mixed DNA profiles using MPS is about the same as in traditional methods (Churchill et al. 2016). Although the quantification of each allele present in the mixture is more realistic, allele sharing is still a common phenomenon observed in a mixture. The determination of the amount of each allele allows a better

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determination of allele ratios in the mixture and, in instances, of shared length-based alleles (i.e., homozygous by length) that differ by sequence (i.e., heterozygous by sequence), the contribution of each unique allele at the locus. Nevertheless, in cases where relatives may be involved, a lack of sequence variation in certain loci will limit the usefulness of MPS technologies (Novroski et al. 2016).

Conclusion Notwithstanding the recent developments in DNA typing methods, the interpretation of a mixture remains one of the most absorbing research areas in the forensic field. Mixed DNA samples arise from the combination of two or more individual bodily uids or secretions: this kind of evidence is common, and even expected, in many forensic investigations such as sexual crimes, large disasters, as well as in products of conception and fingernail cuttings taken by police or at autopsy. The difficulties related to DNA mixture interpretation are closely related to the different starting conditions: different types of materials, number of donors, and different proportions of each component in the mixture, as well as artifacts such as allelic dropout and allelic drop-in. As far as the interpretation of mixed DNA samples is concerned, the objective of this analysis is the identification of major and minor contributors in the mixture profiles, following the national team and international guidelines (Gill et al. 2018). The use of MPS methods has improved the ability to identify the contributors of a DNA mixture by identifying sequence variation through both STR typing and anking region sequencing. Moreover, modern STR-based kits allow the sequencing of several markers with a high degree of diversity; in the same way, the analysis of STR sequence variation in sample population groups highlighted that several current STR loci lack the necessary sequence diversity. In this game of equilibrium, the new methods offer the opportunity to enhance human identification for forensic purposes in DNA mixtures: by using new multiplex kits with highly heterozygous STRs, it is possible to have powerful conditions to solve the interpretation of many complex mixture samples more effectively. In the same way, it is possible to improve the DNA database by inserting more useful information that increases the number of forensic samples uploaded. The current human STR kits use markers with different heterozygosity, showing some highly heterozygous loci, while others are only marginally heterozygous. To improve the discrimination power in mixtures, it is important to search for new candidate loci. There are three criteria routinely used by the forensic science community to evaluate a new candidate STR: the locus should conform to the standard conditions (with particular regard to the amplicon size and composition); the locus must show great resolution power; and it should be evaluated and validated with robust studies. Recent studies have highlighted two important parameters for new forensic STR markers. First of all, they should exhibit a high degree of heterozygosity; moreover, they should show limited allele spread.

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In this context, it is important to note that several highly informative STR markers are not included in the current kits because of technology constraints related to several conditions such as the chemicals used. Moreover, to date, mixtures generated by more than two contributors (three or more persons) remain frequently unsolved because of the difficulties in the evaluation of stochastic effects and/or other technical problems, such as the differences between true alleles and stutter products. For these reasons, future studies are needed to resolve both of these technical and interpretation difficulties. To give an instance, a very recent study compared NGS and traditional methods to evaluate the ability in the identification of major and minor contributors (Ragazzo et al. 2020). Based on the results of this experimental model, NGS represents a reliable and robust method for human identification in standard conditions (starting from optimal/suboptimal amounts of DNA). It is important to note that the discrimination power (meaning the possibility to identify the major and minor contributors) in this system is obtained by setting the default analytical threshold (5% of the total coverage of alleles in each locus). As reported in this study, removing filters, the analysis fell into a range defined “inconclusive,” confirming the importance of the pre-established analytical threshold for these methods. Finally, the importance of statistical methods to establish the “weight of evidence” should be remembered, particularly for the interpretation of MPS multiplex kits for forensic DNA analysis. The interpretation of MPS results remains another interesting research field for the forensic community, particularly in the application of statistical methodologies.

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Ethical and Legal Standards Sachil Kumar, Saranya Ramesh Babu, and Shipra Rohatgi

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . National DNA Databases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . African Region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Middle East . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . European Region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Australasia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . American Region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Asia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ethical and Legal Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Major Ethical Concerns Include . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Recent advances in DNA technologies have made it possible to establish DNA databases for forensic investigations. Forensic DNA databases are currently in operation in about 70 countries. As expected with the tremendous success of the use of forensic DNA databases, many ethical and legal problems arise in the preparation of a DNA database, and these problems are particularly significant when one analyses the legal regulations on the subject. The continuous rise in the

S. Kumar (*) · S. R. Babu Department of Forensic Sciences, College of Criminal Justice, Naif Arab University for Security Sciences, Riyadh, Saudi Arabia e-mail: [email protected]; [email protected] S. Rohatgi Former Faculty/ Research Scholar, Amity Institute of Forensic Science, Amity University Gurugram/Noida, Noida, India © Springer Nature Singapore Pte Ltd. 2022 H. R. Dash et al. (eds.), Handbook of DNA Profiling, https://doi.org/10.1007/978-981-16-4318-7_48

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size of forensic DNA data sets poses concerns about the requirements for inclusion and preservation and suspicions about the efficacy, affordability, and privacy violation of such vast repositories of personal data. In light of its broad reach, the database raised concerns about anonymity, government surveillance, and human rights. The aim of this chapter is to shed light on the current status of forensic DNA databases and their ethical and legal issues. Keywords

Humans · Databases · Nucleic acid · Police · Privacy · Law enforcement · Crime · Data collection · DNA

Introduction The establishment of DNA database began when the standard practice of forensic DNA typing of STR markers was accepted globally. The national DNA database of England and Wales (NDNAD) was the first criminal DNA database to be established in 1995 followed by New Zealand’s database (Williams and Johnson 2008). In the USA, the Federal Bureau of Investigation (FBI) organized the Combined DNA Index System (CODIS) database, and it was from then, most welcomed by the criminal justice system including Parliamentarian, policy makers, police officers, and criminal detectives to solve criminal cases (Amankwaa 2018). DNA databases were started by only collecting the profile of the individuals convicted/offended or suspected in previous crimes and in addition to the DNA profiles gathered from crime scene samples so as to find any hit or lead in other cases (Santos et al. 2013). But later, it began to acquire data from the public to maintain the references sample of the specific population be used in police investigation, and some countries continue to keep up record for specialized missing person’s DNA database including the Missing Persons Relatives, Unknown Humanoid Remains, Y-STR, and mitochondrial DNA analysis (DNA 2020). Forensic DNA databases from different countries vary in their data inclusion/ retention and exclusion criteria based on laws enacted by their government policies (Montague 2011). According to Interpol global DNA profiling survey of 2016 and 2019, out of 194 member countries, only 89 countries reported acquiring DNA profile for investigative purposes of which are 11 African, 13 American, 19 Asian, and 46 European countries, while 70 countries has DNA database of which are 7 African, 10 American, 13 Asian, and 40 European (DNA 2020). The biggest DNA databases are in China with 80 million profile as of 2020 (Wee 2020), followed by the USA which is projected with 19.5 million in 2020 (CODIS – NDIS Statistics 2016) and the UK with 6.2 million profiles in 2018 (NPCC 2017–2018).

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National DNA Databases African Region South Africa’s DNA Criminal Intelligence Database (DCID) was established in 1998, and new legislation for expanding the National Forensic DNA Database of South Africa (NFDD) was passed in 2015. The profiles in NFDD are arranged by the following indexes, (a) Crime Scene, (b) Arrestee Index, (c) Convicted Offender Index, (d) Investigative Index, (f) Elimination Index, (g) Missing Persons, and (h) Unidentified Human Remains Index, and 91,240,168 DNA profiles were available by 2016 (Heathfield 2014; de Wet et al. 2011). Botswana’s and Namibia DNA database contains 3300 and 1338 DNA profiles (Interpol 2011; Global Summary 2020).

Middle East Egypt, Tunisia, Lebanon, Saudi Arabia, and Bahrain have 4162, 17,070, 23,000, 909,745, and 69,609 profiles (Interpol 2019). Israel Police DNA Index System (IPDIS) is the Israeli national DNA database established in 2007. The IPDIS includes elimination bank holding the profiles of the personnel handling the forensic sample and contains 491,380 profiles (Interpol 2019). Iran, Kuwait, the UAE, and Qatar are reported with 10,000, 14,591, 24,370, and 2500 profiles (Interpol 2008). Jordan Database was established in 2000 and contains 14,104 profiles according to Interpol 2008. However, Algeria, Kenya, Rwanda, Uganda, Somalia, and Seychelles are planning to open up international DNA database (Global Summary 2020).

European Region The council of European Union (EU) established DNA database in 1997. The DNA working group of the European Network of Forensic Science Institutes (ENFSI) decided that all EU member state should begin the establishment of forensic DNA database. The ENFSI has also set standards to generate DNA profiles for the DNA database, and Prüm treaty was signed in 2008 to make the database available for the exchange of forensic DNA data among the EU member states (Toom 2018). However, Prüm treaty was not operational in Greece, Ireland, and Italy (Europol 2019). The Netherland has DNA database for criminal cases and missing persons which was by established National Forensic Institute (NFI) in 1997 and contains 351,912 Dutch people profiles as per the 2017 annual report of NFI (Ministerie van Justitie en Veiligheid 2018). The former database has index for traces, suspects, convicts, and

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deceased victims and later has index for unidentified, missing persons and family members of missing persons (Ministerie van Justitie en Veiligheid 2019). MixCal6 a statistical method for assessing the evidentiary proof of mixed DNA traces was developed by NFI and included in CODIS by FBI (Ministerie van Justitie en Veiligheid 2020). Germany’s DNA database Bundeskriminalamt (BKA), Wiesbaden was set up the German Federal Police in 1998 and holds up to 1,213,331 profiles (Interpol 2011). The campaign “Stop the DNA Collection Frenzy!” initiated by various civil rights and data protection organizations to circumvent the increase in DNA data-collection leads to the cancellation of international exchange of DNA data in 2011. Austria’s ministry of interior maintains their DNA database and is composed of 384,098 profiles (Interpol 2011). Banca dati nazionale del DNA is the Italian national DNA database (ITNDNADB) which was formed in 2009 and uses CODIS software for comparison (Biondo and De Stefano 2011); however, concerns are raised by the member countries as the ITNDNADB are not consistent with security measures to protect the data and storage of DNA profiles and biological samples (Marchese et al. 2013). Belgium has two national DNA database in 1999 by National Institute for Criminalistics and Criminology with 54,399 profiles as of 2019 data (Belgium 2020). The criminalistics database for the DNA profiles are derived from crime scene while convict’s database for DNA profiles of persons convicted for offence crimes, and however, no database for suspects DNA profiles were created (De Moor 2018). Similarly, in Denmark’s Central DNA Database was introduced in 2000 and divided into individual database where DNA profile of perpetrator charged with a crime and Trace Database for those obtained from unidentified crime scene samples and currently holds 184,913 profiles (Anker et al. 2017). Estonia and Ireland established its DNA database in 2004 and 2013 currently holding 67,758 and 26,492 profiles, respectively (Interpol 2011). Finnish National DNA Database is formed in 1999 under the custody of National Bureau of Investigation (NBI) Crime Laboratory and contains 193,575 profiles. Both Estonian and Finnish police were authorized to forcibly collect DNA samples from adults and minors suspected or convicted for any offense (Global Summary 2020). The French national DNA database called as the Fichier National Automatisé des Empreintes Génétiques (Automated National File of Genetic Prints) was formed in 1996 and holds 4,247,382 profiles (Interpol 2011) which is 5% of its population; initially profiles were reported only sexually related crimes but expanded to cover profiles from almost all violent crimes in 2003 (Global Summary 2020). Greece began its DNA database in 2008 for convicted individuals, but by 2009 it started to include suspects of adults and children and presently holds 34,647 profiles (Voultsos et al. 2011), while national database were established for Cyprus, Croatia, and Czech Republic in 1998, 2000, and 2001, and each contains 51,093,16,706, and 253,085 profiles, respectively (Interpol 2011). Hungary’s DNA database was set up in 2004 and contains 212,196 profiles, while Bulgaria’s Database was formed in 1999 and holds 17,055 profiles. Portuguese DNA

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databank which was formed in 2005 known as Portuguese Ethics Council (CNECV – Conselho Nacional de Éticapara as Ciências da Vida) has index only for criminal’s DNA profile and holds 5393 profiles as per 2013 Portuguese press report (Borja-Santos 2015). Romania established National System of Judicial Genetic Data (NSDGJ) in 2008 and is subdivided into 1) personal database containing personal data of perpetrators and instigators, 2) investigation database for derived from crime scene stains, and 3) DNA profiles database for profiles of individuals and unidentified crime scene stains (Frank and Găină 2014). According to Interpol 2011 report, 8000 reference DNA profiles are stored in NSDGJ. Slovenia, Slovakia, and Luxembourg reported to have 37,531, 96,203, and 10,358 profiles (Interpol 2011). Sweden and Spain established their DNA database in 1998 and 2007 and have 201,900 and 627,163 profiles, respectively, while North Macedonia holds 27,545 profiles according to 2019 report of Interpol (Interpol 2011). The other non-EU member countries including Norway, Iceland, Switzerland, and Liechtenstein also signed Prüm treaty for the exchange of DNA data (Council of the European Union 2018), while Norway and Switzerland have reported to contain 109,180 and 268,417 profiles, respectively (Interpol 2011). The National Commissioner of Police and the Ministry of Justice in Iceland established two forensic DNA databases in 2008: one contains the profiles of individuals convicted for crimes for the Identification Database and the Trace Database for unidentified genetic profiles found at crime scenes (stjórnarfrumvarp: Erfðaefnisskrá lögreglu 2020). Belarus and FYR Macedonia are currently holding up to 336,408 and 27,454 profiles, respectively (Interpol 2011). In Russia, Federal database of genome information (FDBGI) was formed in 2009 and has voluntary and mandatory genomic registration and contains 734,373 profiles (Interpol 2011). The mandatory database includes profiles of the convicted and imprisoned persons involved in all categories of crimes and unidentified corpses/ persons collected at the crime scene (Perepechina 2019). The UK National Criminal Intelligence DNA Database (NDNAD) was foremost forensic DNA database formed in 1995. Later in 2007, an NDNAD service was reassigned to the National Policing Improvement Agency to maintain the database operations and ensure the integrity of the data. According to the Criminal Justice and Police Act 2001, DNA sampling was done only from individuals charged with any recordable offence. But later, due to the Criminal Justice Act of 2003, samples were collected only from individuals above 10 years of age on arrest for their involvement in any crimes in England and Wales. However, after the Protection of Freedoms Act in 2012, DNA data of more than 1.7 million innocent people and children who were not charged and/or not found guilty have been deleted, and more than 7.7 million DNA samples have also been destroyed. The National DNA Database department in the Police Forensic Science Laboratory Dundee of Scotland disseminates the collected data to the NDNAD in England (Johnson and Williams 2004). Similarly, Isle of Man Constabulary’s Scientific Support Department and the police of Jersey and Guernsey also store their data to NDNAD.

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Australasia New Zealand’s Criminal Investigations (Blood Samples) Act of 1995 initiated the establishment of National DNA Profile Databank (NDPD) in 1995, and ESR (Environmental Science and Research) maintains the operations of the NDPD. Legislation was amended in 2004 to expand the Criminal Investigations (Bodily Samples) Act of 1995 in which samples were obtained from convicted current prison inmates prior to 1995 and mandatory sample collection as buccal swabs for the Databank (Criminal Investigations Bodily Samples Act 1995). It has two databases, namely, DNA Profile Databank (DNAPD) and Crime Sample Database, one for the individual profiles of the registered criminals and volunteers and the other for profiles from unsolved crimes, respectively (Harbison et al. 2001). In 2010, Legislation was amended and expanded for the Enactment of the Criminal Investigations (Bodily Samples) Amendment Bill 2009, and thus Temporary Databank (TD) was created holding profiles of the person arrested or intend to charge, and if convicted, it is transferred to DNAPD (Flaus 2013). In 2013, New Zealand signed a data sharing agreement with the USA, to allow each country legal access to the other’s fingerprint database under specified conditions, for automated searching. If a match was found in either country, further information such as addresses, convictions, known associates, and aliases will be shared (Agreement between the United States of America and New Zealand 2013). From 2014, DNAPD utilizes Globalfiler ® PCR amplification kit for the generation of profiles and plans to expand the database to massively paralleled sequencing technology for the analysis and integration of case samples with the DNAPD and currently maintains 237,269 DNA profiles. In Australia, the Crimes Act 1914 enable offenders, suspects, and volunteers to be sampled for criminal investigations and also standardizes the use, storage, release, and removal criteria in the DNA database system. The Australian Criminal Intelligence Commission established three DNA databases, 1) DNA database for law enforcement purposes operated by the Australian Federal Police, 2) Disaster Victim Identification database, and 3) the National Criminal Investigation DNA database (NCIDD), in 2001. In 2015, the NCIDD started to include familial searching, kinship matching, mtDNA, and Y-STR profiling and reported to held 830,000 DNA profiles as of 2014 (The National DNA Database Is Watching You 2020). Australia has also signed agreement for supported the international exchange of DNA information with the UK, the USA, and Canada in 2014 (Smith and Mann 2015).

American Region The Technical Working Group on DNA Analysis Methods (TWGDAM) in the USA established its first national DNA database by 1989. Later, by 1994 the Combined DNA Index System (CODIS) was created and maintained by the FBI. The CODIS is sub divided to: 1) National DNA Index system (NDIS) for the exchange and comparison of profiles among the national laboratories, 2) State DNA Index System

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(SDIS) for the exchange and comparison among various states laboratories, and 3) Local DNA Index System (LDIS) collecting profile in the neighborhood and then uploaded to SDIS and NDIS. All laboratories are installed with Criminal Justice Information Systems Wide Area Network to integrate and connect all the three levels to CODIS. For the criminal investigations purposes, three indexes are mainly used: 1) the offender index for storing convicted profiles, 2) the arrestee index for arrested criminals, and 3) the forensic index containing profiles collected from a crime scene. In addition, there are also separate indexes for staff index who are involved in handling of case samples, the multi-allelic offender index that have more than two alleles at two or more loci obtained from individual samples, and partial profiles index for degraded or mixtures of multiple individuals. In 2000, the National Missing Person DNA Database, also known as CODIS (mp), was maintained by the FBI at the NDIS level and has unknown human remain index, missing person’s index, and the biological relatives of missing person’s index. The Unidentified Human remains were tested for nuclear, Y-STR (for males only), and mitochondrial analysis by the University of North Texas Center for Human Identification which is funded by the National Institute of Justice to exploit the possibilities of identifying remains (Florida International University – Digital Communications 2020). The Department of Defense maintains a database called Department of Defense Serum Repository for US servicemen with their dependents profile to support in the identification of human remains and has more than 50 million profiles. From January 2017, CODIS adopted to include 20 STR markers in its database where previously it was only 13 markers with Amelogenin and currently contains over 14 million DNA profiles. Since 2004, there is a DNA databank called “Caminho de Volta” for missing kids in Brazil (da Silva et al. 2009). The Brazilian DNA database the Rede Integrada de Bancos de Perfis Genéticos (RIBPG) was formed in 2013 by the Brazilian Federal Police and presently incorporates 20 laboratories from all over Brazil and further aims to connect the remaining state laboratories for utilizing CODIS to solve criminal cases, sexual assault, and missing people searches (Ferreira et al. 2013). According to the 2020 report of RIBPG, 82,000 profiles have been submitted by 18 states laboratories compared to 30,809 genetic profiles produced in 2019 report of RIBPG. The Canadian National DNA Database (NDDB) managed by the Royal Canadian Mounted Police (RCMP) was operational from 2000 by the DNA Identification Act (Milot et al. 2013). The amendment in the Criminal Code of Canada made provision for the collection of blood, buccal, or hair samples from the convicted persons. The NDDB has four indexes: 1) the Convicted Offender Index (COI) containing DNA profiles of offenders in imprisonment and 2) the Crime Scene Index (CSI) containing DNA profiles obtained from crime scenes. The COI are operated at the national level and CSI managed by the local forensic labs: 3) the victims Index (VI) and 4) the voluntary Donors Index (VDI). DNA profiles are stored in the database based on 13 Core CODIS markers and records up to 535,236 profiles.

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Additionally, National Missing Persons DNA Program (NMPDP) was established in 2018 by the RCMP and holds about 500,000 DNA profiles. NMPDP has three non-criminal/humanitarian DNA indexes: 1) Missing Persons Index (MPI) contains DNA profiles obtained from the personal objects of the missing persons, 2) Human Remains Index (HRI) contains DNA profiles obtained from the human remains, and 3) Relatives of Missing Persons Index (RMI) contains DNA profiles obtained from the close relatives of the missing person. The RMI is restricted to search only against MPI and HRI and not to any of the criminal indexes. Chile National DNA Databases Sistema Nacional de Registros de (SNDD) was established in 2004. SNDD is divided into five different databases: 1) Offender Database for individuals convicted of certain serious crimes, 2) Defendant/Suspect Database for individuals charged for serious crimes and subject to the verdict either removed or moved to the Offender database, and 3) Evidence Database unidentified biological material collected at crime scenes. The profiles included in Offender, Defendant/Suspect, and Evidence databases are retained for 30 years, regardless of the offender’s death, parole, or release 4) Victims Database for those confessed of serious crimes and are retained until the perpetrator(s) are identified 5) Database of the Disappeared or executed individuals and profiles are retained until they are identified. The SNDD operates using the CODIS software and has 78,733 profiles. In 2011, new law was introduced in Argentina’s Ministry of Justice to form two independent DNA databases: 1) for the profiles of the individual committed major crimes with confirmed convictions and 2) for the evidences collected from unresolved cases which also has database containing DNA donated by the family of missing children (Penacino 2008). However, in 2016, Mendoza province has created a new database the Registro Provincial de Huellas Genéticas Digitalizadas and contains 40,652 profiles of these 30,507 belongs to prisoners and the remaining are from the person involved in handling the case (Locarno et al. 2019). Since 2006, the Committee of Relatives of Dead and Missing Migrants of El Salvador (COFAMIDE) with Argentine Forensic Anthropology Team (EAAF) and the Salvadoran government established archive for disappeared migrants in Mexico. Lately in 2019, Mexico planned to operate national DNA criminal database with the USA to combat the people against smuggling and human trafficking. Bermuda established DNA database in 2005 and has stored 6620 profiles. It has criminal DNA database and general population database. The inclusion of arrested persons and retention of DNA profiles indefinitely from innocent people were implemented. Colombia’s DNA database was set up in 2008, and their code of criminal procedure 2004 provides provision for DNA analysis and has 6833 profiles. DNA databases are also planned in Costa Rica, Cuba, and Ecuador.

Asia In 2004, the Genetics Laboratory of the Institute of Forensic Sciences started DNA Database in China which is currently the largest known database with 80 million profiles as of 2020 and started 429 forensic DNA labs throughout China for the DNA

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samples collection. This database also contains additional core STR loci for enhanced discriminatory capacity tailored to the ethnic make-up of China’s population (Ge et al. 2013). The following are the indexes, Crime Scene Index, Convicted Offender Index, Suspect/Accused/Arrestee Index, and Unknown Deceased Index present in the database, and plans to include Missing Persons and Victim/Volunteer Index in addition to missing and migrant children database are established. China’s Ministry of Public Security (MPS) established a national Y-STR database in 2017 (Ge et al. 2014). ASPI reports that the Chinese police claims that database would help to establish a link with the evidence and nothing to do with crime and estimated to contain around 35–70 million Y-STR profiles. Hong Kong, a Special Administrative Region of China, started regional Forensic DNA Database using CODIS software in 2001. The Hong Kong Police Force Ordinance provides guidelines for collecting samples only for serious offence and the destruction of DNA samples as soon as the forensic analysis has been completed. According to 2015, there are about 49,466 profiles in the database. Japan’s National Police Agency (NPA) established DNA database in 2004 and proposed the following indexes: 1) crime scene DNA profiles, 2) unnatural death DNA profiles, and 3) suspect DNA profiles. The DNA crime database accounts to have about 1.3 million as of late 2019 which means 1 of every 100 citizen’s DNA has been profiled (Cyranoski 2004). National Research Institute of Police Science plans to start up a first of its kind database to include suspect’s ethnicity, blood type, metabolic enzymes, hair and skin pigment proteins, mitochondrial DNA, and signs of asymptomatic viral infections to distinguish ethnicities. South Korea’s DNA database of missing children were started since 2002; however by the Use and Protection of DNA Identification Information Act in 2010, Forensic DNA Division build up a national DNA identification information database and DNA quality control service. Samples are collected from those accused or convicted of 11 violent crimes, including robbery, arson, drug trafficking, rape, and sexual assault against minors. The DNA Identification Management System maintained by the National Forensic Service has three indexes (arrestee, crime scene, and elimination index), while Korean DNA Database was operated by the Supreme Prosecutors Office including the convicted offender and crime scene index, and both database contain 179,848 profiles. The Forensic DNA Databank of Malaysia (FDDM) was set up lately in 2015 by the Royal Malaysia Police Forensic Laboratory (RMPFL) and police (RMP) under the enactment of the Malaysian DNA Identification and Regulation Acts in 2009 and 2012, respectively. Approximately, database contains nearly 75,000 DNA profiles acquired from suspects, convicted offenders, crime scenes, detainees, drug dependents, missing persons, and volunteers from the members of the missing person and/or FDDM staff (Kumar et al. 2016). Indonesian National Police and the Eijkman Institute for Molecular biology has DNA forensic unit for parentage, disaster victim, and perpetrator identification database containing mtDNA and forensic STRs for many Indonesian ethnics and contains 17,830 profiles. Bangladesh, Sri Lanka, the Philippines, Vietnam, and Thailand plan to set DNA database.

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As of now, India doesn’t hold any repository or DNA database at national level (Kumar et al. 2016). However, a survey conducted by Interpol in 2008 states use of a national design software to operate state database called HID (human identification). In India, DNA Technology (Use and Application) Regulation Bill, 2019 states “that no legal proceedings can be initiated against the Union government or any member of the board of the DNA National Data Bank for any action which is done in good faith.” However, what constitutes “good faith” is vague here that fails to ascertain accountability on the board or the government of any responsibility in cases of security breach. DNA evidence is undoubtedly conclusive and hence Centre is trying for fast implementation of the DNA Technology (Use and Application) Regulation Bill. It is under consideration with the parliamentary standing committee on science and technology, and once it comes into picture, it will go long way in faster and fair trials, standardization of protocols, and also prevention of repeat offence with a help of a regulated DNA databank (Anand 2020).

Ethical and Legal Standards DNA databases all over the world change comprehensively on issues related to access and consent to support of both DNA tests similarly as the modernized profiles produced using them. Nonetheless, none global principles as well as restricted shields are there for ensuring security along with basic liberties. The development of criminological DNA information bases overall is frequently described as improvement in policing on public request. Different nations need essential quality affirmation for research centers or a dependable framework to follow DNA proof from the wrongdoing scene to the court and forestall misunderstandings, tainting, and unnatural birth cycles of equity. US people are effectively advancing DNA information bases, regularly depicted as specialized answers for horror rates. FBI Laboratory has worked in accordance with more than 29 nations by using the CODIS software in order to advance peaceful accords and approving enactment (Williams and Johnson 2004; Issues 2020). The advancement of legal DNA information bases is in no way, shape, or form restricted to governments. A private organization has been created to straightforwardly contract with unfamiliar governments for the construction and maintenance of DNA database, offering strategy suggestions generally displayed on US practice. In the recent time period of 10 years, Life Technologies has prompted more than 50 unfamiliar organizations related to legal DNA enactment, strategy, and special regulations providing a base to foreign countries. Japan Legislature in 2009 normalized DNA assortment and examination for nation’s 47 prefecture research centers utilizing Life Technologies DNA testing frameworks. Life Technologies keeps on offering help to NPA (National Police Agency). In 2009 the Bermuda government marked a huge contract of about 1,000,000 dollar with Trinity DNA (Florida based firm) for setting up DNA database plan (Issues 2020; Williams and Johnson 2004).

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Hereditary information bases for wrongdoing have become a public point after the capture of the Golden State killer, Joseph James De-Angelo. He was captured at the age of 72 years and committed in excess of 50 assaults and 12 homicides. While his capture was commended as a law requirement triumph, a large group of inquiries developed in view of the way law authorization authorities in the end discovered De-Angelo: through a mix of conventional criminologist work and use of information from a publicly supported hereditary information base. For this situation, police looked through GEDmatch. This case boomed various discussions on DNA-related issue and its utilization as a proof. The central issue is that specialists use hereditary information bases to recognize the groups of suspects. In spite of the fact that the open doors for wrongdoing settling by using DNA information base hunts might be huge, new advances and inventive employments of them don’t happen in a vacuum. All things being equal, novel employments of innovation request thought of countless moral issues and order cautious cross-examination of the likely effect of DNA information bases on wrongdoing control (Carracedo 2008). The maintenance of DNA profiles and tests taken from wrongdoing scenes can be promptly defended in light of the fact that they may be valuable if an examination should be re-opened later on (either to convict a culprit or to absolve a guiltless individual). The significant basic freedoms concerns identify with the extending people gathering for DNA collection and handling afterward. The DNA framework is on the grounds that: • DNA can be utilized to follow people or their family members; thereby database of DNA can be abused by who can invade the framework, e.g., governments or any individual. • It can prove to be fruitful in the case of finding a suspect. The stored DNA records are compared to the records stored in other PCs, for example, for the rejection of a visa the captured records can be used as a source. • DNA tests and profiles contain private data about well-being and hereditary connections (counting paternity and non-paternity) (Issues 2020, Carracedo2008, Kobilinsky et al. 2007).

Major Ethical Concerns Include Human Rights and Racial Contemplations Basic liberties concern that includes DNA sample collection without full educated assent of subject’s must be defended in restricted conditions. Director of a Human Rights Watch in China states “DNA assortment can have real policing utilizes in examining explicit criminal cases, yet just in a setting in which individuals have significant security insurances,” as the system in which it functions there have been reported to infringe rights to privacy. Grand (ECHR) Chamber of the European Court of Human Rights in 2008 prohibited the assortment and inconclusive maintenance of fingerprints, cell tests, and DNA profiles. The European Court, in arriving at its decision, contemplated that broad, unpredictable DNA information bases

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abused the privilege to individual protection. It added that DNA assortment might be suitable according to state security and crime deterrence, however just if the assortment framework is intensely managed by setup law and open to the cautious investigation of judiciary. The Supreme Court of the USA decided that the assortment and maintenance of DNA profiles on individuals indicted for rough violations were legitimate given the restricted kinds of assortment, investigation, and utilization of tests gave by rule, in Maryland v. Ruler. In China, the police collection of DNA samples seems to be limiting in nature, Article 130 of CrPC states that over the span of criminal examinations, to “learn certain highlights, states of wounds, or states of being of a casualty or a criminal suspect, an actual assessment might be directed, and fingerprints, blood, pee and other natural examples might be gathered. On the off chance that a criminal presume will not be inspected, the specialists, when they consider it fundamental, may lead a mandatory assessment.” It clearly lacks a legal guidelines and principles on the time limit for the preservation of DNA, how the data can be used and shared by any organization. It is a clear lack of privacy protection measures and gives way to abuses (Carracedo 2008; Kobilinsky et al. 2007). For instance, New York State division in 2007 regarding equity in criminal administrations relaxed family looking strategies. Currently the major focus is on the look, yet just in specific situations, and just under survey. The change had the help of lead prosecutors, in a similar manner of homicide victim dad who was public in his assistance of the augmentation of DNA framework. The ill effect of security issues can be seen on the people who are generally involved in criminal case. Because of the utilization of online media, wrongly charged people may wind up with profound reputational harm or notwithstanding expanded pressure from accepting they might be erroneously blamed for a wrongdoing. Lastly, the most amazing investigations of the usage of data bases for familial DNA looking is the disproportionate impact it would have on dull and Latino individuals who are starting at now caught by the criminal value structure. Greely, Roberts, and Erin Murphy in their study found that racial incoherencies in imprisonment transform into an uneven grouping of African American men from whom DNA was obtained and kept in databases all across the world (Carracedo 2008). In the year 2010, individuals of color represented roughly 27 percent of grown-up captures when the grown-up dark populace was 12 percent. In certain areas, where individuals of color comprises the populace more than 50%, for example, Shelby County, individuals of color establish 85 percent of lawful offense litigants. In the Maricopa County, 30% were ariz and 39 percent of the lawful offense litigant populace was Hispanics. Furthermore, in that very year, the Hispanic populace of Maricopa County, Ariz., was 30%, while Hispanics made up 39 percent of the lawful offense respondent populace. Significantly, while a considerable lot of those captured are dark and Latino, accordingly populating DNA information bases, many are frequently not sentenced (Kobilinsky et al. 2007).

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Privacy (Lack of Safeguard) Privacy is one of the significant setbacks for keeping up a DNA database as the data set comprising a large number of profiles of DNA which relate to biological samples databank. DNA Fingerprinting and Civil Liberties procedures were described by Journal of Law, Medicine, and Ethics in its issue of mid-2006. The issue of protection can be drawn nearer in various manners. For example, to begin with 13 STR loci, CODIS center which lies in non-coding genes of the human genome which has no relationship with any kind of hereditary sickness and henceforth the data in the information base is just valuable for human character testing. Secondly individuals or other portraying data are taken care of at public level with the DNA profiles. Explicit case information is made sure about and constrained by the law authorization organizations that present the information. In this way, just the crime lab that presented the DNA is such a kind of foolproof evidence that can give a positive result with a known individual. Thirdly the information is scrambled and is just open to some limited authorized organizations and CODIS directors. Lastly, government and state punishments regarding ill-advised utilization of DNA tests incorporate heavy fine along with conceivable detainment. If a person utilizes the DNA data for any for say reason except the authorized persons, then he or she has to face severe punishments. The punishments incorporate a fine of 250,000 dollars for unapproved divulgence of data (Herkenham 2006). Familial Looking, Research Use, and Counter-Psychological Oppression Familial looking, a cycle through which specialists search halfway coordinates between DNA profiles obtained from the crime scene and the DNA profiles belonging to people put away on the basis of their DNA information. It is used for recognizing suspected relative with a probability of whom he or she might have met, possibly prompting the speculator’s ID and maybe a fruitful arraignment. Familial looking through prompts a not insignificant rundown of incomplete comparison analysis should be abbreviated by the help of more extensive DNA testing and the police official’s investigations. In UK, the technique is spearheaded as it is assisted with illuminating various genuine wrongdoings. Nonetheless, it raises extra worries for the genuine people security who are not the culprit or suspect, but still their profile matches with the suspected one when specifically talking about non-paternity cases which may coincidentally be uncovered by familial looking cycle. Whenever utilized regularly, familial looking could prompt critical maltreatments by permitting examiners or any individual who invades the information base to find the family members of political protesters or to seek after foes (Maguire et al. 2014). DNA data sets comprise assortments of organic examples (whenever put away), mechanized DNA profiles, and other data (e.g., criminal history and nationality) that might be significant to hereditary analysts. Nonetheless, much examination around there is disagreeable because of the historical backdrop of selective breeding. Specifically, endeavors to interface hereditary attributes to undermined ideas of

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race or to distinguish “qualities for guiltiness” are disputable. Dissimilar to setup of information bases for research purposes, measurable DNA information bases contain information gathered without assent and additionally once in a while with assent for policing purposes as it were. Any attempt to use such databases to make inferences regarding inherited characteristics is thus in violation of established moral principles. Such breaks have just happened with some current information bases (Kobilinsky et al. 2007). The utilization of DNA information bases in criminal examinations requires a person’s personality to be uncovered in particular case where the DNA profiles stored in the databank match with the profile found at the crime scene. Up to this point, employments of DNA information bases were limited to a great extent to searching for matches with wrongdoing scene DNA profiles. Currently it is evolving. In UK, the DNA gathered and held under the act of Counter-Terrorism 2008 would be able to be utilized for “recognizable proof” of the individual from whom the material came. This is an ongoing difference in use which permits organic observation of specific people (e.g., the capacity to utilize a person’s DNA to follow and distinguish them, regardless of whether they are associated with perpetrating a wrongdoing). Obviously, this might be valuable to security benefits; however it is additionally conceivably prone to mishandle. Government of proposition to gather fingerprints and DNA information regularly on capture of an offense and using it regularly for the purpose of recognizable proof (e.g., coordinating the person to their subtleties on unique mark information bases and DNA, utilizing offices set up in malls for such intentions) were dropped in 2008 after open objection. Notwithstanding, this remaining parts a likely use for DNA information bases later on, especially as new innovation creates that can permit DNA constant testing while on the spot of crime scene and coordinating with records of information base (Maguire et al. 2014; Ahmed 2020).

DNA Is Not Secure and Foolproof DNA isn’t foolproof and secure. By some coincidence a false match can occur among the DNA profile of a person with the crime scene DNA profile, or because of helpless lab strategies, and the ramifications of somebody’s profile of DNA at the scene of crime likewise be misconstrued. The chance of a counterfeit match between an individual’s DNA profile and a bad behavior scene DNA profile depends upon the course of action of DNA profiling that is used. With the passage of time, the rules used to make a DNA has changed and vary from country to country: the USA uses 13 STRs at better places in the genetic gathering; anyway most various countries use less STRs. The UK structure (10 STRs) is surveyed to have around a 1 out of a billion “facilitate probability”: this is the likelihood that an individual’s DNA profile arranges a bad behavior scene DNA profile by chance whether or not the DNA at the bad behavior scene didn’t begin from. In spite of the fact that this probability is extremely low, the quantity of bogus matches that happen relies upon the quantity of examinations that are made between various

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DNA profiles. On the off chance that each wrongdoing scene DNA profile is thought about against each put away DNA profile on an enormous information base by theoretical looking, few bogus matches are required to happen essentially by some coincidence. Bogus matches are bound to happen with family members; sibling of any individual who has committed some sort of crime will have in common a portion of the family general’s DNA succession. The issue is exacerbated when only a few crime scene DNA profiles are completed, the risk of a false match increases, which could lead to some wrongdoing at the site. The risk of spurious similarity may increase dramatically if DNA profiles were completed (Maguire et al. 2014; Machado and Silva 2014). The samples of DNA and their nature might change as indicated by the wellspring of the DNA, regardless of whether it has gotten debased after some time, whether the DNA belongs to a single person or more than one. Very small size sample for DNA analysis from the crime scene can make the case investigation move in a wrong direction regardless of the fact whether the person was actually present at the crime scene or not. Conversely, a huge amount of blood found at the location of a homicide or murder will offer dependable outcomes. A combination can be deciphered from numerous points of view, but there exists no such reliable method to tell from which piece of DNA profile originates from which implies that blended DNA database profiles are not entirely clear, especially if a criminological lab is one-sided by attempting to catch hold of the real culprit by having an exact match. This improves the probability of a bogus match with some unacceptable individual. DNA tests can likewise be wrongly examined or stirred up during lab techniques, bringing about a match with some unacceptable individual if there is no proper adaptation of quality affirmation methodology. Routine cross-outskirt theoreticallooking of DNA profiles at crime scenes with the DNA samples captured from the people in different nations is a way prone to hurl a lot more bogus matches than if such ventures are confined toone nation or restricted to justfew suspected people for DNA matching (Machado and Silva 2014). Regardless of whether a DNA coordinate is real, an individual’s essence at any scene of crime does not prove that the person is involved in any kind of wrongdoing. The accurateness of the profile matching ought to rely upon whether there is extra supporting proof. Any person whose DNA data information is already existing may be defenseless in the case proofs are being planted against him or her by the degenerate cops, ground-breaking government offices, or hoodlums. Regardless of whether an unsuccessful labor of equity doesn’t happen, a person who is dishonestly blamed for a wrongdoing because of a DNA match might be exposed to a distressing police request, pre-preliminary confinement, or removal to an unfamiliar nation (Anderson et al. 2011; Maguire et al. 2014).

Errors and Improper Utilization of Police Resource DNA is without a doubt a significant instrument in criminal examinations and has assisted with getting the culprits of some intense violations, including assaults and

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murders. In any case, the possibility of having no more sexual assaults or murders is only when every single individual gets his/her DNA information recorded on an information base. Notwithstanding worries about protection and rights, the primary constraints to this thought are: (i) The troubles in gathering applicable and valuable wrongdoing scene DNA proof (ii) The extremely low probability of the vast majority perpetrating genuine violations for which DNA proof may be significant (iii) The expenses and handy challenges related with gathering and keeping records of DNA profiles and related information from gigantic amounts of people (iv) Open trust consequences in policing England and Wales has a significant venture started by their government in 2000 for extending DNA utilization. The positive outcomes can be achieved by improvising the DNA assortment from scene of crime and accelerating the examination. Be that as it may, there were still genuine down as far as possible in how much useable DNA could be gathered along these lines: regardless of enhancements in systems, DNA profiles are as yet stacked to the DNA information base from under 1% of recorded violations. Numerous wrongdoing scenes don’t uncover any DNA in the purest form (Machado and Silva 2014). On an extreme, a monstrous expansion in the quantity of people’s DNA profiles gathered and DNA information base is set aside in Europe region didn’t improve probability of having the option to indict somebody for a wrongdoing. This gives off an impression of being on the grounds that DNA profiles were gathered and keep distant from large-scale individuals (everybody captured for an offense, whether or not they were at last sentenced). The incorporation of a huge number of guiltless individuals’ records on the DNA information base likewise brought about lost public trust in policing. In spite of the way that it is difficult to assess the impacts, this may have made a couple of infringement all the more difficult to handle by making a couple of individuals less supportive with police assessments. In Scotland, the standards on the support of DNA profiles kept up open assistance, and their DNA information base stayed a successful device in criminal examinations regardless of most blameless individuals’ records being erased (Ahmed 2020). The DNA sample should be collected from the entire population instead from the ones who have been suspected would clearly cost generously more and furthermore raise down to earth and moral troubles about how to gather DNA from everybody without assent. Gathering DNA from unfamiliar guests would add further to the expenses and challenges which can impact sly affect individuals ready to head out to the nation. Gathering DNA from children upon entering the world would raise genuine moral issues about assent and the part of the clinical calling. Likewise, greater information bases – and more correlations between DNA profiles put away in various nations – improve the probability of bogus matches, as portrayed previously. These can burn through police time following bogus leads, regardless of whether they don’t prompt premature deliveries of equity (Kobilinsky et al. 2007).

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Conclusion DNA databases since their inception have been continuously providing help to law enforcement agencies and are very much likely to grow in the coming years. With this, the search will widen the horizon and successfully enlarge the hunt net by including crimes from an extended rundown of wrongdoings which will acquire more DNA samples as new laws become effective. As DNA bases grow in size, various operational reformations will be needed; like additional core loci to avoid extraneous matches, algorithms enabling faster searches will be needed, expanded infrastructure, manpower, training and research resources, etc. There were innocent people who stayed behind bars for years; post-conviction exonerations have become possible for them only with the help of DNA databases. DNA database hits enabled to link serial crimes and helped in locating the perpetrators. This revolutionary tool is open to criticism; however, it holds immense power. Owing to this, it is imperative that it should be handled responsibly and with utmost care to ensure safety of public and their civil liberties.

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Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Insertion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Deletion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . InDel (Insertion/Deletion) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Use of Insertion/Deletion Polymorphism in Forensic Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Commercial Kits Based on InDel Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Use of Gonosomal InDel Loci in Forensic Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Analysis of InDel Loci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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As a part of forensic science, in the investigations of forensic cases, such as murder, sexual assault, and theft, DNA profile, which is obtained from biological materials found at the crime scene, is used to determine whether there is a connection between the suspect people and the offense. Depending on the type of case, autosomal or gonosomal polymorphism, which is obtained from X and Y chromosome, can be used in solving forensic cases. Although STR (Short Tandem Repeats) loci is still widely used in routine forensic genetic analysis, in many cases, typing problems can occur in highly degraded biological samples collected from the crime scene, and results cannot be obtained. In order to solve these problems, forensic scientists have been looking for alternative genetic markers. InDels, which have been used in forensic science in recent years, can achieve more successful results in forensic identification when used together with STR and SNP loci. InDels are the most common type of polymorphism in the genome T. Unsal Sapan (*) Institute of Addiction and Forensic Sciences, Uskudar University, Istanbul, Turkey e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 H. R. Dash et al. (eds.), Handbook of DNA Profiling, https://doi.org/10.1007/978-981-16-4318-7_49

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after SNPs. InDel occurs as a result of insertion and/or deletion of one or more nucleotides, and it is a kind of polymorphism which can be used in population studies since insertion/deletion is seen with a frequency of more than 1% of the population. These loci can be used for identification, biogeographic genealogical research analysis, evolutionary research, and revealing kinship relationships since they allow to do multiplexing PCR study and have high heterozygosity rate and also small amplicon lengths (60–200 bp). Research on the use of InDels in forensic science has gained momentum in recent years. The number of readymade commercial kits for these loci is quite limited. For this reason, researchers create their own InDel multiplex panels to use in their studies. Keywords

Forensic biology and genetics · Human identification · Insertion/deletion (InDel) polymorphism · InDel loci

Introduction Advances in molecular genetics in the 1980s allowed the study of polymorphic traits directly at the DNA level. The use of DNA in forensic science developed rapidly after Alec Jeffrey’s discovery of polymorphic repeat sequences in the DNA molecule in 1985. Since then, the identification of biological samples collected from both individuals and from the crime scenes in the determination of paternity-kinship relations and other criminal investigations has carried out using DNA analysis techniques. In forensic cases, such as terrorism, murder, sexual assault, and theft, it is possible to link up between the suspect and the crime scene by using DNA profiles, which are isolated from biological materials detected at the crime scene (Jeffreys et al. 1985; Robertson et al. 1990; Chan 1992). Although VNTR (variable number of tandem repeats) loci, which were used in the first period of DNA analysis in forensic sciences, have a high discrimination power, it has been replaced by new technologies on account of the need of good qualified (non-fragmented) and excessive amounts (300–500 ng) of DNA, long and laborious analysis times, exposing radioactive materials, and so on. For the last 20 years, STR loci have been widely used in forensic identification (Lee et al. 1994; Robertson et al. 2002). Short tandem repeats (STR) loci have been used as ideal genetic markers in forensic science in recent years due to their small amplicon size, successful results in degraded biological samples, capability to do multiplex analysis, and no requirement of expensive equipment (Weber and May 1989; Edward et al. 1992). Although STR loci have been still used today, typing problems are experienced in extremely degraded biological samples, which are collected from the crime scene, and successful results for comparison cannot be obtained many times. In order to solve this problem, mini short tandem repeats (miniSTRs) that allow typing even in degraded samples were put on the market in the early 2000s (Coble and Butler 2005). In recent years, for the same purpose, researchers have been focused on

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different DNA polymorphisms, such as single-nucleotide polymorphism (SNP), which takes up a very little space in DNA, and InDel polymorphism (Pereira et al. 2009a, b).

Insertion It is a type of mutation that disrupts the natural sequence as a result of addition of one or more bases to the DNA base sequence. This addition can be a base or as much as a whole chromosome (Fig. 1). As a result of DNA polymerase shift, it is usually thought to be formed by addition of base sequences, which are not adjacent to microsatellite sites, to the main sequence (Gelbart et al. 2002; Kondrashov and Rogozin 2004; Rodriguez-Murillo and Salem 2013).

Deletion It is a type of mutation that occurs as a result of the deletion of one or more bases in the DNA sequence. Deletion, as well as insertion, can occur on one or more bases, as well as in chromosome size (Fig. 2). If a deletion occurs when part of the Fig. 1 Insertion on a chromosome level (Gordon and Egner 2013)

Fig. 2 Deletion on a chromosome level. (Gordon and Egner 2013)

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chromosome breaks off and disappears, it can lead to serious genetic diseases (Gelbart et al. 2002; Kondrashov and Rogozin 2004; Rodriguez-Murillo and Salem 2013).

InDel (Insertion/Deletion) Point or gene mutations are defined as changes that occur in the DNA sequence and can be passed on to subsequent generations. These mutations usually occur in one or more nucleotides and cause changes in the structure of the genome. Point mutations can also occur in the form of insertion or deletion (Campbell et al. 2006) (Fig. 3). However, insertion or deletion mutations are much more important changes than point mutations. Because insertion or deletion of one or more bases in the DNA chain usually leads to the shift of the genetic code that starts from the point where the insertion or deletion occurs, this causes polymorphism by creating significant changes in the structure of the gene (Pereira et al. 2009a, b). InDels and somatic/gonosomal chromosome mutations are terms used to express mutation combinations, which include deletion or insertion separately or together, in the studies of forensic molecular genetics, evolution, and population genetics (Gelbart et al. 2002; Kondrashov and Rogozin 2004; Gregory 2004). InDels, which account for 16–25% of all genetic variations in the genome, are the most commonly seen DNA polymorphism after SNPs, and 1.6–2.5 million InDel polymorphisms have been identified in human population studies. This, in turn, suggests that hereditary changes that occur as a result of insertion or deletion (InDel) mutations can be used as a genetic marker since they are seen in the human genome frequently. Even though they are so common, studies with InDels are limited (Hongbao 2005). However, since InDels have power of discrimination and heterozygosity, it is possible to use InDels in studies of human identification, ancestry, and evolutionary and molecular anthropology. For this reason, today, it

Fig. 3 Schematic representation of insertion and deletion on the DNA sequencing. (Gordon and Egner 2013)

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is an alternative polymorphism to SNPs and STRs, which are used in DNA profiling in forensic identification (Reiner et al. 2005; Pereira et al. 2009a, b, 2012; Martínez-Cortés et al. 2015).

Use of Insertion/Deletion Polymorphism in Forensic Genetics Insertion/deletion polymorphism (InDel) is known to be as a length polymorphism, which formed as a result of insertion and/or deletion of one or more nucleotides on the genome. Differences are used to discrimination of two people from each other in forensic identification, which makes polymorphisms the basis of identification (Rodriguez-Murillo and Salem 2013). Polymorphism occurs as a result of successive mutations and is passed down from generation to generation according to Mendelian laws. In 2002, James Weber and colleagues identified over 2000 biallelic insertion/deletion polymorphisms on the human genome. Thus, studies of InDel polymorphism began for the first time in forensic sciences (Weber and May 1989). InDel loci have been used in forensic identification since they are able to study with multiplex PCR and have high heterozygosity and small amplicon lengths (60–200 bp) (Fig. 4). They can also be used to identify disaster victims in mass fatality cases, such as aircraft accidents, terrorist attacks or natural disasters, ancestry determination, evolutionary research, and molecular anthropology (Manta et al. 2012; Martínez-Cortés et al. 2015). More successful results can be achieved in identification by using InDel polymorphism, which are called next-generation genetic variations together with STR and SNP loci (Sanchez et al. 2006; Pereira et al. 2012). InDel loci have been used in forensic sciences, especially in recent years. Commercial kit production for InDel loci is highly restricted. So forensic scientists often create their own InDel panels and try to popularize them (Guangyao et al. 2015).

Commercial Kits Based on InDel Analysis InDel loci have been used in forensic science in recent years. However, InDel kit production is also quite limited. Currently, there are three commercial InDel kits made for the use in forensic science.

Investigator® DIPplex Kit It is a kit containing 30 InDel loci found in somatic chromosomes that have been marketed by QIAGEN firm in the last 2–3 years. The kit also includes the locus of amelogenin. Selected InDel loci are smaller than 160 bp and are specifically designed for the use in the identification or anthropological research. In addition, they are analyzed by ABI 310, 3130, 3130XL, 3500, and 3500XL capillary electrophoresis (Investigator ® DIPplex Handbook 2014).

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Fig. 4 Comparison of PCR product sizes of InDels with STRs

InDelPlex INDEL Polymorphism Detection Kit It is a kit developed by Pereira R. et al. in partnership with the Institute of Molecular Immunology and Pathology of Oporto University and the University of Santiago de Compostela and commercialized with Genomica firm over the past few years. The kit allows multiplex PCR amplification of 38 InDel region and produced for the use of forensic identification and clinical diagnostic purposes. These analyses can be done by using ABI 310, 3130, 3130XL, 3500, and 3500XL capillary electrophoresis (Pereira et al. 2009a, b, 2012). Mentype® DIPplex PCR Amplification Kit It is a kit containing 30 InDel loci found on somatic chromosomes that have been marketed in recent years by the Biotype firm and also includes the locus of amelogenin. This kit restricted amplicon length to ~150 bp, which makes the kit

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perfectly suitable for analyzing critical stains. The kit can be analyzed by using ABI 310, 3130, 3130XL, 3500, and 3500XL capillary electrophoresis (Mentype ® DIPplex PCR Amplification Kit Handbook 2009).

Use of Gonosomal InDel Loci in Forensic Genetics InDel loci exist on both somatic and sex chromosomes. Compared to somatic chromosomes, there are InDel variations in similar ratios on the X and Y chromosomes too. It can be used in addition to autosomal genetic markers in father/daughter relationship in X-linked polymorphism analysis. In the cases of paternity of two relative men (such as father/son), paternity can be determined by the use of polymorphism on the X chromosome if the child is a girl. It is because suspicious fathers will have different X chromosomes since they have different mothers (Szibor et al. 2005; Prinz and Sansone 2001). Since the Y chromosome is transferred unchanged from father to son, it is observed in male members of the same family in the same form, except for mutations and genetic abnormalities. For this reason, population genetics, biogeographic lineage determination, male kinship evaluation or link analysis, and forensic genetic identification studies can be done by utilizing Y chromosome-related polymorphism analysis. In paternity cases, if the child is a boy, it is possible to get results by typing Y chromosome loci of any man (grandfather, uncle, cousin, etc.), who is in the family tree of the father candidate, especially when DNA cannot be obtained for various reasons (such as the father candidate cannot be found, DNA cannot be obtained from his biological material or he is dead, etc.) (Roewer et al. 2000; Corach et al. 2001; Prinz and Sansone 2001). Similarly, in paternity cases, where the father candidate is dead or could not be found, it is possible to get results by analyzing the X-linked polymorphism between the grandmother candidate and child (if the child is a girl) because the child gets one of her X chromosomes that her father gets from her grandmother. As a result, the paternal X chromosome in a girl will necessarily be coming from one of her grandmother’s X chromosomes (Szibor et al. 2005, Prinz and Sansone 2001). Pregnancies that occur after sexual assault crimes can result in abortion. In 6– 8 weeks of abortions, the tissue and maternal blood materials are found together with miscarriage, and it is difficult to separate them microscopically. In this case, if the fetus is female, the presence of the X chromosome, which was inherited from the suspected father, can be determined by X-linked polymorphism analysis (Szibor et al. 2005; Prinz and Sansone 2001). The Y chromosome is also used in illuminating sexual assault crimes. In cases where the victim is a woman, the victim’s vaginal swab sample contains a mixture of DNA belonging to the victim and the perpetrator. Since there is no Y chromosome in women, only the profile of the perpetrator is obtained when identification is performed using Y chromosome loci in the mixture DNA (Roewer et al. 2000; Corach et al. 2001; Prinz and Sansone 2001).

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By analyzing the polymorphism associated with X chromosome, it can also be determined whether two girls’ father is the same, regardless of whether their mothers are different or the same. Since the father has one X chromosome, he will pass on the same X chromosome to all girls. But if their father is not the same, the X chromosome loci transferred from their father will also be different (Szibor et al. 2005; Prinz and Sansone 2001). By using gonosomal and autosomal InDels together with STR and SNP loci, more successful results can be achieved in such cases or in identification of samples, which were collected from the crime scene (Szibor et al. 2005; Prinz and Sansone 2001).

Analysis of InDel Loci Although InDel loci are biallelic, it is analyzed by the method of fragment analysis, such as STRs. These steps are followed in the identification of InDels as in STRs: first, extraction and quantification of DNA sample, then amplification of InDel loci of DNA by using uorescence-marked InDel primers in multiplex PCR, and, finally, obtaining the profile of person by separating these loci in capillary electrophoresis with ABI 310, 3130, 3130XL, 3500, and 3500XL devices. As a result of the analysis of InDel loci in capillary electrophoresis, three types of alleles can be seen in electropherogram because either deletion, insertion, or a combination of both can be transferred from the mother and father. These are expressed as deletion (Del – minor allele), insertion (Ins – major allele), and insertion-deletion (InDel), where both deletion and insertion are transferred together. The base pair size of the identified InDel loci on the DNA is defined by researches. Since it is defined how many base deletions or insertions they undergo, determining only the alleles as insertion or deletion will be sufficient to say the number of base pairs. Before the analysis in capillary electrophoresis, this base pair data is defined in panel manager. It is created with a panel for each dye color (primer) with bins. In this way, the data of all alleles that exist in the population, just like in analysis of STR loci, is defined into the device. In capillary electrophoresis of DNA, if a person’s peak was observed in the first bin, as shown in the figure, this shows the person’s allele is “Del” because it has an incomplete base sequence. If the insertion has been transferred from the parent, the peak will be observed in the second bin, as shown in Fig. 5, which means that the person has the insertion as a result of addition of base, and the allele is “Ins.” If a peak is observed in both bins, it means that the person has received both an insertion and deletion polymorphism from the parent. This, in turn, refers to the third allele, which we call “InDel” (Fig. 5). In this way, the profile of people analyzed with InDel multiplex panel kits in all regions can be determined just like in STR analysis. The number of loci that will reach sufficient (99.9%) discrimination power for human identification of InDel loci differs from STR loci number. In order to use InDels for identification purposes, as in STR analysis, a sufficient number of loci to

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Fig. 5 Electropherogram image of insertion/deletion (InDel) polymorphism. (Fondevila Álvarez et al. 2011)

use are determined, depending on the heterozygosity rates and discrimination power of the locus contained in the multiplex kit or panel. The number of loci contained in InDel kits or panels developed by researchers, which exist on the market today, has a 99.9% discrimination power, and this locus number ranges from 20 to 40 (Ünsal et al. 2017). While expressing D70+ as the insertion allele in the figure, D70- refers to the deletion allele.

Conclusion Currently, STR systems are widely used for human identification in criminal laboratories. Forensic scientists are developing new polymorphic systems that can obtain results from all kinds of biological samples as an alternative to polymorphic systems (Coble and Butler 2005). Insertion/deletion polymorphism occurs in the form of the addition or loss of one or more bases in the human genome, which is causing polymorphism and can be used in human identification and genealogical determination in illuminating forensic cases. Especially in cases where DNA obtained from biological evidence from the

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crime scene is degraded or trace amount; As an alternative to STRs, small-sized systems on DNA are preferred for analysis in forensic sciences. In such cases, successful identification results can be obtained by using InDel polymorphism (Pereira et al. 2009a, b, 2012). The loci that these systems occupy on DNA are quite small (60–200 bp), and the potential for a successful DNA profile is high, even if the DNA is degraded. The amount of DNA that can be obtained from a degraded or trace biological sample is either non-existent or ranges from 100 pg to 1 ng. Since it is known that the amount of DNA required for multiplex PCR used for identification studies is 0.5–1 ng, it has been determined by studies that identification with InDel loci gives successful results.Therefore InDels have been used in forensic science in recent years, and studies continue to develop InDel panels as an alternative or complement to STR and SNP loci in identification (Ünsal et al. 2017).

References Campbell N.A, Williamson, B., Heyden, R.J. (2006) Biology: exploring life. Boston: Pearson Prentice Hall, 11(6): 242–243 Chan L (1992) Advances in molecular biology with applications in clinical medicine. Klin Lab 38: 2–4 Coble MD, Butler JM (2005) Characterization of new MiniSTR loci to aid analysis of degraded DNA. J Forensic Sci 50:43–53 Corach D, Risso FL, Marino M, Penacino G, Sala A (2001) Routine YSTR typing in forensic casework. Forensic Sci Int 118:131–135 Edward SA, Hammond HA, Jin L, Caskey T, Chakraborty R (1992) Genetic variation at five trimeric and tetrameric tandem repeats. Genomics 12:241–253 Fondevila Álvarez M, Pereira R, Gusma L, Phillips C, Lareu MV, Carracedo A, Butler JM, Vallone PM (2011) Forensic performance of short amplicon insertion-deletion (InDel) markers. Forensic Sci Int Genet Suppl Series 3:e443–e444 Gelbart WM, Lewontin RC, Griffith JF, Miller JH (2002) Modern genetic analysis: integrating genes and genomes. W.H. Freeman and CO., New York Gordon A, Egner S (2013) Dynamic biology for high school. Sapling Learning, Austin Gregory TR (2004) Insertion-deletion biases and the evolution of genome size. Gene 324:15–34 Guangyao F, Yi Y, Haibo L, Yiping H (2015) Screening of Multi-InDel markers on X-chromosome for forensic purpose. Forensic Sci Int Genet 5:42–44 Hongbao M (2005) Development application of polymerase chain reaction (PCR). J Am Sci 1(3):1–47 Investigator ® DIPplex Handbook (2014) For multiplex amplification of 30 deletion/insertion polymorphisms, plus Amelogenin. Qiagen. https://www.qiagen.com/us/resources/download.aspx? id¼2e1b66cf-e497-4054-82fc-3e1d2e14faba&lang¼en. Accessed 12 Sep 2020 Jeffreys AJ, Wilson V, Thein SL (1985) Hypervariable “minisatellite” regions in human DNA. Nature 314:67–73 Kondrashov AS, Rogozin IB (2004) Context of deletions and insertions in human coding sequences. Hum Mutat 23(2):177–185 Lee HC, Ladd C, Bourke MT, Pagliaro E, Tirnady F (1994) DNA typing in forensic science I. Theory and background. Am J Forensic Med Pathol 15:269–282 Manta F, Caiafa A, Pereira R, Silva D, Amorim A, Carvalho EF, Gusmão L (2012) INDEL markers: genetic diversity of 38 polymorphisms in Brazilian populations and application in a paternity investigation with post mortem material. Forensic Sci Int Genet 6(5):658–661

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Martínez-Cortés G, Gusmão L, Pereira R, Salcido VH, Favela-Mendoza AF, Muñoz-Valle JF, Inclán-Sánchez A, López-Hernández LB, Rangel-Villalobos H (2015) Genetic structure and forensic parameters of 38 Indels for human identification purposes in eight Mexican populations. Forensic Sci Int Genet 17:149–152 Mentype ® DIPplex PCR Amplification Kit Handbook (2009) Biotype Diagnostic GmbH Pereira R, Phillips C, Cíntia AA, Amorim Carracedo A, Gusma L (2009a) A new multiplex for human identification using insertion/deletion polymorphisms. Electrophoresis 30:3682–3690 Pereira R, Phillips C, Cíntia AA, Amorim Carracedo A, Gusma L (2009b) Insertion/deletion polymorphisms: a multiplex assay and forensic applications. Forensic Sci Int Genet Suppl Series 2:513–515 Pereira R, Phillips C, Pinto N, Santos C, Batista dos Santos SE, Amorim A, Carracedo A, Gusma L (2012) Straightforward inference of ancestry and admixture proportions through ancestryinformative insertion deletion multiplexing. PLoS One 7:e29684 Prinz M, Sansone MY (2001) Chromosome-spesific short tandem repeats in forensic casework. Croat Med J 42(3):288–291 Reiner AP, Ziv E, Lind DL, Nievergelt CM, Schork NJ, Cummings SR, Phong A, Burchard EG, Harris TB, Psaty BM, Kwok PY (2005) Population structure, admixture, and aging-related phenotypes in African American adults: the Cardiovascular Health Study. Am J Hum Genet 76: 463–477 Robertson J, Ross AM, Burgayne LA (1990) DNA in forensic science: theory. Techniques and applications. Ellis Horwood, London Robertson J, Ross AM, Burgayne LA (2002) Properties of hypervariable single locus polymorphism and their application to identity testing. In: Balazs I (ed) DNA in forensic science: theory, techniques and applications. Taylor& Francis e-library, London Rodriguez-Murillo L, Salem RM (2013) Insertion/deletion polymorphism. In Gellman, Marc D., Turner, J. Rick (eds) Encyclopedia of Behavioral Medicine. Springer-Verlag New York 1:1076. Roewer L, Kayser M, Knijff P, Anslinger K, Corach D, Füredi S, Geserick G, Henke L, Hidding M, Kärgel HJ, Lessig R, Nagy M, Pascali VL, Parson W, Rolf B, Schmitt C, Szibor R, TeifelGreding J, Krawczak M (2000) A new method for the evaluation of matches in recombining genomes: application to Y-chromosomal short tandem repeat (STR) haplotypes in European males. Forensic Sci Int 114:31–43 Sanchez JJ, Phillips C, Børsting C, Balogh K, Bogus M, Fondevila M, Harrison CD, MusgraveBrown E, Salas A, Syndercombe-Court D, Schneider PM, Carracedo A, Morling N (2006) A multiplex assay with 52 single nucleotide polymorphisms for human identification. Electrophoresis 9:1713–1724 Szibor R, Hering S, Edelmann J (2005) A new web site compiling forensic chromosome X research is now online. Int J Legal Med 25:1–3 Ünsal T, Filoğlu G, Aşıcıoğlu F, Bülbül Ö (2017) Population data of new 21 mini-InDels from Turkey. Forensic Sci Int Genet Suppl Series 6:e189–e191 Weber JL, May PE (1989) Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. Am J Hum Genet 44:388–396

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The Role of DNA Profiling in Landscape of Human Migration

J. A. Lorente, Christian Haarko¨tter, María Saiz, M. I. Medina-Lozano, X. Ga´lvez, M. J. Alvarez-Cubero, L. J. Martínez-Gonza´lez, B. Lorente-Remon, and Juan Carlos Alvarez

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Definitions and Numbers: The Magnitude of the Problem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Missing Persons and Migrations: A Difficult Crime to Solve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Human Trafficking: The Most Dangerous Scenario . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Role of Forensic Science: DNA Typing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Advantages and Need for DNA Migrant Identification Databases . . . . . . . . . . . . . . . . . . . Missing Children: The DNA-PROKIDS Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Organ Trafficking: The DNA-ProORGAN Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future Perspectives: Enlarging and Coordinating the Databases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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J. A. Lorente Laboratory of Genetic Identification, Department of Legal Medicine, Toxicology and Physical Anthropology, Faculty of Medicine, University of Granada, Granada, Spain GENYO, Centre for Genomics and Oncological Research (Pfizer / University of Granada / Andalusian Regional Government), Granada, Spain e-mail: [email protected] C. Haarkötter (*) · M. Saiz · M. I. Medina-Lozano · X. Gálvez · J. C. Alvarez Laboratory of Genetic Identification, Department of Legal Medicine, Toxicology and Physical Anthropology, Faculty of Medicine, University of Granada, Granada, Spain e-mail: [email protected]; [email protected] M. J. Alvarez-Cubero · L. J. Martínez-González Department of Biochemistry and Molecular Biology III, Faculty of Medicine, University of Granada, Granada, Spain Pfizer-University of Granada-Junta de Andalucía Centre for Genomics and Oncological Research (GENYO), Granada, Spain e-mail: [email protected]; [email protected] B. Lorente-Remon Program International Liaison Officer (ILO), Department of Legal Medicine, Toxicology and Physical Anthropology, Faculty of Medicine, DNA-PROKIDS, Granada, Spain © Springer Nature Singapore Pte Ltd. 2022 H. R. Dash et al. (eds.), Handbook of DNA Profiling, https://doi.org/10.1007/978-981-16-4318-7_50

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Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1064 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1065

Abstract

Human migration in general and forced human migration in particular is an increasing problem worldwide. The significant number of people involved in migrations and the extent of their needs have attracted the attention of criminal networks. However, the most tragic cases are those individuals who disappear, either physically or “virtually.” In both scenarios, forensic science and particularly genetic identification play key roles in solving and preventing such crimes. Human migration and the need for human identification is an immense and growing problem that needs to be addressed using forensic technology such as DNA typing, which has been proven to be effective in solve a large percentage of criminal cases. The aim of this chapter is giving an overview about the internationally accepted terms of human migration as well as the attempts to measure the problem, followed by the discussion of the importance of forensic DNA typing as a powerful tool in order to try to give an answer to this issue. DNA migrant identification databases come as an interesting instrument due to its robustness, power of identification, comparison possibilities, versatility, and managing characteristics. Finally, two implemented programs, DNA-PROKIDS and DNA-ProORGAN, both created by our team in the University of Granada (Spain), will be exposed, as well as a few future perspectives in order to try to give an answer to such a complex problem as human migration. Keywords

Human migration · Human trafficking · DNA typing · Forensic science · Illegal adoptions · Organ trafficking

Introduction Human migration in general and forced human migration in particular is an increasing problem worldwide. As a result of natural catastrophes and human-related causes (such as wars and civil strife), the number of immigrants has grown year after year. However, not all migration is related to violence or natural disasters but rather to the pressing need to search for better futures for oneself or one’s family. The significant number of people involved in migrations and the extent of their needs have attracted the attention of criminal networks. Human smuggling and trafficking organizations can be found in regions and along routes with a constant ow of migrants. A large number of migrants, especially women, are exploited and assaulted during their migration. Sexual abuse and labor exploitation are common during migration, especially for those considered illegal immigrants, who will likely be exploited even after arriving at the destination.

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However, the most tragic cases are those individuals who disappear, either physically or “virtually.” The physical disappearances occur when individuals are killed or die from whatever cause during migration and are buried without identification in mass or single graves, with no record. “Virtual” disappearances are those victims whose identity is changed, such as with children who are kidnapped and offered up for illegal adoption or enslaved for sexual or labor exploitation. In both scenarios, forensic science and particularly genetic identification play key roles in solving and preventing such crimes. Human migration and the need for human identification is an immense and growing problem that needs to be addressed using forensic technology such as DNA typing, which has been proven to be effective in solve a large percentage of criminal cases.

Definitions and Numbers: The Magnitude of the Problem Given that this chapter concerns the technical and scientific aspects of DNA typing, the legal aspects related to human migration will not be reviewed in depth. Due to the complexity of this topic, however, we will cover the internationally accepted definitions and terms that need to be considered to understand and approach this issue. Human migration can occur within a country or across borders and, depending on the reason for the migration, can be voluntary or forced. International migration can be legal if all administrative and border control documents and procedures are duly completed. Migration can be termed illegal if any of the former items are missing, incomplete, or falsified. People can illegally enter another country, usually under the guidance of experienced teams of smugglers belonging to criminal networks, as illegal immigrants or as victims of human trafficking (see Fig. 1). We therefore need appropriate definitions for the terms “migrant,” “human smuggling,” and “human trafficking.” We will therefore follow the internationally accepted consensus definitions. The United Nations International Organization for Migration (IOM) defines the word “migrant” as an “umbrella term, not defined under international law, re ecting the common lay understanding of a person who moves away from his or her place of usual residence, whether within a country or across an international border, temporarily or permanently, and for a variety of reasons. The term includes a number of welldefined legal categories of people, such as migrant workers; persons whose particular types of movements are legally defined, such as smuggled migrants; as well as those whose status or means of movement are not specifically defined under international law, such as international students” (International Organization for Migration 2019). In 2019, the number of international migrants worldwide was estimated at 272 million, approximately 3.5% of the world’s population, 48% of whom were female. In 2018, the global refugee population was estimated at 25.9 million, with more than half of these under 18 years of age. The number of individuals displaced in their own country reached 41.3 million, while the number of stateless persons was estimated at 3.9 million.

Fig. 1 Types of human migration

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Migration patterns differ by region. Most migrants born in Africa, Asia, and Europe remain within their country, while most migrants from Latin America leave their country of birth. The number of migrants is increasing daily, and the patterns of migration are changing (International Organization for Migration 2021). Since 1990, there has been an increase in the number of migrants. In 2020 alone, the IOM identified 22,221,538 individuals (11,651,019 males and 10,570,519 females) as migrants (United Nations. Department of Economic and Social Affairs. Population Division 2020). Human trafficking involves the recruitment, movement, or harboring of people for purposes of exploitation (sexual exploitation, forced labor, slavery, or organ removal). Victims are trafficked through threats or use of violence, fraudulent schemes, deception, and abuse of power. Human trafficking can occur within a country or across borders (United Nations Office on Drugs and Crime 2018). In contrast, migrant smuggling occurs only across borders and consists of assisting migrants in entering or staying in a country illegally. Given that migrants consent to being smuggled by irregular methods into countries, they are not considered victims. However, smuggled migrants are frequently placed in dangerous situations (e.g., crossing rivers, seas, deserts, and wild forests with their respective hazards). Migrants are also often exploited, sexually abused, harmed, and even killed, due to the smugglers’ sense of impunity resulting from the migrants’ inability and/or unwillingness to report them (United Nations Office on Drugs and Crime 2018). Among the many potential scenarios, the most common characteristics of immigrants are their long distance from their homes and their escape from violence, hunger, and extreme poverty. Migrants travel alone or with their families, often times without their personal belongings and without protection, a perfect scenario for unscrupulous individuals to exploit the vulnerable with impunity.

Missing Persons and Migrations: A Difficult Crime to Solve One of the most tragic situations in migration is the case of individuals who disappear during transit. This relevant but underestimated and complex problem presents a real challenge for the international community. In 2014, the IOM described this problem as “an epidemic of crime and abuse” and sought to coordinate efforts and data, creating the IOM Missing Migrants Project (https://missingmigrants.iom.int/), which responds to the needs of the 2030 Agenda for Sustainable Development, specifically 10.7.: “Facilitate orderly, safe, regular and responsible migration and mobility of people, including through the implementation of planned and well-managed migration policies.” (United Nations General Assembly 2015). According to available worldwide data and estimates regarding this crime, 1754 migrant fatalities were recorded in 2021 alone, with 1689 in 2014, 3522 in 2015, 4510 in 2016, 3653 in 2017, 2844 in 2018, 3556 in 2019, and 1457 in 2020. The lower number in 2020 is most likely the effect of the COVID-19 pandemic. Based on

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these data, 22,055 migrants have died from 2014 to the end of June 2021. The main reported causes of death were drowning, hypothermia, dehydration, starvation, vehicle accidents, and shootings. More relevant data divided by region, country of origin, country of destination, age, sex, etc., are available at the referenced webpage (https://missingmigrants.iom.int/). There are no reliable data on the percentage of deceased migrants who have been and who are regularly identified, but the number is low. Forensic science plays a unique and essential role in identifying such victims and establishing the cause and date of death. Coordination between forensic anthropology, forensic odontology, and forensic genetics is needed to solve the problems that arise when a dead body or remains are found.

Human Trafficking: The Most Dangerous Scenario The internationally accepted definition for “human trafficking” is that adopted by the United Nations Convention against Transnational Organized Crime, in resolution A/RES/55/25 of the 15th of November 2000 at the 55th session of the United Nations General Assembly, originally signed in Palermo, Italy, in December 2000, as defined in Article 3 (United Nations 2000): “For the purposes of this Protocol: (a) “Trafficking in persons” shall mean the recruitment, transportation, transfer, harbouring or receipt of persons, by means of the threat or use of force or other forms of coercion, of abduction, of fraud, of deception, of the abuse of power or of a position of vulnerability or of the giving or receiving of payments or benefits to achieve the consent of a person having control over another person, for the purpose of exploitation. Exploitation shall include, at a minimum, the exploitation of the prostitution of others or other forms of sexual exploitation, forced labour or services, slavery or practices similar to slavery, servitude or the removal of organs”; (b) The consent of a victim of trafficking in persons to the intended exploitation set forth in subparagraph (a) of this article shall be irrelevant where any of the means set forth in subparagraph (a) have been used; (c) The recruitment, transportation, transfer, harbouring or receipt of a child for the purpose of exploitation shall be considered “trafficking in persons” even if this does not involve any of the means set forth in subparagraph (a) of this article; (d) “Child” shall mean any person under eighteen years of age.””

According to the 2020 United Nations Office on Drugs and Crime Global Report on Trafficking in Persons, the number of victims detected and reported in 2018 was 49,032 worldwide. Approximately 50% of reported trafficked victims are sexually exploited, 38% are placed into forced labor, 6% are pressed into criminal activities, 1.5% are forced into marriages, and the rest are exposed to various forms of exploitation, including baby selling, organ removal, and other forms of exploitation (United Nations Office on Drugs and Crime 2020). The US State Department Office to Monitor and Combat Trafficking in Persons publishes the yearly Trafficking in Persons (TIP) Report. According to their data,

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140000 120000 100000 80000 60000 40000 20000 0

2014

2015

2016

2017

2018

2019

Prosecutions

Convictions

Victims Identified

New or Amended Legislation

2020

Graphic 1 Number of prosecutions, convictions, identified victims, and legislation (Department of State. United States of America 2021)

there were 109,216 victims identified globally in 2020. That same year, only 9876 prosecutions were conducted, resulting in 5271 convictions. According to the report, the number of cases is growing year after year (from 44,462 victims in 2014 to 118,932 in 2019); however, the number of prosecutions is not growing at the same rate, from 10,051 in 2015 to 11,841 in 2019 (Department of State. United States of America 2021), as shown in Graphic 1. These findings do not re ect the scope of the crime or the efforts of the international community. These cases are difficult to prosecute and subject to corruption and lack of necessary data from the countries of origin. The US State Department’s TIP Report therefore classifies countries into three separate tiers, depending on the seriousness with which they operate internally and internationally to prevent and prosecute this crime. Another concern raised in the 2021 report is related to the COVID-19 pandemic, which has led to significant economic difficulties worldwide, problems that exacerbate the risk for the poor and marginalized, with children and women being especially vulnerable. This pandemic has facilitated human trafficking activities. Alerts and special surveillance need to be enforced in the countries of origin and, in cases of international forced migration, in those countries of transit and origin. From a genetic, DNA typing perspective, there are three situations where DNA is of major interest: identification of the deceased, identification of trafficked children, and identification of trafficked organs. The procedures and existing programs, as well as the challenges, will be covered in sections “Missing Children: The DNA-PROKIDS Program” and “Organ Trafficking: The DNA-ProORGAN Program” of this chapter.

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The Role of Forensic Science: DNA Typing Forensic science faces two main challenges: identifying the victim and determining the cause of death, the solutions to which will depend on the circumstances and laws of the country where the victim is found. In terms of the cause of death, forensic pathologists can offer valuable information if the body is well preserved and if a full autopsy, including complimentary analysis and techniques, can be performed. If the body is not well preserved or if only partial remains are found, forensic pathologists will need to work with anthropologists to determine the cause of death. The cause of death could be easy to determine in some cases (e.g., those with severe skull fractures), while being next to impossible in others. Identifying victims in cases of human migration is a complex problem, mainly due to the lack of data and records with which to compare and create reference databases. Although the international forensic community has the necessary tools for solving most cases, there is a lack of appropriate national and international legislation, insufficient collaboration between agencies, fear among the relatives of victims, and government corruption, which all contribute towards perpetuating this unacceptable situation. The four main disciplines that can help identify cadavers are forensic pathology, forensic anthropology, forensic odontology, and forensic genetics. Depending on their amount and quality, the data obtained from victims can vary in terms of support, usefulness, and implementation. Provided they coordinate the data, these disciplines can be additive and compatible. The only partially destructive discipline is that of genetics, in those cases in which bone or teeth fragments are pulverized for DNA extraction and analysis. Appropriate coordination among the various specialists in terms of work ow is therefore essential. Forensic genetics has a number of advantages when implemented worldwide. Although not intrinsically better than other methodologies, genetics offers unique tools for solving cases of persons gone missing during migration and in humantrafficking crimes.

The Advantages and Need for DNA Migrant Identification Databases We are not proposing building and using only DNA databases for identifying missing persons but rather the generation and storage of all types of records that can be used in the context of forensic pathology, odontology, and anthropology. Forensic DNA does not always yield useful results, and biological samples from relatives are not always available. The main advantages of DNA typing for identifying missing persons during migration are detailed below. For human trafficking that includes missing children, illegal adoptions, and organ removal, DNA offers unique features compared with the other mentioned disciplines. Genetic identification is a robust, reliable, universal

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technology that has a high power of identification, has the ability to compare different relatives, can be used with all types of biological samples, and facilitates the creation of databases.

Robust, Reliable, Universal Technology Forensic DNA typing has become the gold standard in human identification since its introduction in the mid-1980s following the publications by Alec Jeffreys using restriction fragment length polymorphism techniques and especially after the adoption of polymerase chain reaction-based techniques and the use of internationally validated and accepted commercial kits. The International Society of Forensic Genetics (ISFG) has from the start played a key role in harmonizing the technical and scientific advances in forensic DNA typing. Following the ISFG recommendations, commercial companies have developed kits with a common set of loci, and countries now have compatible databases. There are continuous advances being applied to human genetic identification (Parson et al. 2016). The development of the Combined DNA Index System (CODIS) by the US Federal Bureau of Investigation in the mid-1990s was another milestone. This system has been donated to numerous countries and is continually expanding (Ge et al. 2012). Regional forensic networks, such as the Asian Forensic Sciences Network and the Ibero-Latin American Academy of Criminalistics and Forensic Studies and its DNA Working Group (GITAD) have also played a key role in harmonizing scientific protocols (Lorente 2001), with a significant focus on quality and the tracking of database development (Carneiro da Silva Junior et al. 2020). Other countries, such as China, have adapted their scientific developments and loci to be compatible with existing international datasets, such as CODIS and the European Standard Set (Guo et al. 2014). The use of the highest standards and the most demanding procedures, required by ISO certification and accreditation, positioned DNA analysis as one of the most reliable and trusted technologies for human identification. One of the goals of The International Forensic Strategic Alliance, a global network of regional forensic science organizations, is to “encourage the exchange of information related to experience, knowledge and skills between the member networks and other operational forensic experts as appropriate” and could play a role in facilitating data exchange. In short, DNA typing is a reliable, compatible, and universal technology whose data can be easily and anonymously compared among countries. Power of Identification Combining the results of the analysis of autosomal DNA, Y and X chromosomes and mitochondrial DNA should theoretically provide likelihood ratios showing the biological and genetic relationships. There are, unfortunately, certain limitations in the context of missing persons during migration and in human trafficking-related problems. Generating sufficient genetic information from samples recovered at sea, from single or mass graves can be problematic. Samples are at times highly compromised, and the quality and quantity of the DNA is very low. There have been numerous published scientific

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papers on overcoming this problem using novel strategies and approaches (Zeng et al. 2019; Emery et al. 2020). Certain authors, such as David Caramelli et al. at the University of Florence, have focused their efforts on identifying the best source for DNA, suggesting petrous bone as an ideal source (Pilli et al. 2018). Other authors have employed powerful next-generation sequencing-based technologies to generate as much genetic information as possible (Zeng et al. 2019; Elwick et al. 2019). Once forensic data have been generated from unidentified remains or cadavers, the data should be compared with that from potential relatives, which can be a challenge and requires caution. Even in mass disaster cases where most if not all victims are known, a statistical analysis might be difficult, especially if there are biologically related individuals or if the relatives to be compared with the victims are genetically distant (Ge et al. 2010; Ge and Budowle 2021; Marsico et al. 2021). The most difficult situations, however, are those where the human remains to identify are tentatively from many different countries or geographical regions, given that in most of these cases there are few reference samples from relatives (Vigeland et al. 2020; Marsico et al. 2021). There is a need to coordinate efforts in collecting samples from the migrants’ relatives, especially those who have not received news of them for some time. Coordinated national and international efforts need to be reinforced and should focus on the close genetic relatives (parents, siblings, progeny) of the missing persons (Alvarez-Cubero et al. 2012). Nonprofit, humanitarian proposals to generate and maintain DNA databases to compare data between unidentified remains and all those relatives who wish to donate biological material would be a highly welcome measure to help solve this problem. Bruce Budowle of the University of North Texas Center for Human Identification put forth just such a proposal, providing a procedure for receiving genetic profiles and biological samples from relatives of missing persons who might have died while crossing the Mexico-United States border (Budowle et al. 2020).

Comparing Victim’s Data with Relatives’ Data From an operational standpoint, this option basically entails identifying the victim by comparing their data with that from numerous potential relatives. Certain relatives (parents and children) are ideal candidates from a genetic standpoint; however, other distant relatives (grandparents, grandchildren, nephews, nieces, cousins, etc.) could provide relevant information or at least clues for continuing the investigation. Other powerful identification methodologies do not allow for anything other than direct comparisons; for example, a reference sample or record from the missing person, such as fingerprints and dental records. This does not imply that DNA is the best technology, just that it might be the only applicable one in these cases. Possibility to Analyze All Kind of Biological Samples Genetic databases for migrations and for identifying missing persons have two main indexes: (1) profiles in question (from unidentified persons or remains) and (2) reference profiles (from known persons).

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Samples can be obtained from unidentified human remains, usually bones and teeth but also hair, muscles, skin, and even body uids, depending on the preservation status of the remains. If the unidentified victim is alive, as is usually the case with children and mentally impaired adults, reference samples can be taken from epithelial buccal cells, saliva, and blood. Samples can be taken from known sources such as potential relatives of the missing persons and usually include epithelial buccal cell, saliva, and blood. Samples can also be taken from the victim’s personal items such as a hairbrush, comb, toothbrush, razor blade, wristwatch, glasses, rings, and tight-fitting clothes. In a small number of cases, reference samples can originate from hospitals (e.g., tissues previously taken for biopsies and through voluntary donation such as blood, semen, and bone marrow). Despite the broad variety of sources for extracting DNA, the results will be identical, a unique advantage for DNA analysis and genetic databases.

Building, Updating, and Managing Genetic Databases Once the genetic analysis has been completed, DNA databases can be generated from the sets of numbers and letters (depending on the type of analysis), short tandem repeats, sequencing, and single nucleotide polymorphisms. The information that is statistically unique to each individual can be stored in a single row composed of 60–120 characters on average. These data are exact and do not deviate, provided they are generated under the highest standard of quality. The data are easy to store, due to the small space they take up compared with data generated from images (such as anthropological, fingerprint, and weapon images). A million profiles would require between 120 and 180 megabytes of storage. Another advantage is that the alphanumeric structure of this information facilitates rapid comparisons in the search for exact matches and other types of coincidences. There are a number of applications that not only store data but also automatically perform searches and establish statistical calculations in the event of tentative matches, such as CODIS and other commercially available systems, most of which are compatible with CODIS, such as BONAPARTE (https://www.bonaparte-dvi. com/index.php), which was recently chosen by INTERPOL to support its efforts in the search for missing persons (INTERPOL 2021a) in coordination with its I-Familia program (INTERPOL 2021b). Another commercially available application worth mentioning is the Mass Fatality Identification System (M-FISys, https://www.genecodesforensics.com/software/) developed by the Gene Codes Corporation (Ann Arbor, Michigan, USA), which helps identify missing children (DNA-PROKIDS) and works with other missing person databases. When creating these databases, the highest standards of quality (certification, accreditation) need to be followed, and the databases must meet all legal and ethical requirements (data dissociation, restricted access) of the country in which it operates.

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Missing Children: The DNA-PROKIDS Program Of the almost 110,000 victims of human trafficking identified by the US State Department in 2020, a large percentage were children, defined as individuals under 18 years of age. Nevertheless, our research group at the University of Granada (Spain) efforts are focused on the most vulnerable children: newborns (0–2 months), infants (2 months–1 year), and toddlers (1–4 years). At these ages, children are easy targets for traffickers, who exploit them for various purposes, including sex, labor, begging, and illegal adoptions. The lack of identification is compounded by the children’s difficulty or inability to explain who they are or to provide clues that could help identify them or their families. Children are kidnapped, issued fake documents and new identities, and adopted by another family to start another life, while the true biological family remains powerless. As an identification tool, DNA typing could help not only to solve such crimes but also prevent them. Human migrations facilitate these illegal activities, especially in cases of forced migration caused by con icts. Europe has recently been affected by large numbers of migrants from Iraq and Syria, who generally attempt to enter the European Union/ Schengen space through the Greek-Turkish and Italian borders (Olivieri et al. 2018; Pavlidis and Karakasi 2019). DNA-PROKIDS is a humanitarian, nonprofit program created between 2002 and 2004 and run by the University of Granada (Spain) and is supported by the Spanish Government and the Andalusian Regional Government, with the collaboration of Zogbi (a provider of forensic equipment in Mexico), the US Returned.org Foundation, and the QSDglobal Foundation for missing persons (Spain). The first pilot program for DNA-PROKIDS was conducted in Guatemala between 2004 and 2006 (Alvarez-Cubero et al. 2012). The DNA-PROKIDS program is open to collaboration with countries willing to combat the trafficking of children at the origin and supports interested countries in creating a database composed of two separate indexes. The first index (or Questioned Database) is constructed from DNA data from unidentified missing children (usually newborns, infants, and toddlers) who reside in orphanages under the protection of the authorities. The second index consists of DNA data from the relatives of missing children who voluntarily donate biological samples after signing an informed consent. DNA-PROKIDS is currently under development in various countries and is in permanent collaboration with state governments, including Guatemala (Guatemala National Institute of Forensic Sciences [INACIF]), Mexico (Mexico City Attorney General’s Office), Honduras (National Office for Infants, Adolescents and Family [DINAF] and Honduras Institute of Forensic Medicine), and Thailand (Royal Thai Police). So far, more than 1800 children have been identified and returned to their families. Through DNA typing, DNA-PROKIDS can help stem the tide of illegal adoptions.

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Organ Trafficking: The DNA-ProORGAN Program Organ trafficking, considered part of human trafficking, which in turn is linked to human migration, is a difficult crime to track and prosecute but one that could be investigated with the use of forensic DNA analysis. The United Nations Office on Drugs and Crime defines organ trafficking as the “illicit removal of organs.” To help combat organ trafficking, the Declaration of Istanbul on Organ Trafficking and Transplant Tourism was signed in 2008 (The Transplantation Society 2008), with an updated edition signed in 2018 (The Transplantation Society 2018). According to the Global Observatory on Donation and Transplantation (the official WHO observatory supported by The Spanish Transplant Organization), there were 163,141 organ transplantations worldwide in 2019. Due to the restrictions imposed due to the COVID-19 pandemic, there were 79,242 transplantations in 2020. Kidneys represent approximately 65% of all transplanted organs, 5–10% of which could be illegal and thus considered organ trafficking (López-Fraga et al. 2014). Although the data are hard to track due to their criminal and secretive nature, between 5000 and 10,000 kidneys might have been transplanted illegally in 2019. Despite the growing number of transplantations worldwide, organ trafficking persists due to the fact that the number of patients needing organs is much larger than the number of donors and potential transplants. If unavailable locally, patients and their families often attempt to obtain organs from other sources, even traveling abroad (Ambagtsheer et al. 2016). The dramatic reduction in transplantations during the COVID-19 pandemic has two main reasons. First, hospitals rooms and intensive care units were needed for the thousands of infected patients. Second, transplantation requires varying levels of immunosuppression, and SARS-Cov-2 virus infection presents a high mortality risk for immunosuppressed patients. Despite the efforts of the global medical community, the waiting lists for transplantation have increased throughout 2020 and are still increasing. The number of cases of organ trafficking will therefore likely increase in parallel (official data still pending). The lack of prosecution of organ trafficking is mainly due to the lack of documentation and the easy falsification of documents. Organs can be extracted from individuals who might sign informed consent forms under false pretenses, under duress, or with forged documents are, falsifying the names and signatures. Tracking the origins of organs is therefore almost impossible. Genetic analysis can help identify victims and the individuals involved, which might help prevent future occurrences (Lorente et al. 2020). In 2016, the University of Granada started the pilot DNA-ProORGAN program in an effort to develop genetic databases that could help track the identities of organ donors and recipients. The program has been supported since 2017 by the Spanish Medical Association and, since 2019, by the Spanish National Transplant Organization. The program’s efforts are now focused on kidney transplantations, given that they account for an estimated 65% of all transplants and approximately 80% of all illegal transplants. DNA from transplanted kidneys (or graft) can be obtained from

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the kidney recipient’s urine. There is therefore no need to perform biopsies or expensive and dangerous surgical procedures (Lorente et al. 2020).

Future Perspectives: Enlarging and Coordinating the Databases The main challenge as of today is not a scientific one but rather a legal, social, humanitarian one: to use the currently available genetic technology as much as possible. All countries should have a national civil database for identifying missing persons, whose main purpose is to identify individuals as a way of restoring their dignity. Enacting legislation for implementing databases to monitor such criminal activity, focused on identifying those who traffic humans and their organs, could pose difficulties for certain countries. However, databases that focus on human dignity and are based on the voluntary donation of samples by relatives of missing persons should be easier to implement. There are already numerous countries that have passed such legislations, which can act as examples for other countries considering the creation of such databases. These databases need to contain an index with DNA profiles from all unidentified remains and another index with profiles from reference samples (relatives, personal belongings, etc.). National authorities need to pay special attention to the relatives of missing persons. Without reference samples for comparison, identification will not be possible. International organizations and nongovernmental organizations can also help, especially when working on-site. Forensic scientists can also play an additional relevant nonscientific role by informing and convincing people and authorities as to the advantages and limitations of genetic identification.

Conclusion The world continues to face the problems of identifying missing persons and unidentified remains, as well as the kidnapping and exploitation of children for illegal adoptions and other purposes. These problems are intimately connected to the ow of human migration, the perfect environment for human traffickers. Human identification is a multidisciplinary task in the forensic sciences, requiring the coordination of these specialists. These specialists include forensic pathologists, anthropologists, odontologists, and fingerprint specialists, employing tools such as forensic genetics and DNA typing, a powerful, reliable, universal, and compatible scientific technology. There are commercially available kits and software that can help generate databases to aid in these investigations. There is a pressing need to focus on identifying missing persons, creating national and regional databases, and connecting them through the established channels of international cooperation following national and international laws.

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DNA typing specialists should include among their goals the raising of awareness among the public as to the benefits of this technology, advocating its creation and permanent implementation. Although we are faced with this problem, we have the tools that could help solve a large part of this crime.

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Christian Haarko¨tter, María Saiz, M. J. Alvarez-Cubero, Juan Carlos Alvarez, and J. A. Lorente

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inhibition of PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inhibitory Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inhibition Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Degraded DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA Degradation Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA Degradation Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Low Copy Number DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Problems with LCN DNA Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA Contamination Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Laboratory Recommendations for the Analysis of Compromised Samples . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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C. Haarkötter · M. Saiz (*) · J. C. Alvarez Laboratory of Genetic Identification, Department of Legal Medicine, Toxicology and Physical Anthropology, Faculty of Medicine, University of Granada, Granada, Spain e-mail: [email protected]; [email protected]; [email protected] M. J. Alvarez-Cubero Department of Biochemistry and Molecular Biology III, Faculty of Medicine, University of Granada, Granada, Spain Pfizer-University of Granada-Junta de Andalucía Centre for Genomics and Oncological Research (GENYO), Granada, Spain e-mail: [email protected] J. A. Lorente Laboratory of Genetic Identification, Department of Legal Medicine, Toxicology and Physical Anthropology, Faculty of Medicine, University of Granada, Granada, Spain GENYO, Centre for Genomics and Oncological Research (Pfizer / University of Granada / Andalusian Regional Government), Granada, Spain e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 H. R. Dash et al. (eds.), Handbook of DNA Profiling, https://doi.org/10.1007/978-981-16-4318-7_51

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Abstract

DNA analysis is a vital tool in forensic sciences – particularly criminal science – since the introduction of PCR at the beginning of the 1990s. However, many challenges remain that must be solved, such as inhibition of the reaction, DNA degradation, low copy number (LCN) DNA, and DNA contamination. The aim of this chapter is to examine these issues and give the reader not a solution but options for dealing with these challenges. First, inhibitory factors and mechanisms are discussed. Next, DNA degradation and LCN DNA are introduced, followed by issues and possible sources of DNA contamination. Finally, recommendations are provided to identify, treat, or at least avoid these problems. Keywords

Degraded DNA · Forensic DNA challenges · Low copy number DNA · DNA contamination · PCR inhibition

Introduction Frequently, forensic genetic laboratories must work with DNA samples that are in suboptimal condition. These samples might be scarce, fragmented, or poorly preserved or might have been exposed to environmental agents. Consequently, the techniques that are used in forensic laboratories are crucial to optimize the recovery of DNA from compromised samples. Thus, the type of sample that is used in the DNA analysis is critical. The best tissues to analyze ancient DNA are the bones and teeth (Miloš et al. 2007), because they protect DNA from degradation and biological processes due to their physical and chemical robustness. Teeth are the hardest tissue in the human body, due to tooth enamel, and DNA is preserved by calcified tissues (Girish et al. 2010). The tooth is made up of dentin, a connective tissue that forms a large part of the structural axis of the tooth. Dentine in the dental crown is covered by enamel, an extremely mineralized, acellular, and avascular tissue of ectodermal origin. The dentin at the root of the tooth is covered by cementum, another type of calcified connective tissue. The soft tissue in the pulp consists of odontoblasts, fibroblasts, endothelial cells, peripheral nerves, undifferentiated mesenchymal cells, and other nucleated components of blood, making it rich in DNA (Muruganandhan and Sivakumar 2011). The pulp of the tooth is used to recover DNA, where it abounds and is unlikely to be contaminated by nonhuman DNA (Girish et al. 2010) (Fig. 1). Bone tissue is made up primarily of proteins and minerals. The two most abundant proteins in this tissue are collagen and osteocalcin. Approximately 70% of the mineral component of bone comprises hydroxyapatite, which includes calcium phosphate, calcium carbonate, calcium uoride, calcium hydroxide, and citrate.

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Fig. 1 Anatomy of a tooth

With regard to the structural organization of bone, the mineral component provides support for the protein element and thus physically excludes potentially harmful extracellular agents and enzymes (Collins et al. 2002). DNA has a high affinity for hydroxyapatite, and DNA degradation is linked to a loss in the crystallinity of hydroxyapatite, although it has also been associated with the loss of collagen. Bony characteristics are directly related to the survival of DNA and its protection from degradation. Bone density is one of the most important intrinsic factors in bone survival, differing significantly between men and women. In addition, there are disparities in bone density between parts of the skeleton, with the middle region of bones having higher levels. Mechanisms that preserve DNA well in bones are not well characterized (Collins et al. 2002; Pruvost et al. 2007). However, there are several factors that affect DNA preservation, such as temperature, pH, humidity, and time. Many studies have measured success in obtaining an STR (short tandem repeat) profile, based on the type of sample, tooth, and bone (Fig. 2), reporting that the molars, petrous bone, and femur are the best tissues (Miloš et al. 2007; Gamba et al. 2014). Keratinized tissue, such as hair and nails, as well as horn, feathers, and scales in vertebrates, can also be used. Because these tissues originate from living cells that die during biogenesis and undergo natural desiccation, DNA might survive for longer periods. However, these cells have lost their nuclei, reducing the analysis to that of mitochondrial DNA.

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Fig. 2 Percentage of success in DNA recovery and STR typing by bone type (Miloš et al. 2007; Behar et al. 2012)

It is difficult to estimate the conservation of DNA through simple macroscopic inspection. However, there are several features of a good sample. The bones must be hard and heavy and have a compact structure. Long bones are preferable to porous bones, such as the pelvis, scapula, and parts of the skull. The bones must not have cracks or evidence of microbial attack. Especially for human identification, teeth with well-preserved roots are useful. Burnt material or material that has been affect by heat is unsuitable. With a large number of individuals, sampling the same anatomical element is recommended to avoid double-typing.

Inhibition of PCR PCR inhibitors usually bind directly to single- or double-stranded DNA, preventing or reducing amplification. Other inhibitors block the enzyme, primarily by altering the main cofactor, Mg2+. One of the chief limitations in forensic cases is the presence of inhibitors in the samples; thus, it is preferable to know how to eliminate them to ensure amplification of the sample, usually by improving DNA purification methods to remove inhibitors.

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Inhibitory Mechanisms Common samples in forensic cases, such as skeletal remains, hair, and blood, are usually exposed to harsh environmental conditions, resulting in damaged or degraded DNA and samples that contain PCR inhibitors. For example, human remains could contain PCR-inhibiting agents, such as collagen, calcium, humic acid, melanin, and hematin that reduce downstream DNA typing success. Blood samples also have harbor inhibitors, including anticoagulants, such as EDTA and heparin, and calcium, which is commonly co-extracted with DNA from skeletal remains. Most DNA extraction methods are effective in removing high amounts of inhibitors from the blood, hair, and bone tissue in forensic samples (Zeng et al. 2019). However, some inhibitors remain in the PCR process and could interfere primarily, not only in DNA amplification processes binding directly to single- or doublestranded DNA or blocking the enzyme but also with the library preparation in MPS (massive parallel sequencing) analysis or qPCR by quenching the uorescence of the uorophore. Inhibitors could have a negative effect on 50 -30 exonuclease activity and thus lead to inhibition, because the DNA polymerase will be unable to hydrolyze the probe, resulting in underestimation of DNA levels. Humic acid and several molecules in blood, such as hemoglobin and hematin, quench the uorescence of DNA-binding dyes. Consequently, these inhibitors could decrease the amplification and corrupt the analytical data, specifically for samples with low amounts of template and high amounts of background material, which are common in forensic scenes. There are several strategies for avoiding or reducing the effects of PCR inhibitors, such as purification, dilution of DNA extracts, and the use of inhibitortolerant DNA polymerases (Sidstedt et al. 2020). Other inhibitors, such as calcium, suppress STR typing as their concentration increases, whereas humic acid has no inhibitory effect on STR profiling but intercalates in the DNA template, limiting its availability for amplification. Collagen strongly inhibits STR typing, effecting allele dropout with rising concentration. Hematin does not seem to inhibit STR typing, but, as it is a metal-chelating agent that binds to magnesium, it can cause complete allele dropout in qPCR (Kieser et al. 2020). Several reagents for extraction, such as phenols, that are introduced during DNA purification can act as inhibitors by denaturing the polymerase (Sidstedt et al. 2020). Thus, several kits for extraction or amplification in forensic cases are designed to reduce inhibitors, such as the Investigator 24plex, with a higher tolerance to common PCR inhibitors, including humic acid and tannic acid (Lin et al. 2017). Many efforts have been made to improve PCR kits, such as a modified version of AmpFlSTR SGM Plus, replacing AmpliTaq Gold DNA polymerase, with a customized blend of two alternative polymerases: ExTaq Hot Start and PicoMaxx High Fidelity. These changes have improved the results for allele calls and stutter sizes, primarily by increasing the resistance to PCR inhibitors that plague forensic identification (Hedman et al. 2011). Certain substances can also help to avoid the effect of PCR inhibition; bovine serum albumin (BSA) may improve between five and ten times

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inhibitor tolerance (Sidstedt et al. 2019), which has led to the addition of BSA in several commercial kits available nowadays. As discussed, there are several types of inhibitor, depending on the mechanism, for which there are various strategies, based on whether DNA polymerization or uorescence detection is primarily affected (Sidstedt et al. 2020).

Inhibition Factors (i) Soil: Many chemical reactions occur naturally in soil, which contains inorganic and organic compounds that can affect DNA. Many of these substances cannot be removed during DNA purification and can in uence PCR (Alaeddini 2012). Depending on the type of soil, there will be varying proportions of fulvic acid or humic acid, which will have disparate effects on the quality of the DNA. Humic acid predominantly affects the initial PCR cycles, during which DNA is mainly genomic, but not the final cycles, when short amplicons abound (Sidstedt et al. 2017). It is difficult to eliminate with extraction protocols (Opel et al. 2010). Besides, soil pH and humidity levels have a negative effect on ancient DNA conservation, and they may ease the introduction of PCR inhibitors in the bone tissue (Dabney et al. 2013). (ii) Blood: Blood is one of the main samples that are collected for human identification, but many of its components inhibit PCR (Al-Soud et al. 2000). Hemoglobin and hematin in blood samples inhibit amplification and thus impede correct genetic identifications. The inhibitory power of each component has been studied, and it has been concluded that hemoglobin is a more powerful inhibitor, requiring lower amounts to completely inhibit amplification (Ambers et al. 2018); however, unlike hemoglobin, hematin directly alters the activity of DNA polymerase (Opel et al. 2010). (iii) Bone: Inhibitory substances in bone become apparent when amplification is performed with these samples (Loreille et al. 2007). The extraction of DNA from bone is complex and expensive; thus, it is essential to know the compounds with which PCR competes to avoid them (Pajnič 2016). Over 99% of calcium resides in bones and teeth (Beto 2015). But, calcium reduces the efficiency of DNA amplification (Opel et al. 2010), by interfering with the interaction between polymerase and magnesium ions in a PCR reaction (Bickley et al.1996). (iv) Saliva: There are minor inhibitors in saliva samples. Amylase is the most abundant protein in saliva, and its function is to convert complex non-soluble polysaccharides into smaller soluble units (Carpenter 2013). Thus, it could affect amplification, although studies have not confirmed it. During sample processing or DNA extraction, inhibitors can be added, including powder from gloves; salts, such as potassium and sodium chloride; detergents; and other organic molecules, including ethylenediaminetetraacetic acid (EDTA), sarkosyl, ethanol, phenol, and isopropylic alcohol. These substances, in addition to

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being necessary for cell lysis and nucleic acid isolation, can inhibit PCR inhibition at certain concentrations. Ionic detergents, such as sarkosyl, SDS, and sodium deoxycholate, have high inhibitory activity, whereas nonionic detergents, including Triton X-100 and P-40, can suppress PCR reactions at high concentrations. EDTA is a common component in elution buffers and purification kits, but its chelating activity can exhaust magnesium ions and thus inhibit PCR. Even contact between PCR reagents and UV-irradiated plastic tubes and the components of cotton swabs can affect the sensitivity of PCR (Shrader et al. 2012). For these issues, several purification kits are available. The DNeasy PowerClean Pro Cleanup Kit (QIAGEN, Hilden, Deutschland) combines DNA purification reagents and a silica membrane spin column: DNA is captured and purified by being washed and eluted from the spin columns, depending on the percentage recovery of inhibitors. Other kits, such as the RNA/DNA/Protein Purification Plus Kit, which uses silicon carbide columns (Norgen Biotek, Ontario, Canada), can perform both DNA extraction and purification. Organic solvents, such as dimethyl sulfoxide (DMSO) and formamide, have been used as additives to increase the efficiency of DNA amplification, whereas bovine serum albumin has been added during PCR amplification of samples that might contain inhibitors. Consequently, BSA enhances PCR amplification yields when combined with organic solvents, improving their effects with its own effects during the initial PCR cycles (Farell and Alexandre 2012). These PCR additives have beneficial effects on amplification, but it is impossible to establish which agents are useful in each particular context (Gene Link 2014): (a) Betaine (0.1–3.5 M), DMSO (2–8%), and formamide betaine (1–5%). DMSO and formamide reduce the secondary structure of GC-rich templates and aid in their amplification. The combination of DMSO and betaine is superior to formamide use alone. (b) TMAC (tetramethylammonium chloride, 15–100 mM). TMAC increases the hybridization specificity and melting temperature and should be used in combination with DMSO and betaine. (c) BSA and Gelatin BSA. These compounds stabilize Taq polymerase and have been especially effective in amplifying ancient DNA, which contains PCR inhibitors, such as melanin, and reducing enzyme loss by nonselective adsorption to tube walls.

Degraded DNA DNA is constantly degraded in living beings by hydrolysis, oxidation, and methylation, which fracture and alter its primary structure, changing the sequence and thus its inherent information (Lindahl 1993). The cell has various DNA repair pathways (Iyama and Wilson 2013), which cease when death occurs, allowing DNA to continue to be attacked by physical, chemical, and biological elements; however, the ensuing damage is permanent. DNA degradation begins with two mechanisms:

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Fig. 3 Exponential decay of DNA: copy number versus fragment length (Allentoft et al. 2012)

nucleases cleave DNA into fragments, after which DNA is digested by microorganisms, effecting random DNA fragmentation, re ected by a negative exponential correlation (Fig. 3) between the number of molecules and DNA length at a rate of 5.50·106 nucleotides per year (Allentoft et al. 2012).

DNA Degradation Mechanisms DNA damage can be classified into two groups: fragmentation and modification (Pääbo et al. 2004), which occur through the following mechanism simultaneously and randomly: (A) DNA bond cleavage. Due to the degradative activities of microorganisms and postmortem nucleases or other chemical components. Nucleases are phosphodiesterases that cleave one of the two (30 or 50 ) phosphorous-oxygen bridges in a nucleic acid polymer by acid-base catalysis (Yang 2011). DNase I is the most common enzyme that is used in artificial DNA degradation assessments in the forensic literature (Timken et al. 2005). (B) Oxidative damage. Free radicals, such as -O22 and -OH, are produced by metabolic reactions and external sources, including ionizing radiation and decomposing aerobic bacteria. Free radicals react with DNA in a process that is called oxidative DNA damage and can induce DNA modifications, such as base and sugar lesions, strand breaks, and cross-links by reduction-oxidation reactions (Dizdaroglu 2002). Guanine is more susceptible to this type of damage due to its double bonds (Cadet et al. 2017). (C) Cross-links. DNA cross-linking is the formation of a covalent bond between two DNA nucleotides. Cross-links appear with high frequency and can block Taq

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polymerase during DNA amplification, which can be seen by electronic microscopy (Pääbo 1989). (D) Hydrolysis-induced rupture. Hydrolysis affects DNA through the 20 deoxyribose backbone and deamination. Deamination can occur in cytosine, adenine, and guanine, causing the loss of the exocyclic amino group, which transforms them into uracil, hypoxanthine, and xanthine, respectively (Marrone and Ballantyne 2010). These transformations lead to changes in sequence. (E) Maillard reactions. Described by Maillard in 1912, these reactions are wellknown in food chemistry, because they elicit the brown color in beers, meats, and baked goods. The exact mechanism remains unknown, but it has been reported that DNA is broken in glucose and amino acid mixtures (Hiramoto et al. 1993), conditions that are common during decomposition.

DNA Degradation Factors In the environment, there are several factors that can affect DNA preservation (Dean and Ballard 2001): 1. UV Radiation. Solar radiation is a well-known mutagenic agent, and UV light is even used in forensic laboratories for exogenous DNA decontamination. Ultraviolet radiation comprises electromagnetic waves from 10 to 400 nm: long-wave (UVA, 400–315 nm), medium-wave (UVB, 315–280 nm), and short-wave (UVC, 280–100 nm). UV light affects DNA in various manners: UV induces damage (oxidative and hydrolysis damage due to free radicals that are created by UV radiation), pyrimidine photoproducts (such as cyclobutane pyrimidine dimers and 6–4 pyrimidine photoproducts, which affect DNA strand structure); purine photoproducts, and double-strand breaks (Rastogi et al. 2010). 2. Temperature. Likely it is the most determining factor in DNA preservation. Low temperatures preserve DNA, because they slow degradative chemical reactions. High temperatures accelerate such reactions, but very high temperatures lead to tissue desiccation and can protect DNA from degradative processes. Ideal DNA preservation occurs at constant temperature, as reported for bones that have been found in caves (Collins et al. 2002). 3. Humidity. The solvating action of water allows for the presence of organic substances, which have DNA polymerase-inhibiting activity, allowing oxidative and hydrolytic degradation. Water can also help organic components from the environment penetrate into the sample if its surface is porous, as with bones or teeth. DNA levels fall slightly in a more humid environment, but real effects are detected on DNA quality (50% less reproducible alleles (Burger et al. 1999)). 4. pH. An acidic pH affects the oxidation state of the phosphorus content in DNA phosphate groups, and low pH levels fragment DNA into its elemental units (Young et al. 2014). Alkaline pH levels affect the hydrogen bridges between strands, and very high pH levels separate double-stranded DNA by deprotonation

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(Ageno et al. 1969). Neutral or slightly high pH levels – near physiological pH – help preserve DNA. 5. Soil components. The minerals in soil have various effects on DNA preservation. For example, sand – composed primarily of quartz – has adsorbing capacity and sequestering DNA and protects it from the activity of degrading agents (Romanowski et al. 1991). The same phenomenon occurs in clays; thus, analyzing the soil itself to obtain DNA is promising (Alvarez et al. 1998). Humic substances, a heterogeneous group of substances that are derived from plants and animals that remain after decomposition, have also been described as PCR inhibitors. However, humic substances bind to DNA (Zipper et al. 2003), protecting it from other substances (Crecchio and Stotzky 1998), so its possible degradation mechanism is not clear yet. 6. Microorganisms. Nucleated human cells are a material of interest in forensic genetics, but they contain many nutrients that attract the decomposing activity of microorganisms. Hemoglobin is a source of iron and protein for Plasmodium, the calcium phosphate in bones attracts Bacillus megaterium, and collagen attracts collagenolytic bacteria; nutrients in semen or vaginal uids induce rapid bacterial degradation by Escherichia coli and Staphylococcus epidermidis (Dash and Das 2018). Degrading microorganisms liberate various components, such as nucleases, that mutate DNA. To mitigate the analytical problems that are associated to degraded DNA, several amplification strategies have been developed: (a) miniSTR analysis is an amplification strategy that uses a low number of short tandem repeats (STRs) and redesigned primers so that they bind closer to the STR regions in DNA, and it has been used successfully with degraded DNA (Wiegand and Kleiber 2001; Coble and Butler 2005). Several commercial miniSTRs kits have been developed, such as MiniFiler™ (Thermo Fisher), a 5-dye chemistry kit with FGA, CSF1PO, D18, D16, D21, D2, amelogenin, D7, and D13 loci, generating 33–200-bp primers. However, miniSTRs have a major disadvantage: few loci can be multiplexed in the same assay (Butler 2011). (b) Mitochondrial DNA can also be used to analyze degraded DNA. Compared with a single molecule of nuclear DNA in the cell nucleus, there are up to 100,000 copies of mtDNA in a single cell (Butler 2011). In cases in which STR analysis has failed, mtDNA sequencing has been successful (Holland et al. 1993). However, there are several disadvantages: heteroplasmy (different mtDNA sequences in the same individual), maternal inheritance, and difficult statistical interpretation. (c) Next-generation sequencing (NGS) and massive parallel sequencing (MPS) are promising tools for analyzing degraded DNA, sequencing large regions of DNA (or even the whole genome) to search for single-nucleotide polymorphisms (SNPs) for human identification and ancestral and phenotypic assessments (Butler 2011). Groups of SNP markers, known as microhaplotypes [40], have been proposed for degraded DNA, obtaining profiles even with highly degraded

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samples (Turchi et al. 2019). There are two NGS platforms that have been validated for forensic analysis: the Ion S5 System (Thermo Fisher), with a whole-mtDNA genome and control region panel; an STR panel with GlobalFiler markers, 48 ancestry SNPs, and 54 identification SNPs; and ForenSeq™ (Verogen, San Diego, CA, USA), with 27 STR markers, 24 Y-STRs, 7 X-STRs, 94 identity SNPs, 22 phenotypic SNPs, and 56 ancestry SNPs.

Low Copy Number DNA DNA input that is less than 200 pg (Budowle et al. 2009) or even 100 pg (Gill et al. 2000), translating into 15 or 30 diploid cells, respectively, is known as low copy number DNA, low DNA testing, low-template DNA, or LCN DNA. There are at least two scenarios in which LCN DNA typing is essential: touch DNA and challenging samples. Touch DNA is based on the analysis of swabs of various objects or surfaces, looking for skin cells to demonstrate that a person touched an object or a crime scene surface (Oorschot and Jones 1997). In addition to individuals having different tendencies to deposit DNA when they touch an object, several people could have been in contact with crime scene items and surfaces but not involved with the crime, which is why mixtures can be expected in touch DNA analysis. Further, passive transfer of DNA of a person who has touched a suspect’s hands is possible (Lowe et al. 2002). Recently, several touch DNA studies have been performed, based on the increased sensitivity of commercial kits, which have generated similar conclusions: touch DNA mixtures are difficult to interpret, and passive transfer is possible (Cale et al. 2016). Conversely, LCN DNA typing can be used for victim identification when conventional DNA typing has failed. LCN DNA STR typing was successfully applied to four sets of skeletal remains from World War I, World War II, and the Vietnam War (Irwin et al. 2007). Despite its limitations, an optimal DNA extraction protocol and miniSTR technology are promising tools when analyzing challenging samples (Mameli et al. 2014).

Problems with LCN DNA Analysis DNA analysis and interpretation have several well-known issues, which are amplified when the DNA input is in the recommended range. There are four main problems in LCN DNA analysis: stochastic effects, contamination, replicate analyses, and controls (Budowle et al. 2009). (a) Stochastic effects. Stochastic variation occurs when two assays randomly produce different alleles in the same locus (Butler and Carolyn 2010) (Fig. 4). 1. Stochastic detection threshold. This threshold is an arbitrary value that is generated during laboratory validation assays; above this value, single

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Fig. 4 Stochastic effects in LCN DNA typing

alleles are assumed to be homozygous (Butler 2014). Because LCN DNA analysis usually proceeds under this threshold, it cannot be applied. 2. Allele dropout. The preferential amplification of only 1 allele due to stochastic variations can result in fake homozygotes (Butler 2014). 3. Heterozygote peak imbalance. Heterozygote balance is obtained by the arithmetic division of high- and low-peak areas, theoretically resulting in a value of 1; however, generally, the value ranges from 0.6 to 1.67, and LCN DNA has a greater tendency toward heterozygote imbalance (Kloosterman and Kersbergen 2003). 4. Stutter. Stutters are allelic products with 1 less unit than the associated allele, and in LCN DNA analysis, they can have even larger areas than allele peaks (Gill et al. 2000). (b) Contamination. Contamination during LCN DNA analysis comes from reagents and laboratory consumables, laboratory personnel, cross-contamination between samples, and evidence collection (Budowle et al. 2009). It has to be noted that at equal levels of contamination, this would be more noticeable in low copy number samples than in reference samples due to the great concentration disproportion between the contamination and the reference sample and it would not be detectable in most occasions. (c) Replicate analyses. Because random allele variations might be detected in the same locus, various aliquots of LCN DNA extracts should be amplified and analyzed to determine a consensus profile (Taberlet et al. 1996). (d) Appropriate controls. Given that LCN DNA analysis is (or should be) sufficiently sensitive to detect a single DNA molecule and because the purpose of a negative control is to detect gross laboratory contamination, controls must be redefined to detect laboratory contamination in the detected alleles (Gill et al. 2000). To mitigate the analytical problems that are associated with LCN DNA, several procedural strategies have been developed:

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(a) DNA extraction protocol is crucial. Its efficiency will determine the amount of DNA that is obtained and can even improve situations with low copy number DNA. Various extraction protocols have been compared in LCN DNA extraction, of which multi-silica-based DNA extraction protocols succeed, whereas spin columns fail (Mameli et al. 2014). (b) DNA amplification can determine success in LCN DNA analysis. Given the small amount of DNA input, miniSTR primers have been successfully applied (Mameli et al. 2014). Notably, touch DNA profiles are more likely to be obtained with Identifiler ® Plus due the use of Hot Start DNA polymerase, pre-PCR hold at 95  C, or buffer components (Martin et al. 2018). But, the classical solution to LCN DNA analysis is increasing the cycle number to 34 cycles (Gill et al. 2000): the peak area of stutters and the imbalance in heterozygotes increase compared with amplification using 28 cycles with less than 100 pg of DNA. However, it has been suggested that a combination of cleanup of PCR products, concentration, increased sample loading, and increased injection variables can achieve the same or better results with products from 28 cycles of amplification (Foster et al. 2008). In addition, mtDNA analysis and NGS platforms are suitable alternatives if STR typing is unsuccessful; both strategies analyze SNPs and take the advantage of having smaller amplicon size. (c) Post-PCR purification in LCN DNA increases the sensitivity of the PCR products, for which silica membrane-based columns are the best option versus filtration and enzyme-mediated hydrolysis. Further, electropherogram artifacts were similar, and no contamination was observed (Smith and Ballantyne 2007).

DNA Contamination DNA contamination implies the accidental transference of DNA to a sample (Butler 2005). DNA contamination with exogenous DNA is complicated and can be detected when working with challenging samples. The analysis of forensic DNA contamination is a tremendous challenge, for which there remain two main issues (Yang et al. 2019). The first is a minor contributor that cannot be assessed due to stochastic effects and cannot be interpreted. The second is contamination by more than three profiles, which is difficult to distinguish between all contributors. Sources of DNA contamination will vary, depending on the sample and analysis: both endogenous and exogenous DNAs can be co-extracted or coamplified, or only the contaminating DNA can even be amplified and detected. The high sensitivity of PCR and its ability to amplify LCN DNA can generate problems in the management of challenging samples, necessitating extraordinary measures and validation protocols in the laboratory to avoid sample contamination.

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DNA Contamination Sources It is important to determine the circumstances when contamination can occur to prevent it or, at least, be alert. Samples can be compromised by contamination through three periods by direct deposition or secondary transfer (Lee et al. 1998): 1. Prior or during sample collection. Compromised samples can be found anywhere, and there will be many types of biological evidence on surfaces and common items that can be a source of DNA contamination, known as background or environmental DNA (Fonneløpa et al. 2016). Further, inattention during the collection of the sample by personnel can lead to contamination due to passive transfer from the investigator. 2. During sample preparation. Because endogenous DNA of samples is degraded and at low levels, it is important to clean the sample as much as possible so that exogenous DNA contamination is avoided, to prevent mixture and to preferentially amplify exogenous DNA. The first DNA identification study of skeletal remains pointed out the need to distinguish between exogenous and endogenous DNA when analyzing an ancient DNA profile (Hagelberg et al. 1991). There are three main techniques to prepare samples prior to DNA extraction: surface abrasion, chemical wash, and ultraviolet light. Surface abrasion consists of applying a Dremel-type drill to the bone surface to eliminate most exogenous material (Carracedo 2005). Chemical wash of the bone surface and submersion in diluted bleach has been a controversial topic, because it can penetrate bone pores, affecting endogenous DNA; however, there are contradictory conclusions on this issue (Koehn et al. 2020). Finally, ultraviolet light irradiation (Kaestle and Horsburgh 2002) is also used due its ionizing effect, which fragments exogenous DNA, but it is not useful with irregular surface samples, and if the exposure time is exceeded, endogenous DNA can also be degraded. 3. Post-PCR products. Separation of pre-PCR and post-PCR areas is essential to avoid contamination with post-PCR products. Further, laboratory coats can be contaminated easily with PCR products, for which special and exclusive pre-PCR protective equipment for personnel must be used when working with compromised samples, including whole suits, double gloves, shoe covers, and surgical masks. In addition, the inclusion of negative controls in the analytical process is necessary. Good laboratory practices and adequate cleaning of laboratory equipment and tools are mandatory to avoid environmental DNA and cross-contamination in samples (Scientific Working Group on DNA Analysis Methods 2017). It has been reported that carry-over contamination can be regulated by substituting deoxyuridines (dUTP) for thymidines (dTTP) or by introducing uracil during primers synthesis and by treating starting reactions with UDG (uracil DNA glycosylase) followed by its thermal inactivation. This enzyme cleaves uracil of uracil-containing DNA with no effects on thymine containing DNA. With these

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measures PCR carry-over contamination can be treated if contaminants contain uracil in place of thymine (Longo et al. 1990). Next-generation sequencing (NGS) technologies are powerful tools for validating results in ancient DNA studies. Ion semiconductor sequencing is a more precise and lower-cost tool versus molecular cloning for distinguishing between endogenous and exogenous DNA in ancient bones. The Ion Torrent™ PGM ® has been used successfully to verify results obtained in Sanger sequencing of human chalcolithic teeth with a coverage of more than 1000 copies of mtDNA for the amplified fragments. Furthermore, NGS sequencing arises information about the damage of DNA (Palencia-Madrid and de Pancorbo 2015).

Laboratory Recommendations for the Analysis of Compromised Samples There are several laboratory criteria (Poinar 2003; Pääbo et al. 2004) that have been established for ancient DNA laboratory work, including the analysis of compromised DNA samples: 1. Working in a physically isolated area. To avoid contamination, it is important to work in a separate laboratory that is dedicated exclusively to degraded samples and, if possible, another area where DNA work is not performed. DNA extraction and amplification should be performed in this area, and the following steps should be implemented in daily laboratory tasks. Personnel must wear laboratory coats, face masks, and gloves; equipment should be washed with bleach and irradiated with UV light; negative test should be included with every assay; duplicate PCR assays must be run; and all results should be compared against a staff and investigator database (Gill 2001). 2. PCR amplification control. It is convenient to make periodic checks of amplification with blank samples that contain only water and the necessary reagents for PCR, and every analysis should include a negative control so that contamination can be detected. 3. Molecular behavior. The obtained results should be proportional to DNA degradation index: an entire DNA sequence from ancient DNA samples should not be expected; such a case would likely be contamination rather than a successful analysis. 4. Quantification. DNA copy number in samples must be assessed by real-time PCR or competitive PCR; thus, if the molecule number is under 1000, contamination might be impossible to exclude. 5. Reproducibility. The obtained results should be repeated with the same DNA extracts or other DNA extracts from the same specimen. 6. Clone. PCR product must be verified by sequencing and cloning of amplification products (at least ten) to assess if contamination has occurred and to what extent. 7. Replication. Extraction, amplification, and sequencing of separate samples from the same individual by independent laboratories should achieve the same results.

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8. Biochemical preservation. Ancillary information on DNA conservation can be obtained by measuring the quantity, composition, and degradation of amino acids or other residues. If a forensic analysis is performed, other markers, such as oxidation of fats, should be revised. 9. Associated remains. When working with human samples, if animal remains are found to be associated to them, the DNA should have the same degree of conservation in both types of samples; thus, PCR amplification of animal remains is advised. 10. Phylogenetic sense. The obtained sequences should be interpreted phylogenetically with other known haplotypes to confirm the results, especially when mitochondrial pseudogenes are detected (noncoding mitochondrial DNA contained in nuclear DNA).

Conclusions Forensic DNA analysis developed rapidly in the past several decades; however, there are still unsolved issues when compromised samples are involved (see Table 1). PCR inhibition is caused by two mechanisms: the inhibitor binds to DNA to prevent the reaction, or it inactivates the DNA polymerase. Inhibition must be considered when working with blood or bones or if samples have been exposed to such components as soil humic and fulvic acids. An adequate DNA extraction protocol, purification, or certain DNA polymerases are recommended for these issues, and the use of purification kits and PCR additives is recommended. Table 1 Problems with DNA analysis and possible solutions and recommendations Problem Challenging samples PCR inhibition

Degraded DNA

Low copy number DNA

DNA contamination

Possible solutions Adequate sample selection if possible Laboratory requirements Inhibitor-tolerant DNA polymerases Developed PCR kits Purification kits PCR additives miniSTRs mtDNA analysis NGS DNA extraction protocol DNA amplification Post-PCR purification mtDNA analysis NGS Good laboratory practices Chemical wash Ultraviolet light irradiation NGS

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DNA degradation is caused by several mechanisms: bond cleavage, oxidative damage, cross-links, hydrolytic rupture, and Maillard reactions – all of which are mediated by the activity of UV radiation, certain temperatures, humidity, pH ranges, soil components, and microorganism activity. Mini-STRs, mtDNA analysis, and NGS are possible strategies to be considered. Low copy number DNA is a problem that is related to DNA degradation, for which several applications have been recently explored, such as touch DNA and victim identification. Nevertheless, stochastic effects, contamination, analysis replication, and the use of appropriate controls are problems that can arise when working with LCN DNA. Specific DNA extraction and amplification protocols and post-PCR purification are recommended. Finally, DNA contamination can have different sources: prior to sample collection, during sample handling, or after PCR is performed. For avoiding sample contamination, and as a general guideline for all of these challenges, several laboratory criteria have been created for ancient DNA work: working in a physically isolated area, PCR amplification control, molecular behavior, quantification, reproducibility, cloning, replication, biochemical preservation, associated remains, and phylogenetic sense. Forensic DNA analysis has advance quickly in quite short time, but there are many challenges that remain. The aim of this chapter was to introduce possible solutions that are being applied by forensic scientists. However, more research is needed for addressing the problems that are posed by an even more complex society.

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Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . What Is Validation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Why Validate? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . How to Validate? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Method validation is a small but critical step in a forensic DNA laboratory’s quality assurance system. Performing method validation is not only considered to be good science, to substantiate and to test the integrity of scientific processes in the laboratory, but also adheres to establishing quality standards and guidelines as dictated by standardization bodies. By performing rigorous validation, the forensic laboratory achieves confidence in the reliability of a testing method with respect to defining expert witness testimony and admissibility of forensic DNA evidence in the courts. Despite the publication of international standards and guidelines for validating forensic DNA methods, laboratories often struggle to define their validation scopes, resulting in either insufficient validation or, often, over-validation. The

I. Muharam (*) HID Professional Services, Genetic Sciences Division, Thermo Fisher Scientific, Scoresby, VIC, Australia e-mail: iman.muharam@thermofisher.com C. Paintner HID Professional Services, Genetic Sciences Division, Thermo Fisher Scientific, Singapore, Singapore e-mail: Carla.Paintner@thermofisher.com © Springer Nature Singapore Pte Ltd. 2022 H. R. Dash et al. (eds.), Handbook of DNA Profiling, https://doi.org/10.1007/978-981-16-4318-7_52

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authors here will share several important considerations when performing validations, to support the laboratory’s implementation of new technologies and provide a pathway to achieving international accreditation. Keywords

Forensics · DNA · Validation · Quality assurance · Accreditation · PCR · Rapid DNA · Next-generation sequencing

Introduction Forensic DNA evidence has the potential to change an individual’s life to the extent of imprisonment or, in the case of some jurisdictions, even the sentencing of a death penalty. Forensic DNA testing therefore must be performed to the highest standards and survive any legal scrutiny in court. Each technical process in the forensic DNA work ow must be robust, reliable, and reproducible, as demonstrated through a validation process, and performed, analyzed and interpreted by trained and qualified scientists (Butler 2010). To provide confidence in the DNA evidence, each laboratory must determine which validation studies are relevant to the methodology (SWGDAM 2016). It is the laboratory’s responsibility to scrutinize and review their methods through a rigorous and standardized validation process. Knowledge and expertise on method validation and quality assurance systems are accumulated over a period of time, often as a necessity when the laboratory is undertaking preparations for accreditation under an international standard such as ISO/IEC 17025:2017 that specifies general requirements for the competence of testing and calibration laboratories. Accreditation is the formal recognition, often by an accreditation or standardization body, that a laboratory meets or exceeds a list of standards to perform specific tests (QAS 2020). For many forensic DNA laboratories in Australia, Western Europe, the USA, and other countries, operating under an international standard such as ISO/IEC 17025:2017 has been a fact of life for almost two decades, as this is a requirement without which they would not be able to operate. In many countries, accreditation or compliance against an international standard is mandated by a national agency. Examples of accreditation bodies include the ANSI National Accreditation Board (ANAB) based in North America, National Association of Testing Authorities (NATA) in Australia, and National Accreditation Board for Testing and Calibration Laboratories (NABL) in India. In other parts of the world, the concept of standardization and accreditation is new, with many forensic DNA laboratories in Southeast Asia and South Asia achieving accreditation in recent years, and many others are currently in progress. Obtaining accreditation can be a lengthy and expensive process and therefore demonstrates the laboratory’s commitment to quality. Regardless of whether a testing laboratory is officially accredited or not by a standardization body, all forensic DNA laboratories should at minimum be performing good, responsible science, either through following accepted relevant forensic guidelines or standards or operating under Good Laboratory Practice

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(OECD 1998), with the vision to eventually be capable of obtaining accreditation under an internationally accepted standard. A small but significant step in the process of becoming accredited is for the laboratory to test and evaluate their procedures, to determine their efficacy and reliability for forensic analysis through the process of validation.

What Is Validation? According to ISO/IEC 17025:2017, validation is defined as the provision of objective evidence that a given item fulfils specified requirements, where the specified requirements are adequate for an intended use. This standard is broad in scope, as it applies to general testing or calibration laboratories, and not specifically forensic DNA testing facilities. For definitions that are more relevant to forensic DNA testing, we can refer to either the Federal Bureau of Investigation Director’s Quality Assurance Standards for Forensic DNA Testing, or the Scientific Working Group on DNA Analysis Methods Validation Guidelines for DNA Analysis Methods: “Validation is a process by which a method is evaluated to determine its efficacy and reliability for forensic casework analysis. . .” (QAS 2020) “Validation is a process by which a procedure is evaluated to determine its efficacy and reliability for forensic casework and/or database analysis.” (SWGDAM 2016)

The QAS and SWGDAM documents are two of the most comprehensive documents that standardize and guide the forensic DNA laboratory’s validation and implementation efforts and often are referenced as critical resources as part of the laboratory’s quality assurance system. The QAS document specifically describes the requirements that laboratories performing forensic DNA testing or using the Combined DNA Index System DNA database in the USA must follow, to ensure the quality and integrity of DNA evidence generated by the laboratory. The standards were recently updated to include rapid DNA testing, next-generation sequencing, and use of interpretation software. The SWGDAM document provides guidelines on the validation process itself, that is consistent in its messaging with the QAS document, that the laboratory should or may follow. Due to their focus on forensic DNA and their acceptance throughout the international forensic DNA community, these two documents are often used by the forensic DNA laboratory to provide a more granular and standardized approach to meet ISO/IEC 17025:2017 requirements around implementing new methods or technologies. Though not discussed in detail in this chapter, globally there are various standards and guidelines utilized by the forensic community and accreditation bodies. In this chapter, we will highlight the requirements and recommendations from the QAS and SWGDAM documents. It is important for your laboratory to identify the appropriate standards and guidelines based on your governmental or regional requirements.

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For the laboratory’s purposes, validation is performed to test a method for its reliability, limitations, and adequacy to suit its intended purpose. Validation minimizes reinvention of methods in different laboratories. Methods that have been validated are also more readily accepted by the scientific community, more likely to have already undergone scrutiny in a court, and allow results from different laboratories to be compared, either between branch laboratories in a domestic setting or across countries, as crimes are not often limited by international borders. There are two types of validation required to implement or modify forensic DNA testing methods: developmental validation and internal validation. Developmental validation is the acquisition of test data and determination of conditions and limitations of a new or novel DNA method for use on forensic samples, whereas internal validation is an accumulation of test data within the laboratory to demonstrate that established methods and procedures perform as expected in the laboratory (QAS 2020). Developmental validation pertains to the rigorous series of tests performed on a new method or technology preceding implementation. As an example, a new commercial STR (short tandem repeat) PCR kit must be developmentally validated prior to commercial release. Depending on the method or technology, developmental validation studies include characterization of the genetic marker, species specificity, sensitivity studies, stability studies, case-type samples, population studies, mixture studies, precision and accuracy studies, and PCR-based studies (QAS 2020), with the underlying scientific principles of the technology to be published in a peerreviewed publication that is accessible to the community (SWGDAM 2016). A DNA laboratory can refer to another laboratory’s developmental validation studies to reduce the need to reinvent the wheel. This assists with the uptake of new methods and technologies, reducing the burden on each laboratory’s implementation process. To implement a new technology in the laboratory, however, internal validation must be performed prior to using a procedure for forensic DNA applications. To put it simply, a developmental validation is performed by the vendor or creator of a new method, whereas an internal validation is performed by someone using that method in their laboratory. Although not as extensive as developmental validation, internal validation studies include similar experiments such as sensitivity and stochastic studies, precision and accuracy studies, mixture studies, known and non-probative sample studies, and contamination assessment studies (QAS 2020), often building on the developmental validation data. Additionally, an internal validation may accumulate data points that are specific for that laboratory’s testing environment, population samples, or other local conditions, ensuring that the method remains reliable and performs as expected. A validated internal protocol will have an associated validation report detailing the validation process for the specific protocol that can be reviewed by internal stakeholders and external quality assessors. Although there is no requirement to publish an internal validation in a peer-reviewed publication, laboratories are encouraged to disseminate their internal validation by sharing a presentation or poster in a scientific meeting. Again, this approach allows forensic DNA analysts to learn the findings from other laboratories and speed up validation and implementation of that method in their own lab. Importantly, the

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validation findings will form the foundation of the laboratory’s standard operating procedure (SOP) for that specific method, as attributes such as limitations of the method would be determined. Validation should be distinguished from other method-assessment processes, such as performance check or evaluation. A performance check is typically required when a laboratory obtains an additional critical instrument, of the same model already validated in the laboratory, prior to use in the routine work ow. The intent of a performance check is generally to assess a new piece of critical instrument for similarities and differences compared to the originally validated instrument. The performance check studies are usually limited to studies that highlight instrument performance (contamination, sensitivity, precision, and accuracy studies) versus the performance of samples or assays (mixture, non-probative studies). For example, if the laboratory performs their capillary electrophoresis on an Applied Biosystems 3500xL Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA) that they have validated internally and, due to an increase in their sample throughput, decides to bring online an additional 3500xL Genetic Analyzer, then the new unit does not undergo the full validation process but instead will be subjected to a smaller-scale performance check study. A performance check may also be performed when relocating a critical instrument to a new location or another part of the laboratory (Iyavoo et al. 2017). Other critical instruments that will require performance checks include robotic systems, thermal cyclers, and other equipment that perform DNA typing results (QAS 2020). An evaluation is typically an assessment of a modified procedure in comparison to the original procedure, or other procedures, using similar DNA samples. For example, results from a number of DNA extraction methods are compared, using similar DNA samples and conditions, to determine which is more suitable for an intended task, such as success rate with low-level DNA samples (Sturk-Andreaggi et al. 2011), or performance across DNA yield and inhibitor removal (Zimmermann et al. 2009). Although the detailed criteria encountered in a validation are not a prerequisite in these processes, both performance checks and evaluations should be sufficiently documented, reported, and available for review. Depending on the application or intended use, the laboratory may decide to expand the performance check or evaluation testing to an internal validation (Iyavoo et al. 2017). Both the QAS and SWGDAM documents are specific in their language and provide clear guidance on what scenarios require which scope of testing. In this chapter, we will be reviewing the concept of validation as it pertains to internal validation that is relevant to the forensic DNA laboratory in implementing a new method or technology.

Why Validate? Validations are everywhere. Unconsciously, we perform validations outside of the laboratory daily, without having a second thought, before making certain decisions. These tests or studies are vital in helping us reach well-educated, reliable results in

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Table 1 Examples of approaches possible when purchasing a new vehicle Your approach Chose a new vehicle at random, not bothering to take it for a test drive Chose a new vehicle at random and quickly test drive down the road

Research various makes/models of vehicles and thoroughly test drive to compare

Your accumulated evidence The vehicle was tested at the factory. Vehicle features were not personally researched or tested. Quick test drive to test time taken to reach 100 km/ hr. Assess comfort of the seats. Not all vehicle features were not researched or tested. Test the car at low, average, and high speeds. Ensure that the features of the car meet your daily driving needs that are important to you, for example, safety features, gas mileage, air conditioning, etc.

our personal lives. Sometimes, these studies can impact the lives and safety of not only ourselves but also our loved ones. Consider the scenario of a long day at work. After spending the entire day in the laboratory, performing validation studies to implement a new STR kit, you are relieved to call it a day and make your way home to your family. Unfortunately, you are so focused on thinking about the validation results you generated today, you lose control of your car and collided with a tree. Thankfully you are safe and no one is injured, but your car is badly damaged and no longer drivable. After a short insurance claim process, you are now ready to purchase a new car. Reviewing the approaches in Table 1 below, which one would you most likely follow? The sensible approach that most of us will select would be the last option. When making an important decision, we often compare advantages and disadvantages to perform an analysis on specific metrics that are important to us. In the example above, when choosing a new car to purchase, we consider many aspects and features, from safety and security to gas mileage and comfort features, such as air conditioning and heated seats. We need a vehicle that is not only comfortable for the long drives but also, perhaps more importantly, ensures that the occupants safely reach their destination. A car is also a big investment, in some cases costing more than a house, so we rightfully take our time to perform the necessary assessments to ensure we are satisfied with our final decision. If we were not to undertake these assessments, it would be of no surprise if we ended up at the beginning again, with a non-drivable car. In the context of a validation, the car can be equated to a method or technology (e.g., a new STR kit), and our assessments, including testing at low, average, high speeds and assessing safety features, are our validation studies. When implementing a new STR kit, it is not advisable to purchase the kit and immediately start using it to amplify forensic DNA samples without any prior testing. Performing a small test of amplifying a subset of ten samples prior to implementation is a minor improvement but most likely not sufficient for the laboratory to understand the complexity and limitations of the kit. Only through performing a thorough validation study using

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Table 2 Example approaches when implementing a new STR kit, with only the last approach satisfying international standards and guidelines Your approach Immediately amplify samples with the new kit

Amplify a small subset of samples and implement the new kit

Perform thorough validation studies and generate protocols based upon data generated

Your accumulated evidence You use all the data generated and apply the protocol in your laboratory. Although instruments and environments were different, at least they were tested somewhere. Assess the new kit using a few samples within your laboratory. Shows that profiles were generated, so approved for use on casework samples. Test the dynamic range of the kit on each instrument. Appropriate samples/controls are concordant. Results are accurate, reliable, and repeatable.

standardized methods, followed by critical analysis of the data and presenting the data in a report for critical review by other stakeholders, will provide the laboratory with enough confidence to implement the new technology. The validation results would then be used to form the foundations of a new SOP and associated training module. Table 2 below compares various approaches a laboratory may take when implementing a new STR kit, but only the last approach would satisfy international standards and guidelines. Validations enable the laboratory to gain confidence and quality in their laboratory processes and to confirm that a method is robust and reliable and the performance is reproducible under differing reasonable scenarios (e.g., when performed by different operators or using different sets of calibrated pipettes). Validation data can be compared against results published or presented elsewhere, against data from other laboratories, therefore showing consistency in results between labs. The accumulated validation evidence provides the forensic DNA analyst with confidence in the evidence that they are reporting to the court, for use in the justice system to convict a suspect or exonerate an innocent person. By validating methods and technologies, the laboratory is providing the best possible testing service for their clients, the people, and the public in their jurisdiction. In many countries, validation is a key requirement of the ISO/IEC 17025:2017 international standard and must be completed for all methods and protocols in use in a forensic DNA laboratory, in order to obtain accreditation. There is no option to not perform a validation, because this can result in the laboratory losing their accreditation and therefore their right to perform the laboratory testing services. Laboratories that fail to complete appropriate validation testing may also be at risk for loss of funding, such as institutional grants or governmental subsidies. Occasionally, a laboratory may find itself having performed inadequate validation, resulting in an over ow impact on the appropriateness of their protocols, analysis guidelines, and even internal training programs. In the best-case scenario, the laboratory’s quality assurance system will recognize this gap and trigger a reassessment of the validation and identify what additional work may be required. To reestablish the confidence of

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not only the analysts but also the public and the court, a laboratory may need to repeat portions of the validation. The cost of this exercise can be large, depending on the revalidation scope, but the cost to the lab may be much larger if the revalidation is not performed, or if a validation is not conducted correctly in the first place. In the worst-case scenario, for example, inadequate validation can result in inaccurate results and miscarriage of justice, resulting in arrest and imprisonment of an innocent suspect (or conversely, the inadvertent release of a criminal back into the general population). These outcomes can result in media attention and international scrutiny, in many cases resulting in lab closures, cost to lab morale or reputation, and even imprisonment of the forensic DNA analyst. The recent report of the Texas Forensic Science Commission (2018) provides an unfortunate example of the impact on the laboratory when the quality assurance system breaks down and weaknesses were identified in the lab’s contamination control procedures and training programs.

How to Validate? Although many forensic DNA laboratories are accustomed to performing validations, the majority still struggle with the concept due to lack of time, resources, and sometimes knowledge (e.g., if their validation “expert” has left the lab). During periods of transformation, when laboratories adopt newly accepted technologies, such as rapid DNA, next-generation sequencing, or probabilistic genotyping, some laboratories can often find themselves in a continuous cycle of validating, or heavy investment in research because of their unfamiliarity with the new technology, and therefore confusion in determining which validation studies are appropriate. These situations may result in the laboratory experiencing validation fatigue, causing lengthy delays in implementing the new technology. For laboratories that have very little or almost no experience in validation, validating a technology is approached with caution and concern, due to lack of experience and in-house expertise. In either scenario, labs may wind up either under- or over-validating. When creating an experimental design for a validation, performance check, or any other scope, it is important to first have a clear understanding of what standards the laboratory will need to meet. Throughout this chapter, we have focused on the FBI Director’s Quality Assurance Standards and ISO/IEC 17025:2017 documents and therefore will continue to reference these standards. To have clarity on the requirements, firstly, laboratories will refer to the relevant audit checklist documents that assessors will actually use as they perform the accreditation audit and will either assess the lab as being compliant or not, or whether a particular clause does not apply. Secondly, the lab will need to determine how to perform the relevant studies required. Unfortunately, the standards only define what studies must be performed, and not the details of how. To answer the how, laboratories often refer to other supplementary sources of information or guidelines, such as the SWGDAM Validation Guidelines for DNA Analysis Methods (SWGDAM 2016) or Validation and Verification of Quantitative and Qualitative Test Methods (NATA 2018), formerly known as Technical Note 17 and most relevant in Australian testing facilities. These

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guidelines provide the granularity on the validation process, by defining the terminologies used and outlining the experiments that should be performed, including specifics on what data need to be defined and sometimes what samples need to be analyzed. For an internal validation, SWGDAM recommend the following studies: known and non-probative evidence samples or mock evidence samples, sensitivity and stochastic studies, precision and accuracy, mixture studies, and contamination assessment (SWGDAM 2016). Further to that, SWGDAM specifies that the sensitivity and stochastic studies should demonstrate sensitivity levels of the test, by determining the dynamic range, ideal target range, limit of detection, limit of quantitation, heterozygote balance, and signal-to-noise ratio (SWGDAM 2016). SWGDAM does provide exibility for the laboratory to determine the suitability of each study, based on the methodology under examination, and may determine that a particular study is not required and also provides the laboratory with the freedom to evaluate the appropriate sample number and types (SWGDAM 2016). This helps to ensure that the laboratory’s validation is always fit for purpose and as per required to demonstrate the reliability and potential limitations of that specific method. Often, a laboratory still finds it challenging to start their validation project and prefers to seek expert support or outsource their validation project to a consultant with deeper knowledge, who can guide the laboratory on their validation. Alternatively, the lab may work with a vendor to create a bespoke validation support solution that includes providing experimental designs and personnel to perform the actual laboratory work, data analysis, and report writing. The Human Identification (HID) Professional Services (HPS) team from Thermo Fisher Scientific was set up in 2007 to address validation support required by forensic DNA laboratories in the USA. Since then, HPS team has performed more than 700 projects across 36 countries, including Singapore, Thailand, Japan, India, Scotland, Italy, Brazil, Mexico, South Africa, United Arab Emirates, and Australia. The HPS validation scope follows the QAS requirements and SWGDAM guidelines, to help the laboratory achieve ISO/IEC 17025:2017 accreditation, by providing a standardized experimental design together with the kits, reagents, consumables, and expert personnel to perform the laboratory work and generate the requisite data. Data analysis and report writing are completed by trained and experienced personnel, with the results provided back to the laboratory in a comprehensive validation report, followed by teachback training to transfer the validation knowledge to the DNA analyst. This type of solution provides the laboratory with a standardized implementation program with defined studies, timelines, and costs and ensures the validation project does not extend unnecessarily for an unpredictable timeframe or result in a budget blowout. An example of a standardized scope for validating an STR kit, as developed by HPS, is presented in Table 3 below. Also highlighted in this table is the QAS section addressed by each study and how the lab may use those results as part of their validation, foundation for SOPs, or data analysis and interpretation guidelines. For most laboratories, the scope above provides an adequate number of data points to validate a commercial STR kit such as the GlobalFiler™ IQC PCR Amplification Kit (Thermo Fisher Scientific, Waltham, MA, USA). The scope has been optimized to allow data from the same sample set to be used to address more

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Table 3 Example of a standardized scope for validating a commercial STR PCR kit (such as GlobalFiler™ IQC PCR Amplification Kit on an Applied Biosystems 3500xL Genetic Analyzer), including the QAS that is addressed and how the laboratory may use the data for their validation and SOP. This scope was developed by HID Professional Services (Thermo Fisher Scientific). Validation study Minimum threshold and contamination study

Sensitivity and stochastic study

Precision study: repeatability and reproducibility

Scope Negative amplification controls are analyzed at 1 RFU to determine noise of instrument and chemistry. Maximum, average, standard deviation, average plus three standard deviations (limit of detection), and average plus ten standard deviations (limit of quantification) are calculated to determine minimum thresholds to be used for analysis. Contamination is assessed in negative controls and blanks using the minimum thresholds. A genomic DNA dilution series (4, 2, 1, 0.5, 0.25, 0.125, 0.063, 0.031, and 0.016 ng) is amplified in triplicate. Average peak heights, peak height ratios, dropout of alleles/loci, peak height of surviving sister allele, and interlocus balance are assessed. Repeatability of size variation is assessed using allelic ladders injected four times; standard deviation of allele size is calculated by injection, by capillary, and for all data together and evaluated for values greater than 0.15 bp. Repeatability of peak heights is assessed using replicate samples from the sensitivity and stochastic study dilution series. Reproducibility of peak heights is assessed using Control DNA 007.

QAS audit 8.3.1(5)

How this study can be used Assist in the determination of an analytical threshold. Determine how to address instances of contamination.

8.3.1(3)

Establish analytical and stochastic thresholds Develop interpretation guidelines. Evaluate input amounts that minimize pull-up peaks, off-scale data, and other artifacts.

8.3.1(2)

Establish protocols for the number and placement of allelic ladders on plates for injection. Understand variation in average peak heights for samples with the same DNA input amounts.

(continued)

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Table 3 (continued) Validation study

Accuracy study

Known and non-probative sample study

Mixture study

Assessment of non-allelic peaks

Scope Reproducibility of genotypes is assessed using Control DNA 007 and NIST Standard Reference Material™ 2391d Component. NIST Standard Reference Material™ 2391d Component is amplified with 1.0 ng DNA target input. Genotype results are compared to the certificate of analysis 20 known and non-probative samples are supplied by the laboratory and amplified with 1.0 ng DNA target input. Samples are analyzed for concordance and expected performance when compared to previous results supplied by the laboratory. Male-female genomic DNA mixture series (1:0, 1:1, 1:2, 1:4, 1:10, 1:20, 1:40, 1:80, 0: 1) is amplified at 1.0 ng target input. Peak heights are analyzed for mixture ratios and presence, absence, or masking of minor component alleles. Samples are assessed for the following PCR artifacts: minus stutter, plus stutter, incomplete adenylation, and dye artifacts. Samples are assessed for capillary electrophoresis artifacts such as pull-up.

QAS audit

How this study can be used

8.3.1(2) and 8.4

Demonstrate compliance with Standard 8.4 of the Quality Assurance Standards for Forensic DNA Testing Laboratories

8.3.1(1)

Identify parameters for the STR kit, including the amount of input DNA, analytical and stochastic thresholds, and interpretation guidelines. Establish standard operating procedures.

8.3.1(4)

Establish protocols for data interpretation and statistical analyses, including the mixture ratios of components in mixed DNA samples and the amount of DNA to amplify.

N/A

Establish protocols for data analysis and mixture interpretation. Determine whether the default marker-specific stutter filters are appropriate.

than one validation study. For example, the sensitivity study sample set, consisting of genomic DNA dilution series from 4 ng input to 0.016 ng input amplified in triplicate, is used to assess peak height ratios for heterozygous loci (Fig. 1), interlocus balance (Fig. 2), and repeatability of peak heights (Table 4). The peak height ratio data show the peak balance for heterozygous loci across different DNA input amounts (Fig. 1). These results show that peak imbalance increases

Input Rep D3S1358 vWA D16S539 AMEL D8S1179 D21S11 D19S433 TH01 FGA D5S818 D13S317 D7S820 SE33 D10S1248 D1S1656 D12S391 D2S1338 DNA (ng) 1 91% 90% 95% 85% 96% 95% 89% 99% 86% 98% 89% 91% 90% 99% 94% 85% 95% 4 2 81% 95% 94% 96% 98% 98% 98% 100% 85% 97% 94% 88% 84% 93% 87% 100% 99% 3 88% 94% 86% 79% 96% 99% 91% 96% 97% 93% 98% 89% 86% 87% 85% 87% 98% 1 96% 92% 98% 88% 92% 98% 99% 95% 97% 97% 96% 97% 81% 96% 89% 96% 99% 2 2 97% 94% 81% 83% 82% 83% 94% 98% 98% 95% 100% 89% 93% 97% 94% 95% 93% 3 86% 88% 97% 82% 95% 94% 99% 90% 87% 90% 95% 88% 94% 93% 86% 87% 91% 1 87% 93% 94% 94% 94% 89% 71% 88% 92% 97% 94% 74% 92% 96% 91% 78% 86% 1 2 95% 94% 99% 65% 86% 87% 88% 99% 99% 90% 95% 94% 87% 76% 96% 78% 98% 3 99% 98% 91% 81% 77% 88% 98% 90% 91% 94% 86% 96% 91% 86% 87% 96% 98% 1 77% 90% 80% 95% 78% 81% 100% 98% 88% 94% 85% 97% 92% 86% 95% 75% 86% 0.5 2 94% 91% 74% 93% 97% 82% 90% 81% 92% 99% 93% 86% 82% 89% 82% 98% 76% 3 87% 96% 92% 89% 95% 92% 87% 91% 75% 100% 88% 90% 99% 97% 92% 92% 79% 1 76% 73% 92% 90% 66% 85% 84% 96% 80% 94% 77% 88% 92% 85% 86% 81% 57% 0.25 2 88% 99% 68% 94% 88% 62% 79% 85% 99% 91% 90% 97% 68% 51% 88% 82% 51% 3 84% 76% 100% 82% 99% 71% 94% 79% 67% 73% 98% 85% 96% 98% 77% 97% 62% 1 73% 62% 62% 80% 81% 83% 51% 96% 98% 88% 66% 93% 79% 71% 69% 76% 98% 0.125 2 63% 80% 43% 67% 72% 84% 98% 56% 94% 70% 87% 95% 57% 71% 50% 65% 88% 3 98% 62% 86% 83% 87% 100% 62% 86% 97% 72% 83% 59% 80% 80% 87% 64% 93% 1 79% 55% 53% 75% 99% 23% 55% 45% 56% 91% 81% 64% 78% 97% 80% 89% 61% 0.063 2 92% 61% 97% 46% 99% 37% 84% 80% 97% 56% 85% 83% 47% 81% 64% 59% 60% 3 65% 51% 71% 62% 73% 68% 92% 42% 83% 59% 85% 63% 78% 55% 89% 95% 72% 1 96% 33% 54% 53% 79% 58% 21% 58% 24% 83% 29% 58% 89% 81% 41% 86% 54% 36% 98% 92% 18% 65% 90% 43% 18% 14% 24% 38% 17% 60% 0.031 2 41% 57% 60% 3 57% 57% 91% 37% 99% 70% 13% 35% 83% 41% 91% 69% 48% 93% 56% 63% 1 55% 38% 51% 32% 43% 53% 30% 52% 29% 59% 69% 28% 73% 2 94% 76% 38% 40% 84% 55% 37% 32% 67% 13% 97% 24% 63% 67% 57% 97% 0.016 3 73% 78% 22% 29% 80% 98% 39% 54% 86%

Fig. 1 Example of peak height ratio data for heterozygous loci, observed after amplification of the sensitivity study DNA dilution series, in triplicate, when validating the GlobalFiler™ IQC PCR Amplification Kit

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as the input DNA amount is reduced, especially once the input is within stochastic range or less than 0.1 ng. In this example data set, peak height ratios were generally above 70% with input amounts of at least 0.5 ng of DNA and above 50% with input amounts below 125 pg, demonstrating variability associated with low-template stochastic amplification effects, as evidenced by peak height ratios as low as 13% and/or allelic dropout. In addition, when graphing the heterozygous peak heights for each marker in the replicate amplifications of the sensitivity study DNA dilution series, we observe the interlocus balance is uniform across the DNA profile at higher input DNA amounts but

Fig. 2 Example of interlocus balance data obtained after amplification of the sensitivity study DNA dilution series, in triplicate, when validating the GlobalFiler™ IQC PCR Amplification Kit Table 4 Example of data showing repeatability of peak heights from amplification of the sensitivity study DNA dilution series, in triplicate, when validating the GlobalFiler™ IQC PCR Amplification Kit Input DNA (ng) 4 2 1 0.5 0.25 0.125 0.063 0.031 0.016

Replicate 1 16,293 12,504 5608 2702 1416 791 375 206 72

Replicate 2 15,995 10,112 6510 3668 1605 869 442 166 106

Replicate 3 16,740 11,467 5587 3341 1867 634 410 148 96

Average (RFU) 16,343 11,361 5902 3237 1629 764 409 173 91

Standard Dev (RFU) 375 1200 527 491 226 120 33 30 18 Average:

RSD (%) 2 11 9 15 14 16 8 17 19 12

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less so when the input is in stochastic range (Fig. 2). This data set also aids in setting a stochastic threshold at which it is reasonable to assume that sister allele dropout does not occur. The average heterozygous peak heights calculated for the three replicate amplifications of the DNA dilution series were generally similar at all levels of DNA not affected by stochastic effects, demonstrating repeatability of the GlobalFiler IQC kit, with an average 12% variation among replicate samples across all dilutions (Table 4). Based on these results, the laboratory has gained objective data that optimal sensitivity is seen when the target DNA input amplified under standard conditions ranges between 0.5 ng and 1 ng, resulting in optimal peak heights for interpretation of reliable and reproducible DNA profile data. This example might come across as a massive leap from the question of “Why do you need to validate?” to “How do I validate?” If the laboratory is new to the concept of validation, or has not performed validations according to the standards reviewed here, the lab may attempt to devise an approach for smaller-scale validation or performance check projects and, within that, to focus on a subset of more critical studies first and phase in remaining studies over a number of weeks or months, depending on the time available. For example, a laboratory may elect to perform studies that provide them with an appreciation of the baseline performance of their new kit, method, or critical instrument, by focusing on experiments that utilize more standardized or controlled sample sets, such as contamination assessment, precision and accuracy, sensitivity, and stochastic studies that can be achieved using control DNA, allelic ladder, or purified genomic DNA samples. These fundamental studies can provide the laboratory with some immediate insight into the state of their laboratory or the system under investigation, such as: • A precision study based on injecting allelic ladder samples multiple times and examining the variation in the resulting base pair sizes over a period of time can assist in showing the laboratory whether the environmental operating temperature allows collection of data with high consistency and robustness. • A sensitivity study utilizing a range of input DNA can provide the laboratory with objective data on the performance of their system. • A contamination assessment study can identify the presence or absence of either systemic or sporadic contamination in the laboratory and therefore trigger improvements in contamination management. As the laboratory gains confidence, the studies can expand into additional experiments that utilize laboratory-type samples and therefore may aide the lab in creating SOPs, establishing thresholds, or improving data analysis and interpretation guidelines, such as: • A known and non-probative study covering sample types that are routinely processed in the lab can help to demonstrate performance of routine samples using the new method.

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• A mixture study can help to elucidate limitations in the system when dealing with samples containing multiple contributors at different mixture ratios and inputs, resulting in better analysis and interpretation guidance for the DNA analysts. Below are additional suggestions that laboratories can consider as they prepare, plan, and execute their validation projects. 1. Ensure your laboratory has essential infrastructure in place. Forensic DNA facilities must have secure and controlled access, with pre-PCR techniques such as evidence examination, DNA extraction, and PCR setup to be performed at separate times or separate spaces, and separate again from post-PCR areas to reduce contamination risk (QAS 2020). Guidelines exist for the planning, designing, and construction of forensic DNA laboratories (NIST 2013) that can assist in ensuring the facility meets international standards. Before embarking on implementing a new method or technology, such as mitochondrial DNA sequencing using a next-generation sequencing platform such as the HID Ion GeneStudio™ S5 Prime System (Thermo Fisher Scientific, Waltham, MA, USA), the laboratory may need to consider modifying existing space or adding an additional section that will allow proper testing and analysis of mitochondrial DNA, as this work ow requires further contamination control measures to prevent double cross-contamination (NIJ 2018; Edson et al. 2004). In addition to the laboratory facility itself, the forensic DNA staff are also an integral part of the functioning of the laboratory. The QAS (2020) specifies that laboratory personnel shall have the education, training, and experience required to examine forensic evidence and provide testimony and further outlines minimum educational requirements, such as a minimum of a bachelor’s degree, with at least nine semesters covering biochemistry, genetics, and molecular biology. To undertake an analyst role, the personnel requires 6 months of forensic human DNA laboratory experience and to have successfully completed the required internal training program with verification and approval by the technical leader (QAS 2020). Without a critical understanding of forensic DNA analysis and the effect of data quality on analysis and interpretation, the validation project will suffer from a lack of technical leadership. Moreover, in preparing for the validation, the laboratory should ensure they have appropriate PPE, calibrated pipettes, filtered pipette tips, and only use new, unopened, unexpired, and correctly stored kits, reagents, and consumables to safeguard the validity of the validation results obtained. An internal staff elimination database will also be beneficial to rule out laboratory staff as being a source of any contaminating peaks (Forensic Science Regulator 2020; OSAC 2019). 2. Plan your project as a team. In our experience, the success of any project is dependent on the planning that was performed in preparation for the project. Before starting the planning process, consider the project management tool that will best meet your needs. Basic software packages available on most computers, such as Microsoft ® Excel, can be a good starting point and is powerful enough to

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generate plans, schedules, and Gantt charts. If the laboratory is working on larger or more complex projects, various commercial project management software packages can be purchased for installation on a computer, or a subscription can be purchased for online project management platforms, such as Smartsheet (www.smartsheet.com), that can be accessed over the Internet using a browser on your computer or even through a mobile phone app. These software tools may include further functionalities, such as creation of project dashboards, sending alerts/notifications, and even tracking your budget. Projects are rarely an individual undertaking and usually involve a group of people with unique skill sets to address different project requirements, so it is important to define the project team and assign roles and responsibilities. For example, the project manager may be the laboratory manager, and the actual laboratory work is performed by a technician, with data analysis performed by a case reporting scientist and report writing by a scientist from the quality team. The laboratory should think about the project team holistically, starting from project conception to eventual implementation, and consider the stakeholders throughout. A responsibility assignment matrix may be beneficial as well, so the team can associate tasks within the project with at least one role based on who is responsible and accountable and needs to be consulted or informed (abbreviated as RACI). This ensures that the team members have clarity on their roles within the project, who to consult when support is required, and who needs to be informed once the project is completed and implementation can begin. 3. Don’t reinvent the wheel. If the laboratory is performing internal validation, this means that the method has already had developmental validation performed by the vendor and most likely other laboratories have performed internal validation, and possibly published their work. When performing a validation, it is always useful to relate the validation studies back to the vendor’s development validation. The vendor’s validation is usually available as part of the method’s user guide or manual and additionally published in a peer-reviewed journal. Commercial products such as the GlobalFiler™ PCR Amplification Kit undergo this process regularly (Ludeman et al. 2018). By reviewing these publications, the laboratory can become aware of the work already performed. This means that the lab can review previous data and identify any gaps that they may want to address in their own validation. For example, a species specificity study is performed in the developmental validation but often only utilizes common primates, such as chimpanzee, and common domesticates, such as dog, cat, and mouse, and a limited number of bacterial pools (Ludeman et al. 2018). Based on experiences with their own routine sample types, a laboratory may expand the species specificity study to include additional species relevant to their work ow within their own environment or geographical location. For example, additional humanassociated microbial species may be examined due to their prevalence in common forensic samples analyzed in a specific laboratory (Martin et al. 2014). In the case of newer technology, such as rapid DNA testing using the Applied Biosystems RapidHIT™ ID System (Thermo Fisher Scientific, Waltham, MA, USA) or nextgeneration sequencing on HID Ion GeneStudio™ S5 Prime System (Thermo Fisher Scientific, Waltham, MA, USA), there is often a lack of published

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validation guidelines; the vendor’s developmental validation is the best source of information on what validation studies are relevant and what results can be expected (Salceda et al. 2017; Cihlar et al. 2020). Almost all journal publications will include the details of a corresponding author, who can be contacted should the laboratory wish to request further information, to share ideas, or simply to connect and collaborate on future projects. By reviewing the literature, a laboratory can consider approaches taken by other laboratories and create a validation plan that is fit for purpose. 4. Review the validation data holistically and make sure to understand the limitations of the system. When analyzing a complex DNA profile with multiple contributors, a forensic DNA analyst is taught to consider the data in the entire profile and not just a small number of loci (SWGDAM 2017). Similarly, a validation project consists of multiple studies with data sets that build on top of each other and should be considered in totality, with no particular piece of data scrutinized on its own. To calculate analytical and stochastic thresholds, a laboratory will not only need to consider baseline noise obtained from the contamination assessment study from amplifying negative controls but also consider the change in baseline noise when DNA is present in the PCR, average heterozygous peak heights across different DNA input amounts, peak imbalance, performance with case-type samples, and ability to characterize artifacts and differentiate between true peaks and artifact peaks. These additional considerations utilize data from the sensitivity and stochastic study, known and non-probative study, and assessment of non-allelic peaks. The laboratory should make a conscious effort to compare the results obtained against observations in the developmental validation or other published material, to ensure that the method is performing as expected and adequate for an intended use. Further to that, a method or technology will have inherent limitations, just like any other system, and this must be re ected when designing the validation experiments. Through appropriate experiment planning and analyzing the validation data objectively, the laboratory can not only determine the optimal conditions that will result in the best data quality but also understand what impact any adverse conditions may have on their data. For example, most commercial STR kits are optimized for a target DNA input of 0.5 ng to 1 ng amplifiable DNA; however, the lab should understand what to expect when either lower or higher amounts of DNA are amplified instead. Typically, lower DNA input (below 100 pg of total DNA) will result in stochastic effects and increased observations of peak imbalance, allelic dropout, and allelic drop-in, whereas higher input (above 2 ng total DNA) results in an increase in both PCR- and instrument-based artifacts, such as stutter and pull-up. Finally, ensure that the completed validation report is reviewed by key stakeholders in the project, who possibly come with different experiences or from different roles throughout the lab, so they can examine the results from their own lenses specific to their roles. This allows the validation to be reviewed from different perspectives and ensures the eventual implementation considers any concerns or requirements from all sections of the laboratory. Through this alignment across the lab, the implementation process will be smoother, and the benefits of the new method can be realized by all staff members.

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Fig. 3 Internal validation and laboratory accreditation lead to laboratory excellence

Through performing an effective validation process, the laboratory can confirm the robustness, reliability, and reproducible performance of their method, providing confidence in reporting the results for use in the justice system. When combined with a quality assurance system that is independently audited against international standards and accredited by a standardization body, a laboratory shows commitment to quality and performance excellence, as illustrated in Fig. 3. All forensic DNA laboratories, regardless of the absence of any national requirement for accreditation, should strive to work toward the highest standards and continue to drive adoption of forensic DNA testing as an important and effective crime fighting tool.

Conclusion Performing a validation may appear to be a complicated process; however, standards and guidelines exist and are well accepted in the forensic community. Forensic DNA laboratories are encouraged to standardize their approach to validating new methods and technologies and collaborate or connect with vendors and other laboratories to perform effective validation testing. The validation process ensures that methods are standardized, comparable, and fit for the purpose and is therefore a critical part of the laboratory’s quality assurance system.

References Butler JM (2010) Fundamentals of forensic DNA typing, validation. Elsevier, San Diego, p 300 Cihlar JC, Amory C, Lagacé R, Roth C, Parson W, Budowle B (2020) Developmental validation of a MPS work ow with a PCR-based Short amplicon whole mitochondrial genome panel. Genes 11:1345. https://doi.org/10.3390/genes11111345 Edson SM, Ross JP, Coble MD, Parson TJ, Barritt SM (2004) Naming the dead – confronting the realities of rapid identification of degraded skeletal remains. Forensic Sci Rev 16:63–90 Forensic Science Regulator (2020) Code of practice and conduct protocol: DNA contamination detection – the management and use of staff elimination DNA databases FSP-P-302 Issue 2. https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_ data/file/915377/302-Elimination_database_protocol_v2.pdf. Accessed 3 Dec 2020 ISO/IEC 17025:2017 (2017) General requirements for the competence of testing and calibration laboratories. 3rd edn Iyavoo S, Draus-Barini J, Haizel T (2017) Validation of ABI 3500xL genetic analyzer after decommissioned and recommissioned at new premises. Forensic Sci Int 6:e542–e543. https:// doi.org/10.1016/j.fsigss.2017.09.197

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Ludeman M, Zhong C, Mulero JJ, Lagacé RE, Hennessy LK, Short ML, Wang DY (2018) Developmental validation of GlobalFiler™ PCR amplification kit: a 6-dye multiplex assay designed for amplification of casework samples. Int J Legal Med 132:1555–1573. https://doi. org/10.1007/s00414-018-1817-5 Martin P, Fernández de Simón L, Luque G, José Farfán M, Alonso A (2014) Improving DNA data exchange: validation studies on a single 6 dye STR kit with 24 loci. Forensic Sci Int Genet 13: 68–78. https://doi.org/10.1016/j.fsigen.2014.07.002 NATA (2018) National Association of testing authorities general accreditation guidance – validation and verification of quantitative and qualitative test methods. https://www.nata.com.au/ phocadownload/gen-accreditation-guidance/Validation-and-Verification-of-Quantitative-andQualitative-Test-Methods.pdf. Accessed 5 Oct 2020 NIJ (2018) National Institute of Justice Office of Law Enforcement Standards Forensic Laboratories: handbook for facility planning, design, construction, and moving. https://www.ncjrs.gov/ pdffiles/168106.pdf. Accessed 26 Nov 2009 NIST (2013) National Institute of Standards and Technology NISTIR 7941 forensic science laboratories: handbook for facility planning, design, construction, and relocation. https://doi. org/10.6028/NIST.IR.7941 OECD (1998) OECD principles of good laboratory practice. http://www.oecd.org/official documents/publicdisplaydocumentpdf/?cote¼env/mc/chem(98)17&doclanguage¼en. Accessed 3 Oct 2020 OSAC (2019) Organization of Scientific Area Committee for forensic science best practice recommendations for the management and use of quality assurance DNA elimination databases in forensic DNA analysis. https://www.nist.gov/system/files/documents/2020/08/04/Best%20Prac tice%20Recommendations%20for%20the%20Management%20and%20Use%20of%20Quality %20Assurance%20DNA%20Elimination%20Databases%20in%20Forensic%20DNA% 20Analysis_OSAC%20Proposed.pdf. Accessed 3 Oct 2020 QAS (2020) Quality assurance standards for forensic DNA testing laboratories. https://www.fbi. gov/file-repository/quality-assurance-standards-for-forensic-dna-testing-laboratories.pdf/view. Accessed 3 Oct 2020 Salceda S, Barican A, Buscaino J, Goldman B, Klevenberg J, Kuhn M, Lehto D, Lin F, Nguyen P, Park C, Pearson F, Pittaro R, Salodkar S, Schueren R, Smith C, Troup C, Tsou D, Vangbo M, Wunderle J, King D (2017) Validation of a rapid DNA process with the RapidHIT ® ID system using GlobalFiler ® express chemistry, a platform optimised for decentralised testing environments. Forensic Sci Int Genet 28:21–34. https://doi.org/10.1016/j.fsigen.2017.01.005 Sturk-Andreaggi K, Diegoli TM, Just R, Irwin J (2011) Evaluation of automatable silica-based extraction methods for low quantity samples. Forensic Sci Int 3:e504–e505. https://doi.org/10. 1016/j.fsigss.2011.10.003 SWGDAM (2016) Scientific Working Group on DNA analysis methods validation guidelines for DNA analysis methods. https://1ecb9588-ea6f-4feb-971a-73265dbf079c.filesusr.com/ugd/ 4344b0_813b241e8944497e99b9c45b163b76bd.pdf. Accessed 3 Oct 2020 SWGDAM (2017) Scientific working group on DNA analysis methods interpretation guidelines for autosomal STR typing by forensic DNA testing laboratories. https://1ecb9588-ea6f-4feb-971a73265dbf079c.filesusr.com/ugd/4344b0_50e2749756a242528e6285a5bb478f4c.pdf. Accessed 5 Oct 2020 Texas Forensic Science Commission (2018) Final report on complaint by Lisa Gefrides against the Houston Forensic Science Center. https://www.txcourts.gov/media/1442270/17-04-final-reportlisa-gefridies-against-houston-forensic-science-center-07202018.pdf. Accessed 7 Nov 2020 Zimmermann P, Vollack K, Haak B, Bretthauer M, Jelinski A, Kugler M, Loidl J, P ug W (2009) Adaptation and evaluation of the PrepFiler™ DNA extraction technology in an automated forensic DNA analysis process with emphasis on DNA yield, inhibitor removal and contamination security. Forensic Sci Int 2:62–63. https://doi.org/10.1016/j.fsigss.2009.08.162

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Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Quality control is essential to the integrity and scientific validity of the DNA test result in human identification. In all scientific methods, there is room for error, and a process must be in place to guarantee that reagents, equipment, and methods are functioning properly. Four main areas are highlighted in this chapter: DNA collection methods, polymerase chain reaction (PCR) factors, electrokinetic injection considerations, and DNA data interpretation. Forensic science laboratories are provided guidance documents by the DNA Advisory Board and the Organization of Scientific Area Committees designed to improve uniformity in DNA test results nationwide. As a path forward, this is necessary since much of past policy created a situation of variation in methodology and data interpretation. It is highly desirable to have a simple uniform methodology that can be applied globally for sharing of data and consistency in the courts. Keywords

Human identification · DNA · PCR · DAB · OSAC · Quality control · Forensic science

H. Miller Coyle (*) Forensic Science Department, Henry C. Lee College of Criminal Justice and Forensic Sciences, University of New Haven, West Haven, CT, USA e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 H. R. Dash et al. (eds.), Handbook of DNA Profiling, https://doi.org/10.1007/978-981-16-4318-7_53

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Introduction The current oversight for forensic DNA analysis is provided through the National Institute of Standards and Technology (NIST) via review by OSAC, the Organization of Scientific Area Committees. This group facilitates implementation of forensically sound science and technical methods by drafting and evaluating standards that are to be used by forensic science laboratories (www.nist.gov). Previously, the Technical Working Groups sponsored by the Federal Bureau of Investigation (FBI) were performing this role by providing recommendations for implementation of new and current technology in the forensic science workplace. In 1989, the Guidelines for a Quality Assurance Program for DNA Analysis was prepared by TWGDAM and then revised three times after the TWGDAM working group of private and public sector forensic scientists was renamed to SWGDAM, the Scientific Working Group for DNA Analysis Methods in 1999. Prior to 1989, there were no national recommendations for forensic science laboratories to follow. The DNA Advisory Board (DAB) began its 5-year project to create a set of federal quality standards for forensic laboratories in 1995. These standards include the quality manual areas of forensic science laboratory organization, personnel, facilities, evidence tracking and storage, validation, test procedures, equipment calibration and maintenance, technical review and reports, proficiency testing and corrective action, laboratory audits, safety guidelines, and use of subcontractors for testing of backlogged samples (https://strbase.nist.gov/validation/Intro_to_DAB_ Standards.pdf). Two sets of standards were created by the DNA Advisory Board. In October 1998, The Quality Assurance Standards for Forensic DNA Testing Laboratories (Forensic Standards) became active to provide oversight to the activities of DNA laboratories that analyze criminal casework (https://strbase.nist.gov/QAS/ Final-FBI-Director-Forensic-Standards.pdf). The Quality Assurance Standards for Convicted Offender DNA Databasing Laboratories was initiated in April 1999 for the analysis of DNA known reference samples collected from convicted offenders (https://strbase.nist.gov/QAS/Final-FBI-Director-Databasing-Standards.pdf). DNA is a molecule that defines the function of genes but also contains noncoding regions that vary from individual to individual that are useful for human identification. Deoxyribonucleic acid (DNA) is composed of two polynucleotide chains carrying genetic instructions for all known organisms and their functions. DNA is a double helix comprised of two antiparallel strands that form the backbone of the helix by creating a sugar-phosphate structure that are bound together by complementary nucleotide base pairing, adenine (A) to thymine (T) and guanine (G) to cytosine (C). The regions of interest for short tandem repeat analysis have been prescreened and preselected for genetic variability in human populations. The estimated frequency of each allele at any given chromosome locus has been calculated in sample ethnic populations and have been shown to vary slightly in frequency based on original founder populations. The high discrimination power or ability to individualize comes from both the great number of allele possibilities at any given locus and the larger number of loci used in combination to calculate estimated relative frequency of any given DNA profile compared to the unrelated random

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individuals in a population. The level of STR analysis is now so sophisticated that a human DNA profile is unique to an individual unless they have a genetic identical twin. The integrity of DNA data is essential for establishing a link to a correct DNA source. Deoxyribonucleic acid or DNA is the biological molecule used to identify potential donors to biological evidence. It is useful as a forensic tool to indicate associations of individuals to biological evidence such as blood, semen, saliva, urine, and epithelial cells. Although an association can be identified by DNA testing, the timing of deposit and the context to the case often is in question. After evidence collection, DNA is extracted, quantified, amplified, and detected by capillary electrophoresis in the forensic science laboratory. Analysis software is used for the viewing and interpretation of DNA fragments to make a conclusion about a potential inclusion or exclusion of a DNA source. Additional DNA mixture interpretation software may be used as an aid to interpret complex DNA samples. The basic steps used to generate a human DNA profile are carefully quality controlled to be sure that a scientifically accurate result can be achieved. This leaves the court debate to the meaning of the DNA in the case rather than with the scientific validity of the DNA result itself. The basic steps of this process can be broken down into the component parts: (a) DNA collection, (b) DNA extraction, (c) DNA quantitation, (d) DNA amplification, (e) capillary electrophoresis, (f) data interpretation, and (g) statistical assessment.

DNA Methods DNA collection. DNA evidence collection refers to the recognition and collection of biological evidence that may be pertinent to the case. Forms of DNA evidence include blood, saliva, semen, urine, touch DNA, bone, tissue, hair, vomit, feces, perspiration, and tears. These forms of evidence may be found as part of property crimes, homicides, sexual assaults, child abuse cases, robbery, kidnapping, etc. DNA collection strategies include the use of personal protective equipment (PPE) to avoid contamination, and therefore error, in the final DNA results. Saliva can contain droplets with DNA molecules that can fall on evidence or a surface and be mixed inadvertently into the crime scene sample (Aparna and Shanti Iyer 2020; Pandeshwar and Das 2014). Therefore, masks and face shields are essential for prevention of DNA contamination by evidence collection personnel. Likewise, shed epithelial cells from the skin and hairs may be falsely included in DNA evidence should it be deposited on the evidence, and full disposable crime scene suits, gloves, and head coverings are required to prevent this phenomenon (Zajac et al. 2019). The key to high-quality DNA results in criminal casework relies on best practices for quality control at the crime scene for evidence recognition, collection, and preservation (Goray et al. 2012; Szkuta et al. 2017; Pilli et al. 2013). DNA collection methods and techniques vary for DNA recovery rates as does the expected amount of DNA yield from different tissue and uid sources (Tan and Yiap 2009). The estimated DNA yield from various tissue sources is the following: blood

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(200 uL, 4–12 ug), cultured cells (5 million, 15–20 ug), liver (25 mg, 10–30 ug), heart (25 mg, 5–10 ug), and spleen (10 mg, 5–30 ug) (www.qiagen.com). The quantity of DNA recovered from biological evidence is affected by the environment that it is exposed to, and therefore, a range of DNA yield is presented in most forensic studies. The estimated DNA content for forensic biological samples in liquid blood is 20–40 ug/mL, in liquid semen is 150–300 ug/mL, and in liquid saliva is 1–10 ug/ mL (Lee and Ladd 2001). Collection method recovery rates vary but include the following estimated percent recovery for epithelial cells from polyester blend fabric: cotton swab (80%), blotting paper (55%), foam-tipped swab (50%), scotch tape (45%), gauze (20%), fingerprint lift tape (15%), Whatman filter paper (15%), Sirchie lift tape (10%), duct tape (5%), positive nylon membrane (1%), and Post-it Note (0%) (https://www.ncjrs.gov/pdffiles1/nij/grants/236826.pdf). Since the method of recovery of DNA molecules varies from forensic science laboratory to laboratory, there may be some level of error introduced by the sampling method or subsequent processing that could lead to suboptimal or no DNA results dependent on the facility or analyst preference (Garvin et al. 2013; Hebda et al. 2014; Singh et al. 2018). Case documentation. Submission form(s) are included in the case folder to document the requesting agency evidence submissions for testing. On occasion, the incorrect test can be requested for the type of evidence, and one quality control step in both standard DNA and postconviction DNA testing is to evaluate the evidence, the test requests, and the unsubmitted items for potential error. If evidence was never submitted for testing but could have been probative and relevant, there may be the opportunity to reevaluate the case circumstances. Other quality control measures include ensuring pages are labeled with the case number, initials, and date; photographs and diagrams are included and labeled correctly; the proper worksheets are used and filled out completely; and communication and correspondence forms are included in the case folder. For each and every step of the DNA testing process, there should be a quality worksheet that is signed and dated and records the process for the case folder. Peer review internal to the forensic laboratory where analysts review each other’s case documentation and supervisors and quality managers also review documentation is designed as a quality control measure to reduce sloppy and incomplete documentation or typographical errors. Reagent preparation. Quality and technical manuals are required in forensic science laboratories to show analysts how reagents are to be made and to reduce variability in preparation techniques. Receipt of reagents and chemicals by the forensic biology section of the laboratory is documented in a chemical log. The tracking of reagent quality includes catalogue numbers, manufacturer lot numbers, suppliers, and testing of new reagents with old reagent results to ensure that a comparable result is achieved. Once the reagent has been tested and is appropriate for laboratory use, it can be implemented in the general preparation of forensic test reagents thereafter. Expiration dates are checked regularly as well (e.g., https:// www1.nyc.gov/assets/ocme/downloads/pdf/technical-manuals/qaqc-proceduresmanual/reagents.pdf). DNA extraction. The DNA extraction process requires either the manufacturer of the kit or the forensic laboratory (or both) quality controls the reagents used to purify

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DNA. All of the reagents and disposable plastics are required to be of molecular biology grade quality indicating they are DNase, RNase, and protease-free (e.g., https://www.mt.com/dam/RAININ/PDFs/TechPapers/test_protocol.pdf). These enzymes, if present, will damage the recovery of the DNA and, therefore, must be removed from all surfaces, disposable plastics, and reagents to ensure DNA of high quality and purity is recovered from the biological evidence. Even the water used in reagent preparation must be of molecular biology grade to be quality enough to use in the DNA extraction process. DNA is a large polymeric molecule that is too large to enter the cell membrane. In order to utilize external DNA, some bacteria secrete DNases (enzymes that biodegrade DNA by hydrolysis) outside of the cell to digest DNA into nucleotides. The nucleotides can then move into the bacterial cell membrane via transport proteins. The bacteria use nucleotides to make nucleic acids and as a source of nitrogen, phosphate, and carbon (https://microbiologyinfo.com/ deoxyribonuclease-dnase-test/). In addition, a positive DNA sample of known quality and identity is used as a calibration control or reference standard for the DNA extraction process. A negative reagent control is processed simultaneously with the samples and the positive control to test the purity of the reagents and indicate no contaminant DNA is present in the reagents (Morton and Collins 1995). The purpose of DNA extraction is to chemically remove any inhibitory contaminants in the evidentiary sample and purify the DNA molecules away from other cellular components such as proteins, lipids, and carbohydrates so that the template is available for PCR replication. DNA quantitation. This step is to determine the yield of the DNA from the evidentiary sample (Arya et al. 2005). qPCR assays vary, but in general they are a two-step PCR method that amplifies DNA target loci and includes a uorescent dye accumulation step to visualize amplification in real time. This allows the analyst to determine both the quantity and quality of the DNA sample prior to generating a full human DNA profile using STR analysis kits and a three-step PCR cycling process called end point PCR. This step requires a manufacturer certification for qPCR kit reagents as well as internal control samples to the assay to check for proper functionality. Positive and negative PCR amplification controls for qPCR assays are commonly included in the kits or can be purchased separately. Human DNA of known concentration is purchasable from molecular biology suppliers and can be purified DNA from American Type Culture Collection (ATCC) certified human cell lines (www.atcc.org); negative controls can be purchased as purified DNA from other animal and plant species to test for PCR amplification specificity. A negative reagent control is performed for the assay by addition of kit reagents to a sample well minus the DNA template to test for purity and no contamination of reagents. In addition, synthetic DNA (internal positive control, IPC) is added to the reagents in the kit as a PCR amplification and pipetting control to indicate that specific DNA template is able to be replicated with the test reagents regardless of whether the evidentiary DNA is amplifiable. The calibration of reagents across the plate is simultaneously performed with the synthetic DNA as the results should be comparable from sample well to sample well (Ewing et al. 2016; Swango et al. 2007; Raymaekers et al. 2009).

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DNA amplification. The PCR process is a molecular biology technique to replicate DNA synthetically in a sterile tube. The components of a PCR reaction include buffer, free nucleotides, short complementary PCR primers to the region of interest for amplification, purified genomic DNA template, and magnesium chloride as a cofactor for the Taq polymerase enzyme and Taq polymerase itself. In the PCR process, the DNA double helix is denatured by heat, the PCR primers bind to the complementary regions during the annealing step, and then the final new strand synthesis step occurs. This three step thermal cycling process continues for 25– 32 cycles until millions of copies of the original target sequence are represented. The specificity of primer binding is due to DNA sequence homology on the template strand; mutations can result in primer mispairing or absence of replication (Zhu et al. 2020; Green and Sambrook 2019). STR analysis kit details and the history of their development are reviewed in a later section in this chapter. PCR amplification of genetic loci is performed by using manufacturer kits. The evolution of these kits to those with expanded multiplexes has not changed the quality control measures. A positive human DNA sample is included in each kit to verify the kit functions properly, and the positive control genotype is consistent from run to run. A negative control is recommended as a “blank” sample well containing reagents but lacking DNA template and, therefore, should produce no DNA profile. PCR replication can be difficult to achieve with low quantity and low quality DNA templates due to variability in template sampling and preferential amplification of higher-quantity target sequences. This results in stochastic effects of the minor component in low level samples and minor components in a mixture (Miller Coyle 2015). Internal validation studies of new DNA typing kits are critical to show that the manufacturer claims are true regarding efficiency and accuracy. The internal validation studies are performed “in-house” in the current laboratory setting to determine how it performs in the hands of the analyst. Validation studies include performance checks on method reliability, reproducibility, sensitivity, and specificity. For each new kit that is developed, forensic laboratories need to validate and peer-review validation studies to show forensic community consensus as part of meeting court admissibility criteria (Ewing et al. 2016; Zhou et al. 2016; Gopinath et al. 2016). Capillary electrophoresis. Capillary electrophoresis is a semi-automated method for selective size separation of DNA fragments based on size and charge. DNA is heat denatured and placed in purified deionized formamide to maintain a singlestrand configuration to the DNA fragment. Only one strand of DNA has been uorescently labeled in the PCR amplification process, so that is the strand that can be visualized in the capillary electrophoresis step. A liquid polymer is used as the sieving medium to separate the DNA fragments into a uorescently labeled and visible “barcode” that fills the glass capillaries in the capillary electrophoresis system. Allelic ladders and internal lane size standards control for the accurate sizing of DNA fragments by providing systematic sized patterns to compare the unknown DNA fragments against. Capillaries are sensitive to heat and mobility of DNA fragments can vary per capillary; thus, an internal lane size standard (or comigration control) corrects for mobility shift. Beyond the use of these standards, data may not be reproducible from PCR reaction to PCR reaction due to slight

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differences in efficiency rates at each locus and due to template sampling differences in the electrokinetic injection step (Krivácsy et al. 1999; Opekar et al. 2016). Data interpretation. A technical review is performed to check that the reports and conclusions are correct. The review includes that proper controls were used; the controls gave appropriate results, and the conclusions are in agreement with data. The allele calls and statistical calculations are then verified by a second, qualified individual. Supervisor oversight includes review of a percentage of the casework that is produced at the forensic science laboratory. The challenges to DNA reside in complex DNA mixture interpretation, and there is considerable variability in data interpretation from forensic laboratory to forensic laboratory (Butler et al. 2018; Buckleton et al. 2018). Each laboratory has set its own policy to follow for data interpretation above and below an analytical instrument threshold, for establishing the true number of contributors to a DNA mixture, and for assessment of contaminant alleles. Analyst bias has been recognized as a subconscious or conscious attempt to include an individual in a DNA result simply because an individual has been identified as a candidate suspect. Bias is represented by selective inclusion and/or nondisclosure of other candidates observed in the data either due to error, intent, or laboratory policy. Most importantly for trial, scientific accuracy is needed to provide effective interpretation of the data and effective counsel. Improvements in data interpretation with mixtures have been made with probabilistic genotyping software analysis systems (e.g., TrueAllele, STRmix) (Greenspoon et al. 2015; Perlin et al. 2015; Bauer et al. 2020; Perlin et al. 2011; Bright et al. 2019; Buckleton et al. 2019; Moretti et al. 2017). While computer software can assist in making statistical inferences, they do also need to be fully understood to recognize the benefits, limitations, and inherent error rates associated with each program. Precision is the ability to obtain the same result every time a test is run; however, the test result may not be accurate. So the ability to obtain both a precise and scientifically accurate result is the desired objective, and finding the optimal method is key while realizing technology may go through several iterations before arriving to the point of optimum. A case in point, here, is the use of probabilistic genotyping software that is designed to eliminate confirmation bias when analyzing data by eye; however, the analyst may have bias in establishing the number of contributors to specify for the software analysis: a situation of concern. Less has been written about confirmation bias in DNA analysis; however, some good forensic studies have been done (Skellern 2015; Mattijssen et al. 2016; Dror 2012, 2015; Dror et al. 2015; Nakhaeizadeh et al. 2014; Brauner 2012; Dror and Hampikian 2011). Certain aspects such as the effect of adjusting the analytical threshold (an invisible line above which alleles are reported and below which alleles are not reported) and determining the effect on reporting number of contributors appear absent in many forensic laboratory validation studies and would affect the interpretation of whether or not an individual may be included in a DNA mixture (Kirby et al. 2017). The setting of the analytical threshold is established in historical policy when forensic laboratories first established their DNA units; however, this will be worth revisiting as probabilistic

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genotyping software programs are installed and validated now, with new and better technology. An article by Gill et al. (2006) clarifies for the DNA analyst factors for identifying the number of contributors in the mixture. “The number of alleles observed per locus, circumstances of the case, and the possibility of related contributors go into deciding how many contributors to condition on.” The allele counting method (assuming a heterozygote as donor) yields a minimum estimate of number of contributors per genetic locus. Scientifically speaking, the maximum alleles observed at any given locus should yield the minimum estimated of detected number of contributors to the DNA mixture. Given the allele counting method, this article goes further to explain the issue of number of contributors and how that assessment relates to determining a likelihood ratio (LR) for probabilistic genotyping. “It is not always easy to specify hypotheses in complex cases where multiple perpetrators or victims may be present. The DNA result itself may indicate that different explanations may be possible. Furthermore, it is possible that Hp (prosecutor hypothesis) and Hd (defense hypothesis) could be very different from each other. For example, under Hp we might consider (victim and suspect); whereas with Hd we might examine more complex scenarios with 3 contributors (3 unknowns, U0 + U1 + U2). There is a common misconception that the number of contributors under Hp and Hd should be the same. There is no requirement for this.” The article suggests “the smallest number of unknown contributors needed to explain the evidence are usually the ones to maximize the respective likelihoods.” In courtroom testimony, often a DNA analyst will state that a DNA result is conservative (“an assignment for the weight of the evidence that is believed to favor the defense”); however, the maximum number of contributors may not be assigned correctly to the DNA mixture due to the policy of consensus profiling. Evidence of additional contributors in replicate PCR amplifications, alleles detected below the analytical threshold, spurious alleles, contamination, and detection of minor DNA elements by differential electrokinetic injection all amount to the same thing: additional alleles are present that cannot be accounted for by the standards submitted and should be disclosed in reports, testimony, and statistics. The effect of including the maximum possible contributors based on allele counts in any one of the PCR replicates (duplicates or triplicates) is to, first, acknowledge the scientific observation and, second, to generate the best possible probability estimate when using probabilistic genotyping methods. Historically, forensic laboratories were greatly concerned over reporting out trace levels of DNA contamination. The development of new DNA standards has been recently completed to address the validation of DNA probabilistic genotyping software systems and for DNA mixture interpretation to improve consistency in results determination from forensic science laboratory to forensic science laboratory. While PCR amplification and capillary electrophoresis artifacts and parameters still present a problem for data interpretation to DNA analysts, the new standards should be an aid to increase uniformity in DNA interpretation of mixtures from biological evidence. History of STR analysis. Approximately three million nucleotide bases (noncoding regions) with multiple copies of short tandem repeat sequences construct the

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DNA backbone (e.g., CAGTCAGTCAGT; three repeats). These regions are called “variable number of short tandem repeats (VNTRs).” If a sufficient number of STR loci are tested (profiled), then the evidence of a person’s identity (and unique STR identifier) is enhanced because the likelihood of two unrelated people having the same number of repeated sequences in these regions becomes vanishingly small (https://nij.ojp.gov/topics/articles/what-str-analysis). The original form of STR analysis used single PCR reactions to test per locus information, multiplexing to combine loci rapidly followed. The first multiplexes had three or four loci per PCR reaction. The primary commercial suppliers for the early systems were (a) Promega Corporation (Madison, WI) that used a silver staining method to visualize loci (CTT: CSF1PO, TPOX, TH01; CTTV: CSF1PO, TPOX, TH01, VWA; FFV: F13A1, FESFPS, VWA; FFFL: F13A1, FESFPS, F13B, LPL; GammaSTRTM: D16S539, D7S820, D13S317, D5S818) and (b) Applied Biosystems (Foster City, CA) that used a uorescent dye technology AmpFlSTR Green I (Amelogenin, TH01, TPOX, CSF1PO and AmpFlSTR Blue: D3S1358, VWA, FGA (https://strbase.nist.gov/multiplx.htm)). Expanded multiplexes quickly followed with the Promega Powerplex series: PowerPlex ES: D3S1358, TH01, D21S11, D18S51, SE33, Amelogenin, VWA, D8S1179, FGA; PowerPlex 16 HS: D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, Amelogenin, VWA, D8S1179, TPOX, FGA; PowerPlex 18D: D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, Amelogenin, VWA, D8S1179, TPOX, FGA, D19S433, D2S1338; PowerPlex ESX 16: Amelogenin, D3S1358, TH01, D21S11, D18S51, D10S1248, D1S1656, D2S1338, D16S539, D22S1045, VWA, D8S1179, FGA, D2S441, D12S391, D19S433; PowerPlex ESX 17: Amelogenin, D3S1358, TH01, D21S11, D18S51, D10S1248, D1S1656, D2S1338, D16S539, D22S1045, VWA, D8S1179, FGA, D2S441, D12S391, D19S433, SE33; PowerPlex ESI 16: Amelogenin, D3S1358, D19S433, D2S1338, D22S1045, D16S539, D18S51, D1S1656, D10S1248, D2S441, TH01, VWA, D21S11, D12S391, D8S1179, FGA; PowerPlex ESI 17: Amelogenin, D3S1358, D19S433, D2S1338, D22S1045, D16S539, D18S51, D1S1656, D10S1248, D2S441, TH01, VWA, D21S11, D12S391, D8S1179, FGA, SE33; and PowerPlex 21: Amelogenin, D3S1358, D1S1656, D6S1043, D13S317, Penta E, D16S539, D18S51, D2S1338, CSF1PO, Penta D, TH01, VWA, D21S11, D7S820, D5S818, TPOX, D8S1179, D12S391, D19S433, FGA. Also, Applied Biosystems “filer” series: AmpFlSTR Profiler Plus: D3S1358, VWA, FGA, Amelogenin, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820; AmpFlSTR Profiler Plus ID: D3S1358, VWA, FGA, Amelogenin, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820; AmpFlSTR COfiler: D3S1358, D16S539, Amelogenin, TH01, TPOX, CSF1PO, D7S820; AmpFlSTR Sinofiler (available only in China): D8S1179, D21S11, D7S820, CSF1PO, D3S1358, D5S818, D13S317, D16S539, D2S1338, D19S433, VWA, D12S391, D18S51, Amelogenin, D6S1043, FGA; AmpFlSTR Profiler: D3S1358, VWA, FGA, Amelogenin, TH01, TPOX, CSF1PO, D5S818, D13S317, D7S820; AmpFlSTR SEfiler: D3S1358, VWA, D16S539, D2S1338, Amelogenin, D8S1179, SE33, D19S433, TH01, FGA,

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D21S11, D18S51; and AmpFlSTR SEfiler Plus: D3S1358, VWA, D16S539, D2S1338, Amelogenin, D8S1179, SE33, D19S433, TH01, FGA, D21S11, D18S51. These kits were a series integrated for the Combined DNA Indexing System (CODIS) core 13 loci required for comparisons with the National DNA Index System (NDIS) DNA database of convicted offender samples. The latest generation kits are large megaplexes and specialty application kits. Included in the Promega series are PowerPlex 16: D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, Amelogenin, VWA, D8S1179, TPOX, FGA; PowerPlex Fusion (includes 22 loci, amelogenin for gender identification, and a Y chromosome locus): Amelogenin, D3S1358, D1S1656, D2S441, D10S1248, D13S317, Penta E, D16S539, D18S51, D2S1338, CSF1PO, Penta D, TH01, VWA, D21S11, D7S820, D5S818, TPOX, DYS391, D8S1179, D12S391, D19S433, FGA, D22S1045; and the Y chromosome specific STR kits: PowerPlex Y: DYS391, DYS389I, DYS439, DYS389II, DYS438, DYS437, DYS19, DYS392, DYS393, DYS390, DYS385a/b; and PowerPlex Y23: DYS576, DYS389I, DYS448, DYS389II, DYS19, DYS391, DYS481, DYS549, DYS533, DYS438, DYS437, DYS570, DYS635, DYS390, DYS439, DYS392, DYS643, DYS393, DYS458, DYS385a/b, DYS456, Y_GATA_H4. Applied Biosystems megaplexes include AmpFlSTR Identifiler: D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, VWA, TPOX, D18S51, Amelogenin, D5S818, FGA; AmpFlSTR Identifiler Direct: D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, VWA, TPOX, D18S51, Amelogenin, D5S818, FGA; AmpFlSTR Identifiler Plus: D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, VWA, TPOX, D18S51, Amelogenin, D5S818, FGA; AmpFlSTR NGM: D10S1248, VWA, D16S539, D2S1338, Amelogenin, D8S1179, D21S11, D18S51, D22S1045, D19S433, TH01, FGA, D2S441, D3S1358, D1S1656, D12S391; AmpFlSTR NGM SElect: D10S1248, VWA, D16S539, D2S1338, Amelogenin, D8S1179, D21S11, D18S51, D22S1045, D19S433, TH01, FGA, D2S441, D3S1358, D1S1656, D12S391, SE33; AmpFlSTR GlobalFiler: D3S1358, VWA, D16S539, CSF1PO, TPOX, Yindel, Amelogenin, D8S1179, D21S11, D18S51, DYS391, D2S441, D19S433, TH01, FGA, D22S1045, D5S818, D13S317, D7S820, SE33, D10S1248, D1S1656, D12S391, D2S1338; AmpFlSTR VeriFiler: D10S1248, D1S1656, Amelogenin, D2S1338, D22S1045, D19S433, TH01, D2S441, D6S1043, D12S391; AmpFlSTR MiniFiler: D13S317, D7S820, Amelogenin, D2S1338, D21S11, D16S539, D18S51, CSF1PO, FGA; AmpFlSTR Yfiler: DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385a/b, DYS393, DYS391, DYS439, DYS635, DYS392, Y_GATA_H4, DYS437, DYS438, DYS448. Promega Corporation and Applied Biosystems are no longer the only STR kit suppliers; manufacturer’s now include QIAGEN N.V. (Venlo, Netherlands) and Biotype (Dresden, Germany) (https://strbase.nist.gov/ multiplx.htm). The STRBase website compiles relevant information on current scientific literature for autosomal and Y chromosome STR marker systems, fact sheets from various

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kit manufacturers, statistics on tri-allele patterns, mutation rates observed for each chromosome locus, the sequence information for each short tandem repeat for motifs/nucleotide base combinations, genomic map positions, and allele size ranges (https://strbase.nist.gov). This website of compiled useful STR marker systems has been available since 1997. Each chromosome locus is fully described and links to the original publications, and authors are conveniently provided. PCR primer sequences for each kit are published, and concordant studies comparing various kit results have been compared for consistency between genotyping STR kit manufacturers. Where non-concordance was detected, NIST worked with the manufacturers to refine the science so that all data sets would be comparable for uploading into the NDIS DNA database system. Specialty STR kits have been optimized for the detection of alleles from highly degraded DNA samples. The ability to type degraded DNA specimens was improved by redesigning the STR marker amplicons so that a smaller-sized polymerase chain reaction (PCR) product was created at each locus. This kit was called the AmpFlSTR MiniFiler PCR Amplification Kit. The kit contains reagents for the amplification of eight miniSTRs which are the largest-sized loci in the AmpFlSTR Identifiler PCR Amplification Kit (D7S820, D13S317, D16S539, D21S11, D2S1338, D18S51, CSF1PO, and FGA) (Mulero et al. 2008; Bright et al. 2011). The MiniFiler kit was validated for casework use (Luce et al. 2009; Hill et al. 2007) and has been used on degraded skeletal remains and cigarette butts (Ip et al. 2014) and for war remains identifications successfully (Marjanović et al. 2009). A Chinese forensic STR kit called Sinofiler was specifically released for the Chinese forensic science laboratories (Shuqin Huang et al. 2010). The kit includes the STR loci: D8S1179, D21S11, D7S820, CSF1PO, D3S1358, D13S317, D16S539, D2S1338, D19S433, vWA, D18S51, D6S1043, D12S391, D5S818, and FGA. After evaluation for the Chinese Han population, the kit was deemed suitable. There was no statistical departure from expectation of Hardy–Weinberg Equilibrium (HWE) for all loci but D6S1043. There was no linkage disequilibrium in all pairs of loci examined. This kit was validated for this particular population group and country (Liu et al. 2014). Future directions for STR technology. Two new trends are present in STR analysis. The first is to revive kinship analysis using DNA test methods and place it in a larger category called forensic genealogy. Interesting genealogy studies include a study of surnames and the founder male lineages (27 total) for the Old Order Amish males in Pennsylvania (Pollin et al. 2008). This study could account for 98% of the male lineages associated with the Lancaster area. Also, surname analysis and YSTR tests have been successfully applied for predictive value in Chinese Han populations to the level of 65% accuracy and increases to 80% accuracy for top four surnames (Shi et al. 2018). This approach can be useful as a “dragnet” concept where surnames can be predicted from Y chromosome haplotypes. Geography and restricted populations fare better with this approach as Y chromosome ancestral “footprints” are detectable in some populations to the level that they stretch back to 500 AD to an ancestral founder population; others are more difficult to use predictively but could still have usefulness for DNA investigative leads (Whiting and Miller Coyle 2019).

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Familial DNA search methodology is different than ancestral surname searches since surname searches rely on the inheritance of paternal lineages by Y chromosome ancestry. Y chromosome ancestry is not one hundred percent accurate as there are some common Y chromosome haplotypes that are not apparently traceable back in historical records to the same family group. While seeking ancestors to your family genealogical tree can be useful with this method, it still requires some verification by investigation of property records, birth, marriage, and death certificates and other genealogical records to confirm a likely family relationship. The concept of a coincidental match in haplotype analysis using STR markers has to be considered as a fortuitous occurrence or the association was not documented back many generations ago in the family tree (https://www.ancestry.com). The coincidental match can be challenging to interpret using YSTR technology, but the DNA method is still highly valuable as an exclusionary tool. Familial search, however, is a search of DNA databases by law enforcement for similar but not exact DNA profiles indicating a genetic relative that can provide a possible investigative lead in the case (https://criminal.findlaw.com/criminal-rights/ familial-dna-searches.html). This DNA database search technique was able to identify through a DNA relative and investigation the Golden State Killer in 2018, approximately 30 years after his homicides began. This was the only effective manner in which he was identified and apprehended. Traditional DNA searches are performed to identify an exact high stringency DNA match, but with familial search techniques, partial matching at reduced stringency is permitted in the software-driven search to provide a candidate list of possible DNA matches. These individuals are then further evaluated and traditionally investigated to establish their possible role in the crime. Most state and federal authorities continuously collect known reference DNA samples from convicted offenders that are continuously uploaded into searchable DNA databases. If there is no familial search match, it may be due to the fact that there are no genetic relatives apprehended and convicted; therefore, there are no leads in that particular database. Law enforcement, however, does have the opportunity to access private ancestry-related DNA databases and uses those reference populations to also search for candidate leads. There are some justifiable concerns by civil liberties advocates that criticize familial DNA search technology as an invasion of personal privacy. The argument is that familial DNA searching affects the privacy of unconvicted genetic relatives, who are innocent of the crime, and this violates the Fourth Amendment in the Unites States Constitution which protects individuals against unreasonable searches. For this reason, some states such as Maryland have banned the use of familial search technology from being used; others like California have passed state legislation to allow familial searching in violent crime with policies in place for correct application and usage. The second trend in human identification technology is to update and revise new DNA standards to fit with current technology and quality management strategies. The newest policies for year 2020 can be found at this website: https://www.nist.gov/ news-events/news/2020/05/two-new-forensic-dna-standards-added-osac-registry. The Organization of Scientific Area Committees or OSAC is an organization with professional and scientific members that have expertise in 25 different forensic disciplines and

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have expertise in the areas of peer-reviewed scientific research, measurement science, statistics, and legal policy. The role of OSAC is to build the foundational science of DNA methods and promote the use of DNA standards for evaluating and implementing new technology. In 2015, OSAC began by drafting new DNA standards that were then submitted to the Academy Standards Board (ASB) of the American Academy of Forensic Sciences (AAFS) and evaluated again by OSAC prior to posting on the registry. The new standards are (1) ANSI/ASB Standard 020, Standard for Validation Studies of DNA Mixtures, and Development and Verification of a Laboratory’s Mixture Interpretation Protocol and (2) ANSI/ASB Standard 040, Standard for Forensic DNA Interpretation and Comparison Protocols. The verification of a laboratory’s mixture interpretation protocol must demonstrate that a laboratory’s protocols produce consistent and reliable conclusions with DNA samples different from the ones used in the initial validation studies. This implies that a test period is required on adjudicated casework samples to validate that the research samples and the authentic casework samples are similar and function appropriately with the new protocol. The new standards also limit forensic science laboratories and prevent them from interpreting DNA mixtures that exceed validated methods especially regarding use on increasingly complex mixtures with additional contributors. The majority of current DNA units in forensic science laboratories still only interpret three person DNA mixtures from biological evidence. The new OSAC DNA standards are still complementary to the FBI’s DNA Quality Assurance Standards. They also build upon the Scientific Working Group on DNA Analysis Methods work, the SWGDAM guidelines. There still may be forensic science laboratory compliance issues as implementation of the OSAC DNA standards remains voluntary. Many forensic science laboratories are already working toward proper implementation, however, as it is best practice to follow the latest edition of DNA standards by OSAC to promote quality and scientific integrity in laboratory practices for forensic human DNA identification methods.

Conclusions Quality control preserves the integrity of the DNA test result in STR analyses. Without reagent, equipment, and method checks and balances, there may be “protocol drift” that allows the analyst to deviate from the written document or method protocol thus creating variance from case to case. Part of public confidence building in forensic science laboratories is to convince the public that the scientific method is valid, accurate, and consistent from case to case regardless of who is on trial. Some of the most spectacular forensic fraudulent cases stem from this problem, where laboratory administration review and oversight is not sufficient to catch incorrect documentation or inconsistent DNA results. The Swecker and Wolf report (http:// wwwcache.wral.com/asset/news/state/nccapitol/2016/09/07/15994768/254822Swecker_Report.pdf) documents the type of quality assurance and quality control issues that can arise in presumptive and confirmatory blood identification tests and

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also created discrepancies in blood serological and DNA reports that became evident in the courtroom to defense counsel and science experts alike. The exhaustive fifteen thousand or more case reviews by outside experts and a three judge panel identified two hundred and thirty cases that resulted in retrials, acquittals, and exonerations simply based on poorly written and poorly enforced protocols. A key element to this issue was the fact that “inconclusive” was not written into the blood identification procedure as a test result option; when forced to choose, sometimes the analyst selected the incorrect result. The Inspector General of New York State issued an executive summary of a leading forensic science serology and DNA unit as well that found fraud in sexual assault kit evaluations for semen evidence. Along with poor quality screening of evidence by a fraudulent serologist, the DNA unit was inconsistent in the manner in which DNA mixtures were interpreted (https://ig.ny.gov/ sites/g/files/oee571/files/2016-12/OCMEFinalReport.pdf). The manner in which the analyst decided the true number of contributors (NOC) to the DNA mixture was at issue; some were calculating NOC based on alleles called by the analysis software; others were including evidence of additional contributors below the analytical threshold. All scientific methods have an error rate, and a process must be in place to guarantee that reagents, equipment, and methods are functioning properly to reduce the error rate. The four main areas highlighted in this chapter (DNA collection methods, polymerase chain reaction (PCR) factors, electrokinetic injection considerations, and DNA data interpretation) are detailed to indicate there are checks and balances to the procedures that are built into the forensic science laboratory procedures. Forensic science laboratories are also provided the guidance documents by the DNA Advisory Board and the Organization of Scientific Area Committees designed to enhance DNA test result consistency. Still, there are some criticisms to the quality control process in forensic DNA testing. “Negative controls also can’t rule out contamination of individual samples” (http://www.injusticeinperugia.org/ viewfromwilmington.html). This statement is true. Most of the quality control in the DNA unit is designed to detect gross contamination events or reagent and equipment failures, but it is possible to have a single independent tube become contaminated, and it may go undetected in the surveillance system of the quality manager. However, there are internal forensic science laboratory DNA databases of the laboratory personnel that DNA results are screened against prior to providing a DNA report to a submitting agency to attempt to screen out accidental DNA contamination during the laboratory processing steps. Crime scene personnel and emergency services workers must be subpoenaed to provide an elimination known reference DNA sample for comparison to casework samples. In addition to OSAC standards, enforcement by laboratory administration, and the personal ethics of the individual analyst, laboratory auditing is another method for reviewing the quality of a forensic laboratories work product. The American Society of Crime Laboratory Directors Laboratory Accreditation Board (ASCLADLAB) (www.ascld.org) is a not-for-profit professional society of crime laboratory directors and forensic science managers whose function is “to foster professional interests, assist the development of laboratory management principles and

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techniques; acquire, preserve, and disseminate forensic based information; maintain and improve communication among crime laboratory directors; and to promote, encourage, and maintain the highest standards of practice in the field.” ASCLD inspects forensic laboratories and accredits their practices and facilities every 5 years to review and maintain the integrity of the scientific processes used to evaluate forensic evidence in criminal casework. The ASCLD auditing procedure reviews the forensic laboratory process as a whole and makes recommendations for quality management improvement; however, the reviews are randomized and based on whatever becomes evident during the audit as the two week process can only provide a “spot check” on the complexities and total volume of casework that is processed in all of the units of the forensic science laboratory. Many forensic science laboratories have also applied for and been granted ISO17025 accreditation status which is an external auditing process that is provided to all clinical and manufacturing laboratories. ISO/IEC 17025 standardization enables “laboratories to show that they operate competently and generate valid results, thereby promoting confidence in their work both nationally and around the world. It also helps facilitate cooperation between laboratories and other bodies by generating wider acceptance of results between countries. Test reports and certificates can be accepted from one country to another without the need for further testing, which, in turn, improves international trade” (https://www.iso.org/ISO-IEC17025-testing-and-calibration-laboratories.html). These claims are true, and the need for global standardization is very helpful for sharing DNA data between country borders. If the DNA data is of high quality and can be shared, increased casework can be solved and more missing persons identified through quality controlled global DNA databases and STR analysis methods.

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Contents Inception of DNA Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Forensic DNA Phenotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Science Behind DNA Phenotyping Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA-Based Prediction of Externally Visible Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Eye Color . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA-Based Inference of Non-pigmented Traits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Face . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hair Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hair Loss/Baldness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Age . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Body Height/Stature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Currently Available Forensic DNA Phenotyping Test Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Legislations for Forensic DNA Phenotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Legal and Ethical Aspects of Forensic DNA Phenotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Case Studies: DNA Phenotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Future of FDP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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K. Vajpayee (*) DNA Fingerprinting Unit, Forensic Science Laboratory, Bhopal, Madhya Pradesh, India School of Arts and Sciences, Ahmedabad University, Ahmedabad, India Biological and Life Sciences, School of Arts and Sciences, Ahmedabad University, Ahmedabad, Gujarat, India R. K. Shukla Biological and Life Sciences, School of Arts and Sciences, Ahmedabad University, Ahmedabad, Gujarat, India e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 H. R. Dash et al. (eds.), Handbook of DNA Profiling, https://doi.org/10.1007/978-981-16-4318-7_54

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Abstract

Identity is a set of individual characteristics. It is crucial to criminal justice system as it links the crime to the perpetrator and the victim. Since long there have been many developments in shaping out the systems of identification like bertillonage, serological methods of identification, etc. Later with the advent of DNA profiling technique using Short Tandem Repeat (STRs), the process of individualization became more robust. With the advancements in the field of genetics, a novel technique was introduced to Forensic DNA technology called DNA Phenotyping. It is the technique where the phenotypic traits are translated from the genotype of the individual and a “Snapshot” of a suspect is created. Although the technology is new, it is growing at a faster rate. The present chapter unfolds the science behind this novel technique. It talks about the DNA-based studies on the prediction of phenotypic characters, legislations, and the notable case studies. Keywords

DNA Phenotyping · SNPs · SNaPshot Technology · Parabon NanoLabs · IDentify

Inception of DNA Technology Identification is crucial to criminal justice system. Identity could be defined as a set of characteristics or attributes, physical or functional, which are unique to an individual. Knowing these unique features helps in identification and individualization of the miscreant. Identification helps in establishing the relationship between the crime, victim, and the criminal, aiding in criminal trial. It also helps in tracing the victims of mass disaster. Since long the criminal justice system has been working to develop a system of identification which is easy to understand, scientifically and statistically sound, saves time, and is cost-effective. Sooner, the very first identification system called the Bertillon System was developed by Sir Alphonse Bertillon in 1879. The system considered measurements of 11 body parts, photography, and individual’s descriptive features, collectively called as Bertillonage System. Advancements in science and technology kept on improving the existing identification systems. It can be seen clearly that one system of identification succeeded the other. Following this, the anthropometric system of identification was replaced later by the fingerprint system of identification (Pierre Piazza 2006). Parallel to this, forensic biology which includes forensic serology and forensic DNA technology was also developing. It is well known that the deoxyribonucleic acid (DNA) is the informational basis of life. The twentieth century observed the development of DNA profiling technology (1984) by Sir Alec Jeffreys where the polymorphism in DNA was taken as markers to establish individuality (Matheson 2016). Since then it has become a robust tool for forensic community. Initially Restriction Fragment Length Polymorphism (RFLP) technology was developed which was highly discriminatory and

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Table 1 Extended set of 20 CODIS core loci

CSF1PO VWA D8S1179 D21S11 D10S1248

FGA D3S1358 D13S317 D1S1656 D12S391

1127 THO1 D5S818 D16S539 D2S441 D19S433

TPOX D7S820 D18S51 D2S1338 D22S1045

scientifically sound. It could analyze the single and multilocus markers for individualization. But the analysis would require large amount of DNA sample (Murnaghan 2019). Due to many such limitations the RFLP methodology of DNA profiling failed. It was then taken over by more sound and advanced technology which used Variable Number of Tandem Repeats (VNTRs) as markers. The technique consumed comparatively less amount of sample DNA. Using VNTRs analysis of degraded DNA also became possible (Gaensslen et al. 2008). But this was not the end. The experts of forensic community tried to introduce a system where even lesser amount of DNA could be used without hampering the reliability of results. With the invention of PCR by Kary Mullis in 1983, PCR-based markers for DNA analysis came into practice. This created the sense of enthusiasm among the researchers. Sooner, Short Tandem Repeats (STRs) became markers of choice by the experts (Goodwin et al. 2007). The DNA profiling technique using STR markers became popular across the laboratories of the world. The method proved to be relatively sensitive, less time-consuming, and required almost one tenth of the nanogram of sample. Further the analysis of Y-STR markers, mtDNA (mitochondrial DNA) markers, familial searching developed making the DNA profiling technology more robust and reliable (Laan 2017). In the year 1994, the US congress brought a bill named DNA Identification Act (Public Law 103, 322) under which Combined DNA Index System (CODIS) was established. With the immense success of UK National DNA Database (UK NDNAD), the concept of databasing the DNA profiles emerged. FBI created its own (US) DNA database National DNA Index System (NDIS) later in 1998. Hence, DNA profiling technology found a new height with the development of DNA databases throughout the world. The formation of CODIS led to the standardization of 13 Core loci which was then extended to the set of 20 core loci (Butler 2010; Li 2008; Goodwin et al. 2008). These CODIS core loci are given in Table 1. Thus, we can see that the identification systems have been evolved gradually from traditional anthropometric methods to technically more advanced, sensitive, and reliable DNA profiling technology. Although the DNA profiling or the DNA fingerprinting technique is being practiced worldwide for individualization, it has also got few limitations. DNA profile generated from the questioned biological sample is taken for comparison with a reference sample in accordance with the law of comparison to look for a possible match. The reference profile is either generated from the sample collected from the suspects or the reference profile drawn out from the DNA databases. The problem with DNA profiling arises when the reference profile is unavailable. In such cases DNA Dragnets method is used where the whole population residing in the area under investigation is screened for DNA. The culprit is expected to participate in the

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mass screening, but the probability is far less than expected. If not the perpetrator then his/her close relative may have participated and could be investigated through the familial searching method using Y-STR and mtDNA technology. But then there are cases where these DNA Dragnets too fail (Kayser 2015). These limitations of relative DNA technology (DNA Profiling) gave birth to a new domain in DNA technology called Forensic DNA Phenotyping.

Forensic DNA Phenotyping Introduction Forensic DNA Phenotyping (FDP) is a novel technique introduced to Forensic DNA technology. It is still developing and growing at faster rate. FDP can be understood as the technique where the phenotypic traits are translated from the genotype of the individual and a “Snapshot” of a suspect is created. Since the information predicted from the genotype cannot be exact, the inference is, however, made by considering the likelihood ratio and presenting it with significant certainty (Samuel and Prainsack 2018). The main objective of the DNA Phenotyping technique is to identify and create a “probable” picture of an unknown suspect, a missing individual, or a victim of mass disaster where the traditional DNA identification systems fail due to unavailability of ante-mortem DNA profile or profile of any relative for reference. The technology uses the molecular genetic markers (DNA isolated from the biological evidences collected from the crime scene) to assist investigators in characterization and investigation process (Kayser and de Knijff 2011). Thus, it can be rightly said that the FDP technology acts as “biological witness” – more reliable than traditional eye witnesses. Besides helping the criminal justice system in identifying the unknown perpetrators, FDP is also expected to aid anthropologists and paleo-genetic researchers in reconstructing old human remains using ancient DNA analysis. Moreover, FDP is considered to answer questions related to bio-geographic ancestry; however, there are cases of mixed ancestry which cannot be solved by only considering the physical traits (King et al. 2014). The notion of applicability of molecular biology – DNA technology – to forensic science came relatively late and progressed slowly. It was in the late 1990s and early 2000 when experts started applying the concepts of DNA technology to forensic caseworks. Forensic DNA Phenotyping as compared to other DNA methodologies was introduced even later because the knowledge about the predicting appearance still remains limited. Although there have been many researches in the field of human genetics, unfortunately most of them were directed toward the gene-disease relationships (Stranger et al. 2010). Scientists worked in several projects; even the milestone project “The Human Genome Project” was intended to decode the human disease genetics by sequencing the whole genome. Very less has been studied about the genes responsible for a particular visible trait/physical appearance. Possible reasons could be the funding for genetic disease-related projects is high compared to the researches on genetic in uence and human variation (Kayser 2015).

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Fig. 1 The basic working methodology of Forensic DNA Phenotyping, where DNA is genotyped and processed by applying statistical models to formulate a probable snapshot of the unknown individual (“Parabon ® Snapshot ® DNA Analysis Service – Powered by Parabon NanoLabs,” 2020)

Apart from this it is well known that these visible characteristics or the phenotypic traits are multi-faceted. They aren’t only controlled by the genetic markers alone but are the result of coordinated mutual interaction between the environmental factors and genetic markers (Kayser 2015). DNA Phenotyping is basically of two types – indirect and direct. Indirect approach to DNA Phenotyping infers the external features of individual by accessing his/her geographic origin. However, the direct DNA Phenotyping analyses the genome/genotype of the individual to infer external physical characteristics of the individual for identification. For Forensic DNA Phenotyping to be concerned both the approaches are followed. The technology utilizes PCR-STR and identifies the SNPs associated with the EVCs (Fig. 1).

Science Behind DNA Phenotyping Technology To understand how a human population varies in terms of physical appearance, it is important to first understand few basic concepts of genetics. In a diploid organism for a given gene, there exist two allelic forms which interact with each other and produce a particular physical trait. The expression of these alleles producing the physical trait is called as its Phenotype, whereas the genetic makeup of the organism which includes all the expressed and non-expressed groups of genes is termed as Genotype (Bartee et al. n.d.). Another term associated with the DNA Phenotyping is SNPs. SNPs or the single nucleotide polymorphism can be understood as a type of (most common) genetic variation in which there exists a change at the level of single nucleotide. A person’s DNA can have 4.5 to 5 million SNPs. They tend to occur after every 1000 nucleotides and act as markers. They can

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Fig. 2 Figure showing the difference between Genotype and Phenotype

Fig. 3 Figure showing the SNP at 4th Nucleotide position (shown in red)

occur within a gene and may play a direct role in gene function (“Genomic Research,” 2020) (Figs. 2 and 3). As per the studies, scientists have found that the different kinds of variations like SNPs and insertions and deletions commonly called as InDels depending upon their position on regulatory gene directly in uence the protein translated. They do so by altering the sequence of amino acid chain. If in case this altered protein is associated with some phenotypic character, then a changed/varied external feature is observed. Thus, these variations in genome of an individual (genotype) tend to in uence the physical characteristics (phenotype) (Marano and Fridman 2019). Presently, the scientifically and statistically sound methods of genotyping for the Pigmented Physical traits, eye color, hair color, and skin color, are under practice for accurate prediction (Mehta et al. 2016; Phillips 2015). Additionally, studies have shown that traits like biological age, morphology of face and hair, freckling, body

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Table 2 Group of researchers who were behind the development of FDP technique (Matheson 2016; Pollack 2015; Murphy 2013; Enserink 2011)

S. no. 1.

Name of the scientist Dr. Manfred Kayser

Institution Forensic molecular biology department, Erasmus University Medical Center in Rotterdam, Netherlands

2.

Dr. Tim Spector

Genetic Epidemiology, Kings College, London, England, and the Director of the TwinsUK Registry

3.

Dr. Susan Walsh

Forensic geneticist, Indiana University-Purdue University, Indianapolis, Indiana

4.

Dr. Mark D. Shriver

Professor (anthropology and genetics), Pennsylvania State University

5.

Dr. Peter Claes

6.

Dr. Wojciech Branicki

Researcher in morphometrics at the Medical Image Computing laboratory at KU Leuven in Belgium Researcher, Department of Genetics and Evolution, Jagiellonian University, and a DNA expert at the Institute of Forensic Research, located in Krakow, Poland

Research area Laid the foundation of many advances in biological analysis, which includes identification of male DNA using the Y chromosome The Inheritability of visible traits as demonstrated in biological twin populations is recognized as a milestone work. His work has given a new direction toward the study of relationship between genetic markers and EVCs Working upon DNA intelligence tools to aid criminal justice system in identifying the unidentified deceased body Dr. Shriver along with Dr. Claes developed a complex mathematical method to represent face which was based on measuring the 3–D coordinates of more than 7,000 points on the face. It is the basis for FDP imaging computer software Worked along with Dr. Shriver on developing a complex mathematical method to represent faces Identified the genetic markers which are responsible for the expression of EVCs

height, baldness in males, and genetic diseases can also be predicted using the genotype analysis (Murphy 2013). The panel of eminent scientists whose contribution to FDP is cherished has been summed up in Table 2. Their studies and observations have laid the foundation and gave the strength to the field. Although the field is new, the immense work of these scientists have put together so much knowledge already. It has drawn the attention of many researchers and scientists toward the utility of phenotyping traits.

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DNA-Based Prediction of Externally Visible Characteristics Eye Color The very first publication on DNA-based prediction of eye/iris color came out in the year 2007. The year saw studies from two well-known expert groups – Frudakis et al. and Sulem et al. Frudakis et al. (2007) worked upon OCA2 gene and used 33 SNPs. With their observations they were able to classify eye colors (8%) of more than 1000 samples considered. At the same time Sulem et al. (2007) conducted GWAS studies on pigmentation traits. He used 9 SNPs from SLC24A4, KITLG, 6p25.3, TYR, OCA2–HERC2, and MC1R genomic regions. Although out of all the individuals (Europeans) chosen as samples, 60% were brown eyed, and the observation was made with 0.5 probability. In 2008, there were many experts who were simultaneously working on the DNA-based eye color prediction. The major groups were Sturm et al., Eiberg et al., and Kayser et al. Sturm et al. and Eiberg et al. inferred that HERC2 rs12913832 plays an important role in color prediction of eye (Sturm et al. 2008; Eiberg et al. 2008). However, Kayser et al. in their GWAS studies included other HERC2 SNPs like rs916977 as major SNP in color prediction (Kayser et al. 2008). Their group further performed studies on DNA-based prediction of eye color and used 3 SNPs: HERC2 rs916977, OCA2 rs11855019, and OCA2 rs7495174. Upon statistically (receiver characteristic operating curve) analyzing, they found that the accuracy reaches 0.8, where 0.5 stands for random prediction level and 1.0 shows complete accurate prediction. Moreover, HERC2 rs916977 alone provided the eye color predictive value mostly. Later in 2009 major work in the area was done by Liu et al. (2009). Liu et al. did a thorough and extensive study on DNA-based eye color prediction. In their study they took SNPs from previous studies and did exhaustive study on their prediction ability. The number of SNPs was taken to be 37 from 8 pigmentation genes, and more than 6100 Dutch Europeans were sampled for study. They also created and validated a model based upon 24 SNPs from 8 pigmentation genes. The results, however, provided the accuracy level of 0.93, brown; 0.91, blue eye color; and 0.73 for intermediate eye color. The study also shows that as predicted by earlier studies SNP HERC2 rs12913832 to be more informative about the eye color, alone achieved the predictive values of 0.899 and 0.877 for brown and blue, respectively. Thus, after validation of the model and analyzing the results from the prediction curve, Liu et al. suggested following SNPs from 6 pigmentation genes: HERC2 rs12913832, OCA2 rs1800407, SLC24A4 rs12896399, SLC45A2 rs16891982, TYR rs1393350, and IRF4 rs12203592 (Liu et al. 2009). This set of SNPs provided the values of 0.93 for brown, 0.91 for blue, and 0.72 for intermediate eye color when tested among more than 2300 Dutch Europeans. In Year 2010, Valenzuela et al. (2010) published a study where they tested 24 pigmentation genes for 75 SNPs with regard to their effect on pigmentation traits skin, hair, and eye. The study considered European as well as non-European

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population. However, mixing these European and non-European subjects in analysis questioned the authenticity of the study – since ancestry too had a role to play when it comes to two different populations. Although the study on one hand got success in proving that the SLC24A5 rs1426654 could not be considered to have its effect on pigmentation trait (19, 20). Lately, Mengel-From et al. (2010) suggested the value of HERC2 rs12913832 in in uencing the eye color pigmentation trait. Based upon the earlier studies and exhaustive observations of Liu et al., Walsh et al. (Walsh et al. 2011a) developed the first DNA-based eye color prediction system. It was intended to be used by forensic community in identification purpose. The system was named as IrisPlex. The IrisPlex system is sensitive and compatible to the SWGDAM guidelines (Walsh et al. 2011b). The system works by multiplex genotyping all the 6 SNPs demonstrated by Liu et al. (HERC2 rs12913832, OCA2 rs1800407, SLC24A4 rs12896399, SLC45A2 rs16891982, TYR rs1393350, and IRF4 rs12203592). The system was then crossed checked for its validity under different situations and was approved for use. The application uses interactive user experience providing the Excel sheet for user input of data. Thus, being validated at all possible levels, the system was said to be accurate worldwide, and the biogeographic origin does not have much in uence to it. In the year 2014, an advanced and technically sound version of the IrisPlex was developed. The advanced technology was even accessed by EDNAP and ISFG, and as per them the module is reliable and easy to use (Chaitanya et al. 2014). For the IrisPlex a commercial tool was invented named as Identitas V1 Forensic Chip. It allowed inference of all the 6 SNPs IrisPlex, ancestry analysis, etc. from the genomic sample (Keating et al. 2012). The tool can be searched under the link http://identitascorp.com/. Studies related to hair color/eye color prediction can be done using the IrisPlex model (Keating et al. 2012). The development of such system has never failed to invite debates on SNP sets included in the system especially regarding the prediction of the intermediate eye color pigmentation. There were groups of experts, Spichenok et al. and Pneuman et al., who insisted inclusion of few more SNPs (Spichenok et al. 2011; Pneuman et al. 2012). One such attempt was done by Allwood and Harbison (2013) who proposed 19 SNPs and emphasized on including 4 SNPs (SLC24A4 rs12896399, OCA2 rs1800407, TYR rs1393350, and HERC2 rs1129038) for eye color prediction. Later a 23 SNP-based system of eye color was proposed by Ruiz et al. (2013); the authors highlighted the addition of HERC2 and OCA2 SNPs. Finally a set of 13 SNPs – 6 IrisPlex SNPs, 4 HERC2 SNPs (rs1129038, rs11636232, rs7183877, and rs1667394), and 3 OCA2 SNPs (rs4778241, rs4778232, and rs8024968) – were accepted on the basis of accuracy level (Kayser 2015). Currently, there are many scientific groups who are still working for predicting eye color pigmentation with accuracy. Some are emphasizing upon limiting DNA-based eye color prediction only to HERC2 rs12913832, while others are debating over effect of gender on in uencing the accurate prediction (Kayser 2015). Conclusively it can be said that the field concerning with the eye color pigmentation is still expanding and a lot of studies are required to establish the markers in uencing the trait with accuracy.

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Hair Initially studies on hair color were centered to red hair only. Hair color as an EVC has been studied in detail by several expert groups, and the major ones are Grimes et al., Branicki et al., Sulem et al., and Valenzuela et al. Grimes et al. (2001) are known for their contribution in introducing the very first DNA test which could predict the hair color. The DNA test was based upon 12 MC1R DNA variants (Grimes et al. 2001). Branicki et al. in the year 2007 sequenced the MC1R gene in more than 180 individuals with hair color variations which included 40 red-haired ones and 36 blond hair individual. They, thus, developed red hair prediction assay on the basis of 5 MC1R DNA variants (Branicki et al. 2007). Sulem et al. (2007) in the year 2007 started a GWAS on pigmentation and published the first DNA prediction assay for hair colors belonging to all category. The authors predicted the red hair individual with >0.5 probability by using 2 MC1R SNPs (rs1805008 and rs1805007). In another study the authors predicted all the other pigments except red using 9 SNPs from 6 genes. But they fail to produce accurate result. Year 2010 saw publications from Valenzuela et al. (2010) who claimed to report 3 SNPs (SLC45A2 rs16891982, SLC24A5 rs1426654, and HERC2 rs12913832) for total hair melanin. Later in the year 2011, Branicki et al. used 46 SNPs associated with hair color to evaluate their predictive power in Europeans of Poland. Further they developed a model which involved 22 SNPs having AUC values of 0.93 for red hair, 0.87 for black, 0.82 for brown, and 0.81 for blond, respectively (Branicki et al. 2011). However, they failed to confirm the role of SLC24A5 rs1426654 in predicting hair color. On the basis of all the previous studies especially the SNP prediction ranking by Branicki et al., a DNA test system for predicting hair and eye colors was developed (2013). This test system is known as HIrisPlex system and is known to have a single multiplex genotyping assay for SNPs associated with eye and hair color. The system thus includes all 6 SNPs from IrisPlex two prediction models, one for hair color and one IrisPlex model for eye color. The 24 SNPs included in the HIrisPlex system are MC1R SNPs (one indel, Y152OCH, N29insA, rs1805006, rs11547464, rs1805007, rs1805008, rs1805009, rs1805005, rs2228479, rs1110400, and rs885479), two from SLC45A2 (rs28777 and rs16891982, one from KITLG (rs12821256), one from EXOC2 (rs4959270), one from IRF4 (rs12203592), two from TYR (rs1042602 and rs1393350), one from OCA2 (rs1800407), two from SLC24A4 (rs2402130 and rs12896399), one from HERC2 (rs12913832), one from ASIP/PIGU (rs2378249), and one from TYRP1 (rs683) (Walsh et al. 2013). For model-based hair color prediction, SNPs TYR rs1393350 and SLC24A4 rs12896399 were used, whereas 6 IrisPlex SNP were used for model-based eye color prediction. HIrisPlex was then validated for the forensic purpose in 2014 and has been described to be compatible with SWGDAM guidelines. Furthermore, the assay can also be applied to access the degraded DNA samples (Draus-Barini et al. 2013; Walsh et al. 2014).

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Presently, HIrisPlex system is being commercialized as Identitas V1 Forensic Chip. It includes 22 SNPs for hair color prediction used in the HIrisPlex system. This tool is available at http://identitascorp.com/. It also provides access to other information relevant to forensic investigations like eye color/hair color prediction. However, 4 MC1R SNPs (N29insA, Y152OCH, rs1805007, and rs1805009) are not included in the system due to several other reasons (Keating et al. 2012).

Skin Presently, there is little knowledge about the skin color variation trait as compared to what we have for other EVCs. This lack of knowledge could be associated with the fact that the global distribution of the skin color variation trait is heterogenous in European population which is unfavorable for GWAS studies, whereas the other traits – eye color and hair color –are homogenous among the population. Hence an inference for these traits can be drawn easily (Kayser 2015). Research on skin pigmentation trait using the same traditional multi-ethnic study group was conducted by Valenzuela et al. (2010). The authors found accuracy level of only 45.7% for skin re ectance. The study emphasized 3 SNPs (SLC45A2 rs16891982, SLC24A5 rs1426654, and ASIP rs2424984) in uence the skin pigmentation. Later, on the basis of 7-SNP set (the 6 SNPs for eye color, SLC24A5 rs1426654) and 2 SNPs as described for skin color by Valenzuela et al. Spichenok et al. (2011) predicted the non-white and non-dark skin pigmentation. Pneuman et al. (2012) on their detailed study assessed and evaluated the abovestated 7-SNP set and found 1% error. On a similar study Hart et al. (2013) emphasized on 6 SNPs which included the 7-SNP set by Spichenok et al. except IRF4 rs12203592. They concluded that there occurred no error. Maroñas et al. (2014) articulated their first comprehensive study in which they included 59 SNPs which were also associated with skin, eye (Ruiz et al. 2013), and hair color in European and non-European populations. In this study they framed questions and measured the skin re ectance. These sets of SNPs differentiated white skin-colored individuals from intermediate/black skin-colored individuals. The authors highlighted the following SNP set, SLC45A2 rs16891982, SLC24A5 rs1426654, KITLG rs10777129, ASIP rs6058017, TYRP1 rs1408799, and OCA2 rs1448484, for skin pigmentation prediction. They further gave the additional SNP set including SLC45A2 rs13289, SLC24A4 rs2402130, TPCN2 rs3829241, and ASIP rs6119471. Since previous studies have included only a small number of samples, it becomes essential to include more data from the individuals so as to form a reliable opinion about the predictive accuracy. It could also be suggested that as with other EVCs skin pigmentation trait should also be considered for quantitative prediction.

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DNA-Based Inference of Non-pigmented Traits Face Identifying the person from the facial sketch is easy. One can expect predicting the facial characteristics from DNA using FDP technique. And if possible this will be regarded as a gold standard technique in the field of forensic science. But the reality of predicting facial features using DNA markers is still a far reaching dream. Liu et al. (2012) studied about the markers in uencing the facial traits/shapes in their GWAS. At the same time, Paternoster et al. in their GWAS (Paternoster et al. 2012) too were studying the same markers. The GWAS published by Liu et al. (2012) and by Paternoster et al. (2012) is the only study where the genes involved in expression of the facial characteristics have been described in brief. Liu et al. (2012) in his genome-wide study worked upon 10,000 Europeans and finally came up with a set of 5 genes PAX3, PRDM16, TP63, C5orf50, and COL17A1. Their study stated that these genes are involved in human facial shape variation. Using 3D magnetic resonance images (MRI) of the head and of 2D portrait pictures, they automated the facial landmarks and had their measures (facial distancing). Of these 5 gene set three (PAX3, PRDM16, and TP63) have already been recognized in a study related to craniofacial development and disease. The only gene to be identified for in uencing a facial character (nasion) is PAX3 (Paternoster et al. 2012). Both the GWAS studies revealed that for determining a facial trait, a large number of DNA variants are involved. Liu et al. (2012) further suggested that for TP63 rs17447439 the largest effect can be seen, where the heterozygote carriers had a 0.9 mm and the homozygote carriers a 1.8 mm reduced eye-to-eye distance. Later, Claes et al. (2014) used complex but advance approach to infer facial traits. They studied SNPs from craniofacial genes in three populations (US Americans, Brazilians, and Cape Verdeans). The researchers highlighted 20 genes and 24 SNPs. Of all, more stress was given on three SNPs, namely, SLC35D1, FGFR1, and LRP6. Recently a group of researchers have questioned the statistical approach used by Claes et al. (Hallgrimsson et al. 2014). Since very little is known about the facial variation in humans, the field needs more involvement and research. It seems there is a long way for the technique to get established and to provide significant predictive markers to be used by the forensic experts.

Hair Structure As per the independent studies by Fujimoto et al. (2007) and Medland et al. (2009) in Asian and European population, respectively, three genes have been reported to be associated with the human hair morphology (Fujimoto et al. 2008). In a genomic scan study on Asian population, Fujimoto et al. described FGFR2 and the EDAR gene to be involved in hair thickness (Fujimoto et al. 2009). In similar studies

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conducted by Medland et al. (2009), the gene associated with the straight hair variant in Australian Europeans is found to be TCHH. Since the hair structure in Asian population remains uniform, the significance of applying Human Hair Structure in identification process remains null. However, in European population the gene TCHH explained the 6% variation observed. The authors briefed that the minor T allele, rs11803731 (a coding, nonsynonymous variant in exon 3 of TCHH), is absent in the populations of East Asia, Oceania, sub-Saharan Africa, and in Native Americans, but is found across Europe and neighboring regions such as North Africa, the Middle East, and West Asia (Medland et al. 2009a). Medland et al. also studied the candidate genes selected by Fujimoto et al. and found that they have an association with WNT10A (Fujimoto et al. 2007). Both the studies on hair morphology suggested that there exists a heritability rate for curly variant, but the genes responsible were not identified (Medland et al. 2009b). Since there is a great diversity for hair morphology among European (straight, wavy, or curly), FDP will have a significant impact only when there will be more predictive genes for this particular trait.

Hair Loss/Baldness Presently as per the studies conducted over the phenomenon of baldness, 12 genomic regions have been identified. These genes are found to be associated with the early onset of androgenic alopecia (AGA). Androgenic alopecia is commonly found in male and is associated with baldness. The genes found to be associated with this particular trait are AR/EDA2R, TARDBP, HDAC9, AUTS2, SETBP1, PAX1/ FOXA2, WNT10A, 17q21.31, 3q25, 5q33.3, and 12p12.1 (Richards et al. 2008; Li et al. 2012; Heilmann et al. 2013). A study has highlighted AR/EDA2R to be strongly associated with the trait and is located on X chromosome. The female pattern hair loss (early onset) is a rare phenomenon. But it has been observed that it shares some of the genetic basis of early baldness with male (as regard to the X chromosome). But no studies have shown the association of the other genes. Thus, the etiology still remains unclear (Redler et al. 2012). The hair loss trait seems to be promising, but since all the studies have been done on early onset pattern and the general population have late onset pattern, the genetic study becomes less vocal. Thus, there is need to establish studies on late onset hair loss pattern which is more likely to have strong connection with the environmental effects too.

Age There are several traits which indicate age indirectly, but as it seems the age in itself is an externally visible characteristic. There have been several studies in predicting

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the genes responsible for aging. Many studies have established the fact of decrement in the T-cell numbers with age (sjTREC). Based upon this knowledge Zubakov et al. in 2010 introduced a method of age estimation by quantifying the sjTREC (Zubakov et al. 2010). The process of DNA Methylation is also considered to be one of the processes behind aging. The field of epigenetics has evolved in recent years and has expanded its knowledge system about DNA methylation and dependency of aging on it. It has also enhanced our understanding over highly promising CpG candidate markers. Expert groups like Bocklandt et al. (2011) studied these CpG markers for age estimation and concluded that promoters of EDARADD, TOM1L1, and NPTX2, two CpG markers, are responsible for 70–73% of variation in age. Further authors predicted the age of an individual with average accuracy of 5 years. Later, Garagnani et al. (2012) published a study on CpG sites in 3 genes ELOVL2, FHL2, and PENK. Out of these three genes, the authors highlighted the ELOVL2 gene as a strong age prediction marker. In a similar study, Weidner et al. (2014) established a relationship between DNA methylation with chronological age. Further, they introduced age estimation calculator. As we all know chronological and biological age of an individual can differ, the DNA methylation is seen to be associated more with biological age. Thus, before applying the theory of DNA methylation in forensic case works, it becomes important to understand the underlying principles behind them. Thus, in the near future one can see the application of age prediction based on DNA methylation in forensic science (Weidner et al. 2014). Parallel to this Yi et al. (2014) studied eight loci and their relation with aging process. The authors included a small sample set (65 individuals) but failed to provide any validation of the markers’ involvement in biological age. Later several studies have provided the evidence that ELOVL2 gene is a better marker till date for age estimation.

Body Height/Stature There is a large data available for the body height. There have been several genomewide studies which had used body height as a common complex trait. Previously, there have been several GWAS studies by expert groups like Aulchenko et al. and Liu et al.; Genetic Investigation of ANthropometric Traits (GIANT) consortium on human height trait. GAINT consortium investigated >183,000 individuals and identified hundreds of SNPs at 180 genetic loci significant height association (Lango Allen et al. 2010). Aulchenko et al. (2009) did the very first systematic study on body height in 2009. The authors predicted top 5% of tall individuals from >1000s of Dutch Europeans using 54 height-associated SNPs. Liu et al. (2013) in 2010 proposed that the 180 genetic loci identified by GIANT have significant association with the normal height.

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However, the knowledge bank on body height signifies that a lot of information is still missing and there are more than a thousand of SNPs associated with the body height trait in humans.

Currently Available Forensic DNA Phenotyping Test Systems Currently only a handful of software has been developed by the research groups which upon analyzing the data provide reliable results on physical traits. Apart from physical traits, these softwares have been developed to infer genetic ancestry, freckling, and facial shape. The milestone softwares aiding the criminal justice system are IDentify by Identitas and SNaPshot Software by Parabon NanoLabs. IDentify is an advance software. Using around 800,000 SNP database and applying rules of genetics upon them generates the external visible traits. It also infers the biogeographic ancestry. The predictions are highly reliable. Forensic community many a times faces the issue related to mixed DNA sample. Taking this into consideration Identitas is working toward developing a software that could discriminate between the mixed samples having 10 sources (“Identitas – Forensic Phenotyping,” 2020).

Fig. 4 Figure showing a sample SNaPshot prediction composite profile (“Parabon ® Snapshot ® DNA Analysis Service – Powered by Parabon NanoLabs,” 2020)

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Parabon NanoLabs situated in Virginia has released a software that works on the programming developed by Drs. Shriver and Claes (Pollack 2015; Mehta et al. 2016). The software is called SNaPshot and is taken care by the US Department of Defense. The SNaPshot utilizes the Next Generation Mini-Sequencing technology and processes 20,000 DNA markers, applies complex algorithms to finally create a file, and compares it with the donor profiles. It then uses the reverse engineering to prepare a snapshot of the suspect out of the raw data (Laan 2017). The SNaPshot system also has provisions which uses the Forensic Facial Reconstruction technique to create 3-D model of the suspect and then adds the non-biological characteristics like tattoos, eye glasses, hair style, etc. with the help of an artist (Laan 2017; Mehta et al. 2016). Like IDentify, SNaPshot analysis can infer genetic ancestry apart from phenotypic characteristics (“Parabon ® Snapshot ® DNA Analysis Service – Powered by Parabon NanoLabs,” 2020) (Fig. 4).

Legislations for Forensic DNA Phenotyping With the advancements in science and technology, the law keeps on amending itself. It can be seen that with the introduction of new technology, the new legislations are being formed or a new bill is framed to guard the use/misuse of the invented technology. Thus, scientific technology and law evolve simultaneously with their effect on each other. Since the development of DNA technology, there have been rules and legislations framed in different parts of the world. Every country has its own legislation regarding application of DNA by criminal justice system. With respect to the legislations for forensic DNA Phenotyping, not every country has got its own legislations framed, or otherwise some countries have a limited provision for regulation of forensic DNA Phenotyping. The countries with common law systems – the UK and the USA – have provisions that allow the DNA testing unless it is not specifically prohibited by law (Kayser and de Knijff 2011). The UK has legislations that allow the analysis of the physical appearance of the perpetrator, but the same technology cannot be used for red hair testing and ethnic inference (MacLean 2013). On similar lines, the federal statute of the USA has put a restriction on DNA Phenotyping technology only to identification purpose, although identification purpose has not been clearly defined in the statute (Murphy 2013; Kayser and de Knijff 2011). Since, in the USA law enforcement agencies have the tendency to slowly adapt the new technologies as compared to the other countries, thus the USA lags in regulating forensic DNA Phenotyping technology (Matheson 2016). Meanwhile some States do have a DNA processing regulation in their jurisdiction. The only state authorizing difference in effective Phenotyping testing which includes physical characteristics and genetic diseases is Texas (MacLean 2013). States like Florida, Michigan, South Dakota, Vermont, Utah, and Washington don’t have any restrictions on FDP or external visible characters, but they do prohibit DNA testing in case of genetic disorders or other medical-based studies. As a representative legislation,

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these five states have Vermont statute. The statute states that “[the] analysis of DNA samples is authorised... to type the genetic markers from the DNA samples for law enforcement identification process...[but] analysis of DNA samples obtained pursuant to [the statute] is not authorised for identification of any medical or genetic disorder” (MacLean 2013). The States prohibiting the determination of physical traits from DNA include Indiana, New Mexico, Rhode Island, and Wyoming (Laan 2017). As per Indiana statute the DNA profiles stored in DNA database are prohibited for obtaining information about external visible characteristics (MacLean 2013). Similarly Rhode Island’s legislation theory states that the DNA samples are the DNA profiles which have been recorded as per the statute and will only be used by the law enforcement agencies for identification purpose. This identification purpose could include the identification of missing persons, but the submitted DNA samples or the profiles shall not be used for obtaining information about physical traits (MacLean 2013). The Wyoming legislation has been built on similar lines where obtaining information about EVCs is restricted whereas the DNA genotyping is permitted (MacLean 2013). Countries like Canada, Belgium, Spain, and South Africa only allow non-coding regions of DNA for typing meanwhile genotyping is allowed in Australia. Germany has the legislations where DNA testing is only permitted for determining gender source parent age; however, they forbid the application of FDP for other purposes (MacLean 2013; Laan 2017). The only country having regulations regarding forensic DNA Phenotyping is the Netherlands. The legislation of the Netherlands restricts the testing only to physical traits. The Netherlands has the Dutch act “determining externally perceptible personal characteristics from cell material” which defines the DNA investigation. As per the legislation “DNA investigation [as] the research of cell material which is only targeted at comparing DNA profiles or determining externally perceptible personal characteristics of the unknown suspect.” Further it states that “if it is uncertain that the source knows about the trait it may not be investigated...[and] any physical characteristic must be (1) externally perceptible; (2) visible; (3) present at the time of and since birth; and (4) publicly perceptible” (Enserink 2011; MacLean 2013; Kayser and de Knijff 2011). In India, since “The DNA technology (Use and Regulation) Bill, 2017” is still pending, the legal position of DNA testing remains dicey.

Legal and Ethical Aspects of Forensic DNA Phenotyping Since the DNA Phenotyping technology assesses the genomic content of an individual, it triggers the legal and some ethical issues with itself. Few ethical issues concerned with forensic DNA Phenotyping are: 1. The slippery slope theory: Since the FDP technology is very new, a lot of information is fed into the knowledge bank daily. This undoubtedly increases

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the scope of forensic DNA Phenotyping technology in the near future. There is a group of scientists who fear that this advancement in capabilities of FDP will lead to the misuse of information inferred. According to them if with advancements assessing the information from one form of FDP will be allowed, then eventually all the forms of FDP restricted/unrestricted will be used. Thus, controlling only a part of FDP to be used for information purposes will become a tough task (Kayser and Schneider 2009). 2. Right not to know: This is a universal right and is also a part of universal declaration of the human genome and Human Rights declaration. Within this an individual enjoys his rights “no to know” his own that medical information- all the information gathered from genetic testing. As per the experts, the information like hidden parentage or genetic diseases could have drastic effects on the individual. Thus, the investigation must be limited to analysis of physically visible traits and all the associated information must not be told to the individual himself. Advancements in the technology have revealed that there exists a relationship between the genetic markers and the behavioral traits of the individual. The genomic testing could thus reveal the behavioral aspect of the individual relating it directly or indirectly to the person’s criminal behavior, for example, aggression, anxiety, pedophilia, etc. The experts are concerned that if such information will be revealed to the person or be accepted by the court of law, the aspects like parole eligibility and preventive detention would be affected to a greater extent. A person may take a benefit of this information to make an excuse for his deeds. These are the characteristics of which an individual in himself is aware of, and hence analysts must refrain themselves from publication of such traits so as to avoid any future harm (mentally and socially) to the individual and/or his family (Murphy 2013; MacLean 2013; Kayser and Schneider 2009). 3. Individual freedoms and privacy: Breach of right to privacy is the foremost concern related to forensic DNA Phenotyping technology. DNA testing infers a lot more information about the concerned individual. This information can be abused in later period of time leading to hamper his right to privacy. Does the information that is released in public should be limited, and loan additional information that is beyond requirement should be derived from FDP testing; the testing such as genetic disease behavior propensities should remain confidential, or otherwise the donor individual should be given a choice of requesting the full profile. To avoid the privacy breach FDP data should be disposed as soon as the identification process has been completed (Murphy 2013; Toom 2012). 4. Racial profiling: Since FDP technique can be used to access the geographic ancestry, this clearly means that while identifying the individual from its EVCs his/ her ancestry can also be revealed. This would, in turn, create sense discrimination among the population. The society could label the respective race or the community with a criminal tag, thus encouraging the prejudice. This will be an injury to the complete society and could create fear among minor communities. A belief would be reinforced that a specific community is of a criminal mindset. Hence, not limiting the access of FDP for investigation can cause racial discrimination (Matheson 2016; MacLean 2013; Enserink 2011; Koops and Schellekens 2006).

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However, the solution to these emerging issues would be restricting the use of FDP technology. The legislations put forward by the Netherlands could be followed at global level. The FDP technique should only be implied where the identification cannot be achieved by using traditional means of identification. Last but not the least the advancements in the field of forensic DNA Phenotyping technique should never be blanketed due to emerging concerns; rather a solution to such concerns should be put forward (Laan 2017).

Case Studies: DNA Phenotyping Currently forensic community is using advanced technologies and working in collaboration with software companies like Identitas and Parabon NanoLabs in analyzing the evidences having limited leads (Laan 2017). Pilot studies are being carried out in the Netherlands, Poland, and Australia (Matheson 2016). Further, the active cases in Ontario are being investigated using the novel Forensic DNA Phenotyping technique (Laan 2017). A milestone case in the field of identification by FDP technique was of Delroy Easton Grant (Marcus and Monique 2015). FDP technique has also played an important role in predicting the ancestry of the Baton Rouge Serial Killer, Derrick Todd Lee, who before getting arrested claimed the lives of seven women in 2003. In the year 2004, the case of Eric Copple in Napa, California, was also assisted by FDP technique (MacLean 2013). The infamous case of Robert Barnes of 2008 – the person behind the rape and murder of Meghan Landowski – in Virginia was also solved using the biogeographical ancestry inferred from the DNA collected from the body of the victim (“‘48 Hours: NCIS:’ To Catch a Killer,” 2020). The law enforcement agencies in the USA have been using SNaPshot technology since long. Using SNaPshot technique in combination with Familial analysis using DNA and Y-STR technique aided the identification of José Alvarez, Jr., the person who murdered Troy and LaDonna French (February 2012, North Carolina) (“Parabon NanoLabs: Engineering DNA for Next-Generation Therapeutics and Forensics,” 2016). Moreover, cases like the Bennett family in Aurora, Colorado, in 1984; Lisa Ziegert in Springfield, Massachusetts, in 1992; Sierra Bouzigard in Calcasieu Parish, Louisiana, in 2009; Candra Alston and Malaysia Boykin in Columbia, South Carolina, in 2011; and identification of an unidentified male victim in Glen Burnie, Maryland, in 1985 were resolved using facial reconstruction technique provided by SNaPshot systems (Augenstein 2016; Greenwood 2016; Pollack 2015). The results from the SNaPshot and IDentify analysis assist the investigation where traditional techniques fail to produce any leads. There are numerous cases where the DNA database could not find a match and the case was held stalled. Such cases have been re-examined/investigated using SNaPshot systems where the probable picture of the culprit was created and resumed the investigation. It can be said that the FDP technology is a promising technique to forensic community (“Parabon NanoLabs: Engineering DNA for Next-Generation Therapeutics and Forensics,” 2016).

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The Future of FDP Since the Forensic DNA Phenotyping technology is novel, many research groups are working together to improve and further develop the technique. In such effort The US Department of Defense has granted around $2 million to Parabon NanoLabs (Laan 2017). The US Department of Defense has also signed an agreement upon development of a new technology called Keystone with Parabon NanoLabs. The Keystone module is being created with an aim to formulate a system which being the first platform will be able to integrate NGS tools for analyzing the DNA sample under a common umbrella. It is thought to be a platform where all the essential DNA analytical tools will be available to the forensic community. Keystone technology will soon be commercialized as a product for laboratories (Laan 2017). Apart from this there are the groups of researchers who are continuously decoding the human genome to broaden the horizons of Forensic genetics. Studies are being done in the areas where the additional traits like hair morphology, age determination, facial structure, and adult height could be inferred from DNA analysis (Laan 2017). Furthermore, for the holistic development of the FDP technique, there is an urgent need of developing the analytical domain of research. It is now essential to improve and invent advanced new generation tools and methodologies which can offer parallel sequencing and DNA Genotyping in a short span of time while maintaining the quality of results (Phillips 2015; Kayser and de Knijff 2011). Researchers are also gaining interest over other EVCs like handedness, chin and cheek dimpling, and earlobe attachment (MacLean 2013). In most of the cases only the teeth and blood are being taken as sample; experts today are more focused toward using other biological evidences (semen, saliva, etc.) as sample (Kayser and de Knijff 2011). The main goal of FDP technique is identification of an unknown individual – victim or suspect. Determination of the biological age, as a factor for identification, from the DNA molecule is also achieving interest (Hamano et al. 2016). Since studies related to the FDP technology is still under process, one can imagine how powerful the technology would be in the coming years when all these projects will get commercialized. Surely, the technology would enhance the working capacity of the forensic laboratories around the world. Therefore, there is no doubt in accepting the fact that the FDP technology is a technology of the future.

Conclusion Identification is crucial to the criminal justice system. The science of identification in the past years has seen tremendous advancements and developments. It has evolved from the use of anthropometric methods of identification (Bertilionage) to the very advanced DNA profiling technology, developed by Sir Alec Jeffreys in1984. Since then, the DNA profiling technique proved to be the gold standard technique in establishing individuality across the globe. Since science is ever evolving, a very

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novel technique called forensic DNA phenotyping (FDP) was introduced to forensic genetics. This technique can thus identify and create a “probable” picture of an unknown individual. From its inception, the technique has attracted several scientific experts from the forensic community. Studies are now being conducted on investigating the SNP sets for skin, hair, and eye color pigmentation genes. Systems like IrisPlex, IrisPlex model, and HIrisPlex system have been developed in this regard for forensic purposes. FDP technology is also being applied to the nonpigmented traits like hair structure, baldness, face, body height/stature, and age. The milestone softwares like IDentify by Identitas and SNaPshot Software by Parabon NanoLabs are now available in the market along with few upcoming proposed projects for commercial purposes. As the technology is expanding, countries are formulating new laws with regards to the latest technology in use. Similar to other genetic research works, FDP also poses some ethical issues which are being considered before applying the methodology in forensic case works. Thus, the FDP technique can be seen as a sensitive technology, aiding the forensic community in all its possible forms. It can be foreseen as a promising technology of the future.

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Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Growing Demand on Forensic Laboratories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rapid DNA Testing as an Investigative Tool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Turnaround Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transportability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Key Concerns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Privacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cognitive Challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Function Creep . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Going Forward . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Recent advancements in swift forensic DNA technologies have allowed for faster convictions and exonerations within the criminal justice systems. One particular technology, known as “Rapid DNA,” allows law enforcement to generate a DNA profile in less than 2 h from a buccal (cheek) swab. These devices have a number of potential applications for law enforcement agencies, as well as for military, forensic, homeland security, and intelligence purposes. While the prospective use of Rapid DNA in criminal investigations has garnered major interest, the increased use of DNA analysis has also led to an increase in DNA profiles uploaded into databases, which has surfaced concerns around privacy and the reliability of the machines being developed by private companies. This chapter will broadly outline the costs and benefits of Rapid DNA as a forensic tool within a law enforcement context. J. Simpson (*) Victoria University of Wellington, Tirohanga, New Zealand e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 H. R. Dash et al. (eds.), Handbook of DNA Profiling, https://doi.org/10.1007/978-981-16-4318-7_62

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Keywords

Rapid DNA identification · Forensic analysis · Crime-scene analysis · Short tandem repeat · Forensic laboratories

Introduction The use of Rapid DNA machines in law enforcement is a growing trend internationally. Also referred to as “magic boxes” (Murphy 2019) or “swab in–profile out systems” (Buscaino et al. 2018), this technology operates by producing a DNA profile from blood, saliva, or other biological matter in as little as 90 min, using a simple “sample in–answer out” method (Mapes et al. 2016). Due to their portability and ongoing success, Rapid DNA machines today can be found in a wide variety of places, including at border crossings, embassies, and in traditional forensic laboratories (Butler and Willis 2020). Until recently, conventional laboratory testing conducted by highly trained and qualified personnel was the only option available for analyzing a DNA sample (Della Manna et al. 2016). Today, by some accounts, Rapid DNA machines have the potential to lead to the most significant change in forensic DNA processing since the commencing of the Combined DNA Index System in 1998 (Carney et al. 2019). With the ability to produce swift results, be operated outside the physical boundaries of the traditional laboratory, and be used without any technical expertise, Rapid DNA machines present themselves as an attractive deployment tool for law enforcement personnel. Rapid DNA in the USA received a significant boost in 2017, with the passing of the Rapid DNA act which has fueled law enforcement’s enthusiasm for using this technology to analyze the growing backlog of forensic evidence (Murphy 2019). The goals of this enactment were to better inform decisions about pretrial release or detention, solve and prevent crimes, exonerate the innocent, and prevent DNA analysis backlogs (Rapid DNA Act of 2017, Public Law 115-50). At present, Rapid DNA machines can be introduced in three locations within a law enforcement context. These include inside forensic laboratories, within crime scene units, and at police stations (Wilson-Wilde and Pitman 2017). While there is growing evidence that Rapid DNA might play a crucial role in improving the efficiency of law enforcement procedures, there are several important issues raised by this new form of testing which cannot be ignored.

Growing Demand on Forensic Laboratories For many law enforcement agencies, conducting DNA analysis requires a major commitment of finite resources. While the swift, autonomous process of Rapid DNA testing is still less sensitive than standard laboratory procedures, the growing demand for police investigative units is driving a need for a more efficient process. The turnaround time of urgent crime scene DNA samples is often far longer than desired, which leads to increased pressure on police investigatory units to produce a

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turning point in the investigation of the sample (Buscaino et al. 2018). Similarly, laboratories around the world are experiencing a “ ood of sample submissions” (Butler 2015, p. 370) in the wake of the growing success of DNA analysis in solving crimes. As a result, backlogs and extended waiting periods have become routine throughout most DNA typing laboratories in the USA (Dash et al. 2020). This alone raises an important issue around unsubmitted samples, with a growing amount of evidence stockpiling in police storage facilities that have not been sent to labs for testing. Because of this, many states have passed laws requiring police to review their evidence rooms for unsubmitted evidence, further increasing laboratory workloads. Illustrating this, the number of untested cases throughout the USA has grown by 85% in the past 6 years (Jackman 2019). The recent uptake of Rapid DNA machines in forensic laboratories and police stations has shown the potential to help fix this growing problem. A recent example of success can be seen throughout Illinois with State Police Director Brendan Kelly recently reporting a 33% overall backlog reduction in DNA samples (Governor’s Task Force on Forensic Science Report 2020). It is believed that Rapid DNA implementation played a significant role in achieving this reduction (Spinelli 2020).

Rapid DNA Testing as an Investigative Tool In response to the growing demand for criminal justice systems, there has unsurprisingly been a strong drive for efficient, portable forensic technology (Morrison et al. 2018). It is believed that the main drivers of demand for this growing need for service have been the increased amount of DNA evidence that is collected in criminal cases, as well as the expanded effort to collect DNA samples from convicted felons and arrested persons (National Institute of Justice 2010). Perhaps the most salient and documented benefit of Rapid DNA systems is the speed at which they can analyze a sample. Rapid DNA drastically cuts down wait times which could otherwise be weeks to a period of under 2 h. Fast results are of particular importance in homicide cases, where the first 24-h period is crucial to an investigation (Wilson-Wilde and Pitman 2017). The value of real-time forensic information produced by Rapid DNA is well summarized by Van Asten, who argues that the availability of fast data directs the investigation and allows police to use their “scarce resources efficiently and effectively, ultimately solving more crimes” (p. 33).

Turnaround Time Rapid DNA machines allow law enforcement officials to generate rapid intelligence, which provides great benefits to solving investigations. Such machines have been specifically developed in a way to combine the four stages of extraction, amplification, separation, and detection of a DNA sample, all without the need for human input. This autonomous process operates in contrast to the conventional, labor-intensive processes of laboratory analysis procedures, which can often take

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months to produce a profile (Steward 2016). In many cases, a single sample can last 5 months or longer in storage before being turned around (Neuhauser 2019). In response to this growing issue, Rapid DNA devices have become quickly adopted with an overarching objective to help reduce this backlog of samples. So far, Rapid DNA systems have received praise for their efficiency from law enforcement officials and academics alike, including the US National Association of Police Organizations. The speed of Rapid DNA analysis is beneficial for law enforcement in several ways. As well as decreasing backlogs, Rapid DNA systems also have the potential to provide an early lead in the investigation, which is particularly beneficial for police who are constantly limited by their finite resources during investigations. This is especially relevant for New Zealand’s current situation, as failure to investigate was the number one complaint made against New Zealand Police in recent times. Processing a sample in real time during an investigation is additionally beneficial because stations typically have a 24–96 h holding window on suspects before having to formally charge them of a crime, or otherwise release them. The testing can work to help ensure the apprehended individual is not linked to any unsolved crimes before their release. Another benefit of Rapid DNA testing within law enforcement is its applicability to a wide range of different crime types. Although originally touted for its potential to revolutionize rape investigations (Schuppe 2019), Rapid DNA’s applicability to lower-level crimes has also been noted by academics who argue that the need for fast DNA analysis is actually of less importance for significant crimes. This is because a suspect of a serious crime would likely be remained in police custody, regardless of available DNA evidence. For less significant crimes, however, there is the possibility that a cold link to a serious matter could be obtained, which better allows investigators to link crimes and could in uence decisions around whether to retain the person in custody. Studies from Wilson et al. (2010) also allude to the benefits of Rapid DNA testing in burglary cases by highlighting the noteworthy percentage increase of a successful identification across routinely low-clearance types of offending, such as burglary. This has also been demonstrated by a series of randomized controlled trials by Roman et al. (2008) across the USA, showing a two- to threefold increase in the percentage of a successful resolution for property crimes when analyzing collected DNA from the crime scene.

Transportability Transportability is another key advantage of Rapid DNA devices. Historically speaking, forensic DNA identification methods have suffered from high latency and low portability (Zaaijer et al. 2017), meaning that analysis has been limited to specialist laboratories and often suffers from long delays in producing information. With the ongoing success of forensic DNA analysis, however, it is not surprising that the use of Short Tandem Repeats is broadening to more efficient measures beyond the laboratory (Grover et al. 2017). Khanna et al. (2020) note that the turnaround time for processing a sample is further reduced by the capacity for Rapid DNA

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devices to be stored at police stations, or even be taken at the scene, as opposed to having to send evidence away to a laboratory. About the size of a large desktop printer (Jackman 2018) and weighing 60 pounds (Tucker 2015), their portability factor means that the devices can also be used by first responders in the field for a wide range of disasters, such as earthquakes or other mass casualty events to identify victims. Indeed, this is an area that Rapid DNA machines have already proven to be highly effective in. In particular, Rapid DNA played a significant role in victim identification during the 2018 Californian wildfires. Nevertheless, despite their wider successes in a broad array of circumstances, some express concern that the devices could be used for purposes beyond their intended means. For example, FBI chief biometric scientist Thomas Callaghan believes Rapid DNA machines are currently best suited to analyzing large amounts of DNA from one person, as opposed to crime scene use which commonly contains the DNA of multiple people (Jackman 2018).

Contamination Contamination of a DNA sample is a major issue in forensics, as it can result in greatly reduced genotype quality for sequencing studies (Jun et al. 2012). The limited existing literature has shown mixed results regarding the risks of contamination when using Rapid DNA devices. Scholars like Busciano et al. (2018) have alluded to the diminished risk of contamination from the use of these devices, although this is contested (see Dolan 2019). On the one hand, the autonomous processing of a sample decreases the likelihood of external contamination, as it requires no human input. Zhang et al. endorse this line of thought, arguing that by autonomously combining the steps of DNA analysis, the risk of analysts accidentally introducing their DNA is eliminated. On the other hand, the reality that non-laboratory professionals are now responsible for gathering and processing samples through the devices increases the risk of polluting or damaging the sample. This is because they lack the extensive education and training with forensic equipment that laboratory professionals have. We know that crime-related traces may easily be confused with traces produced by events before and after the crime. Further to this, the first extraction phase is the step where the DNA sample is most susceptible to contamination. If a sample is not installed correctly and/or becomes contaminated beforehand, this runs the risk of leading police to the wrong person in an investigation or jeopardizing the case as a whole. In one instance, incorrectly processed DNA led to the incrimination of an impossible suspect, a man who had been dead for 2 years (Murphy 2015). Such inaccuracies have also charged at least one innocent man with capital murder (Worth 2018). It is also crucial to note that many Rapid DNA machines are currently stored in offices as opposed to sterile laboratories (Balk 2015), which in itself heightens the risk of contaminating the sample. One criticism pointed out by Salceda et al. (2017) is that most Rapid DNA machines can only process one sample at a time, which suggests that their applicability to crime scenes may be limited. This is because such environments often

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contain a mixture of DNA from several individuals, thus increasing the likelihood of sample contamination or a false match. This is noted by Romsos et al. (2020), who advocates for Rapid DNA’s use strictly for single-source samples only. In sum, the limited set of existing literature is inconclusive regarding the risk of contamination when using Rapid DNA devices. However, even traditional DNA analysis undertaken by professionals in recognized laboratories have led to drastic errors. Whether or not the expansion of DNA testing beyond the laboratory increases the number of inaccuracies remains uncertain. Future success and accuracy rates of Rapid DNA will largely depend on the operators of these devices receiving high-quality training in evidence collection methodology.

Key Concerns Privacy At the heart of most concerns with DNA in criminal investigations are issues around privacy. Academics such as Murphy (2018) and Butler (2015) have raised concerns about privacy, equality, and abusive government surveillance stemming from autonomous forensic DNA testing. In the USA, recent law changes have allowed police officers to obtain DNA samples from arrestees without the need for a warrant (Maryland vs King 2013). Due to the high degree of law-abiding citizens who have encounters with police, the increasing use of Rapid DNA systems puts innocent people at risk of prosecution based on their DNA, eroding the presumption of innocence (Quick 2019). Showcasing their impact in recent times, it is estimated that forensic DNA analysis has identified more than 440,000 previously unknown suspects over the past 20 years (Jackman 2019). Although evidence exists that the growth of DNA databases does have a net deterrent effect on convicted offenders (Doleac 2017), several concerns are raised by Rapid DNA in law enforcement and the subsequent expansion of forensic databases. As the growing number of profiles in databases increases, the likelihood of finding an offender, the chances of inaccuracies and false matches also rise. Some have commented that the enthusiastic, increasing deployment of Rapid DNA machines threatens the tradition of protecting the privacy and rights of individuals who would not otherwise be under suspicion (Flaus 2013). While the goal of clearing backlogs has achieved some success already, there is concern around the increasing use of DNA within the criminal justice system resulting in a high number of innocent people’s information being stored in databases. With record-high reports of cybercrime in recent times (Healy and Mcgrath 2019), the threat of this data being hacked could lead to serious privacy breaches. In Samuel and Prainstack’s (2019) study around stakeholder perceptions of forensic DNA profiling, around half of the interviewees described FDP as “intrusive” or “invasive.” Furthermore, it has been pointed out that these machines are also likely to incentivize the growth of rogue DNA databases, which are maintained with far fewer quality, privacy, and security controls than federal databases (Eidelman and Stanley 2019). Also, others have expressed worry that providing genetic material to

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the government allows unscrupulous analysts to reveal health conditions and other sensitive information (Doleac 2017). The increasing use of forensic DNA testing raises important ethical and social policy discussion points around how much authority we give law enforcement officials in these types of scenarios.

Cognitive Challenges Rapid DNA is widely considered to be accurate, though imperfect (Kloosterman et al. Kloosterman et al. 2014). At present, it is still in the early stages of being accepted as a reliable alternative to standard DNA tests (Cino 2017). In 2018, testing found that Rapid DNA devices were accurate approximately 85% of the time, with the potential for increased accuracy when operated by laboratory experts. Its applicability to a law enforcement context has faced some criticism for the “time/success rate trade-off” that Police and Scene of Crime Officers face when using this technology. In law enforcement agencies that use Rapid DNA machines, Scene of Crime Officers are confronted with a dilemma of whether to utilize this fast-acting technology or forward the crime sample to the laboratory, where the rate of accuracy will be greater. It remains the case that this technology is less still sensitive than traditional methods carried out at the forensic laboratory (Mapes et al. 2016). Because of this, Rapid DNA instruments are not, at present, sufficiently matured for use in analyzing crime scene evidence for automatic submission to CODIS (Hares et al. 2020). In one previous study, the sensitivity rate between laboratory and Rapid DNA testing differentiated by 5% in favor of laboratory testing (Schroeder and White 2009) when examining a sample from a ski mask. Rapid DNA profiling has, however, proved to be especially accurate in certain scenarios, such as trace detection of bloodstains at crime scenes (Vittori et al. 2016). Cino (2017) advises that the devices are currently best suited to quickly rule out or further investigate an individual suspected of a crime. She further recommends that a prosecutor seeking to use DNA evidence during a trial should be required to revert to traditional laboratory analysis to argue his/her case in the courtroom. This is because the validity of Rapid DNA still could be brought into question during a courtroom case, simply due to its novelty (Seaone 2019). Although this technology has made considerable improvements since its conception, it is not expected that rapid analyses will reach the precision of laboratory testing any time soon (Mapes et al. 2019), meaning that this cognitive dilemma faced by law enforcement officials is likely to persevere. However, due to the urgent nature of criminal investigations, we can expect frontline officers to favor rapidity over sensitivity when considering the high demand for their services (Mapes 2017). Helsloot and Groenendaal (2011) highlight that the internal factor of emotion can greatly impact an officer’s discretionary powers in deciding to employ a Rapid DNA test, especially in instances where the “desire to see justice done” (p. 897) is stronger. Furthermore, early studies from Tversky and Kahneman (1974) have argued that people are inclined to act quickly when faced with uncertain situations, which can lead to errors when an officer’s categorizations are incorrect (Gigerenzer et al. 1999).

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Function Creep Ease of use combined with affordability is a recipe for overuse or “function creep.” In a society that favors certainty over accuracy (Stevens 2013), there is a high potential for this technology to be overused by law enforcement officials, or be used for reasons beyond their intended purpose. Unlike skilled laboratory technicians, frontline officers who use Rapid DNA machines are trained for a couple of hours and lack any distinct protocols when using the devices (Hawkins 2020). Although Rapid DNA testing is autonomous, the human factor plays an important role even when complete trace analyses are conducted by machines (De Gruijter and de Poot 2019). This is especially relevant as DNA is often considered the “gold standard” of a forensic investigation, making officers even more eager to obtain incriminating evidence from a suspect. Furthermore, as Gruijter et al. (2016) suggest, the use of Rapid DNA devices at crime scenes could potentially cause a shift in investigatory focus toward finding and analyzing alleged perpetrator-related traces, resulting in other crime-related traces being overlooked. The direction of an investigation could be in uenced if crime scene officers prioritized the search for specific traces that are compatible with Rapid DNA machines. This narrow focus would mean that noncompatible traces might be labeled as irrelevant (Wyatt 2014) if they do not fit the requirements for a Rapid DNA device. De Gruijter and colleagues make note of several miscarriages of justice stemming from the negative consequences of “tunnel vision” (p. 850) during an investigation. It remains the case that crime scenes can and should be examined in multiple ways (De Gruijter et al. 2017). The measures taken to protect against tunnel vision or contamination mean that some work processes may be set up less efficiently, for example, more protocolled, to avoid the risks of bias (Groenendaal and Helsloot 2015). Another key concern of Rapid DNA’s use in law enforcement is that databasematches may have too much in uence on an investigation, leading to scenarios being based on database-matches without considering the relevance of the traces that did provide a match. This is especially problematic considering the high volume of nonoffenders who may have a profile in Combined DNA Index Systems. If used improperly, the potential for error and breach of public trust is extremely high (Hoey 2019). Weiss (2020) summarizes that the future may be bright for Rapid DNA systems, but only if quality and integrity are maintained at both organizational and frontline levels. While Rapid DNA by design is geared toward efficiency and simplicity, they do present new cognitive challenges for users around whether to employ the machines and to what extent they should direct an investigation. Little research has been conducted on CSI decision-making (De Gruijter et al. 2016), yet it is arguably more important now than ever to understand the growing development of potentially behavior-changing technologies. The accuracy of Rapid DNA is expected to increase with time, yet it should not be assumed that the benefits of forensic DNA will necessarily override the social and ethical costs (Roewer 2013). Looking into and addressing some of the cognitive challenges faced by frontline employees at a training level should not be ignored as a route to future success.

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Going Forward To weigh these issues against the potential value of Rapid DNA machines, several approaches should be considered. Firstly, quality of training is essential as it is crucial for law enforcement officials to recognize the full capabilities of Rapid DNA testing, including its potential downsides. Operators of these devices should be competent not only in a technical manner but also in ensuring the nondisclosure of private information from the results produced. Revisiting the IACP (2001) study on the implementation of police in-car cameras, researchers argued that “as with any new technology, failure to properly train officers in its use, operation, and legal implications of improper use can result in disaster” (p. 19). While Scene of Crime Officers (SOCOs) have strict procedures and protocols in place for collecting evidence, Wyatt (2014) observes that such investigators routinely go beyond the provided guidelines and utilize their discretion in decision-making. Mapes et al. (2019) similarly note that the majority of SOCOs’ decisions in practice are likely based on intuition. Therefore, it is crucial to recognize that policy or protocol alterations do not guarantee changes in operational behavior. Instead, positive changes should look to be driven by proper and regular training procedures. The increasing use of swift DNA technologies could also facilitate the growth of other forensic DNA techniques such as familial searching, which similarly has shown the potential to be a valuable investigative tool. Perhaps in the future, we could see the development of machines that can combine rapid technologies with other forensic analysis results, such as familial searching and phenotyping. However, growth in any form of forensic DNA analysis further expands databases to include innocent individuals. If forensic DNA systems are employed more frequently, public trust in the technology is vital (Samuel and Prainsack 2019). It is also necessary that the high levels of trust extend to law enforcement officials, who in recent times have come under intense public scrutiny by citizens, politicians, and advocacy groups due to allegations of misconduct (Ufford 2019). Today, growing calls to “defund the police” have emerged, largely due to ongoing incidents of excessive use of force and alleged racial bias of officers on duty. This has spotlighted the perceptions of many that police officers are untrustworthy, with consistent media portrayals of tense confrontations between police officers and citizens. Stemming from the high levels of distrust in recent times, especially from minority groups, there have been greater calls for oversight and review of police decisions while on duty. To be effective in their duties, law enforcement officials are highly dependent on public support and cooperation (Skogan and Frydl 2004). Concerns around differential policing and officer misconduct when using Rapid DNA could be addressed by body-worn camera use for officers. As frontline officers largely work without direct supervision, there is potential for their high levels of discretion to be used in a discriminatory way, which could undermine police legitimacy (Lum 2011). With the absence of empirical evidence, it is unlikely that such officers will be held accountable for their actions. However, studies have demonstrated that the presence of video recording technology in law enforcement may have a “civilizing effect” on police-citizen encounters (Ariel et al. 2015). Others report behavioral improvements in police

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officers from their deployment (Ufford 2019). Furthermore, officers equipped with this technology are shown to be significantly less likely to have a complaint sustained against them (Katz et al. 2015; Ariel 2016). Such findings fundamentally relate to the Hawthorne Effect Theory, which argues that individuals behave differently when they are being watched (Adair 1984). The adoption of body-worn cameras could be a beneficial accountability tool to ensure that law enforcement officials are using swift DNA technologies in a legitimate manner and context. Unfortunately, many law enforcement agencies are hindered by financial constraints in adopting the latest technologies. Despite this, the costs of running a sample through a Rapid DNA machine are expected to reach all-time lows in the near future. For instance, Weiss (2020) estimates that Rapid DNA will soon be estimated at $100 per sample, compared to $500 in a forensic laboratory. However, it is important to recognize that the cost of swift DNA technologies does not just relate to the equipment and consumables but also the infrastructure (e.g., waste disposal) and logistical maintenance (Wilson-Wilde and Pitman 2017).

Conclusion Rapid DNA, even in its developing state, is useful. In the past, successful DNA testing has relied on the skill of experienced laboratory analysts. Today, Rapid DNA is an exciting adaption to help law enforcement and criminal justice meet the growing needs of communities. The significance of this new technology to aid in criminal investigations largely derives from its efficiency, portability, and ease of use. However, research into its effectiveness in a law enforcement context is still in its infancy, with unanswered questions remaining around its accuracy and applicability to forensic investigations. Additional concerns lie with the cognitive challenges and bias that law enforcement officials may face when choosing to employ the technology. Perhaps the biggest key to the future success of Rapid DNA will involve ensuring that adequate training is provided for officers who may be charged with the operation of the devices. Over the next decade, it is expected that DNA testing will become more rapid, more informative, and more sensitive (Butler 2015). As the amount of time taken to analyze a sample continues to decrease, we might see the expansion of Rapid DNA machines to other areas of law enforcement, for instance, in police vehicles. To avoid future scrutiny around ethical and privacy concerns, it is of crucial importance that these technological advancements do not become used as a surveillance tool rather than for their original intended purpose as an aid for criminal investigations.

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Tracing of Human Migration and Diversity by Forensic DNA Analysis

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Nithyanandam Mahalaxmi, Avinash Chand Puri, Pawan Kumar Chouhan, and Alka Mishra

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA as a Palimpsest of Human Migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General Markers Used in DNA Profiling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Short Tandem Repeat (STR) Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondrial DNA (mtDNA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Y Chromosome Marker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Human Migration: Origins in Africa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Out of Africa and the Conjunction with Neanderthals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Peopling of Europe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Colonization of Asia and Oceania . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Populating Americas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Denisovans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Human migrations have changed the face of lands and continents, racial, ethnic, and linguistic composition of their populations. Many people perish on perilous treks around the world in search of a better life. However, there is not much being done to track down these people. DNA profiling has made it possible to track these migrants with its advanced techniques and established protocols. DNA profiling is still a gold standard technology for human identification. In the last three decades, genetic research has become increasingly applicable in the study of human history. Several advancements in our knowledge of human migration have N. Mahalaxmi Forensic Sciences Department, Chennai, Tamil Nadu, India A. C. Puri (*) · P. K. Chouhan Regional Forensic Science Laboratory, Indore, Madhya Pradesh, India A. Mishra Regional Forensic Science Laboratory, Gwalior, Madhya Pradesh, India © Springer Nature Singapore Pte Ltd. 2022 H. R. Dash et al. (eds.), Handbook of DNA Profiling, https://doi.org/10.1007/978-981-16-4318-7_65

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been made possible by developments in the sequencing and study of both current and ancient peoples’ genomes. They have helped to prove that anatomically modern humans initially arose in Africa around 250,000–350,000 years ago and then spread to other provinces of the world, and the humans’ genetic adaptations to local environmental conditions are among them. Analyses of this original genetic data are transforming our understanding of humans’ migration history and adaption. We may now collect whole-genome sequences from a variety of existing and prehistoric Africans using today’s sequencing technologies. In the coming years, significant discoveries will be made in the study of human migration. Keywords

Haplotypes · Mitochondrial DNA · Y chromosome marker · Human migration

Introduction When Alec Jeffreys of the University of Leicester in the United Kingdom discovered remarkably variable and heritable patterns from repetitive DNA studies using multilocus probes, his “Eureka” cry shook England and was heard across the world. He named this technique “DNA fingerprinting,” a first breakthrough that opened a new area of science (Jeffreys et al. 1985; Roewer 2013). Genetic analysis, in its most basic form, entails comparing DNA from different groups of people, such as those with or without a specific type of disease or people from different parts of the world. Since the sequencing of the complete human genome, our ability to sequence DNA has improved considerably (Johnston et al. 2019). We do not need to look at all of the three billion letters in the human genome because persons differ by only one letter in a thousand on average. Instead, we can compare persons who have known genetic differences, which are referred to as genetic markers. Millions of these markers have been identified, and this, along with genetic sequencing technology that allows us to look at these markers in a large number of people at a low cost, has resulted in a massive increase in the amount of data available to geneticists. Dr. Kary Mullis, a scientist at the Cetus Corporation in Emeryville, California, created the polymerase chain reaction (PCR) in 1985, which was the second major advance in molecular biology. By allowing for the amplification of specific sections of highly polymorphic DNA, this stunningly simple technology revolutionized forensic DNA analysis. This not only gave forensic analysts the sensitivity they needed to extract valuable DNA profiles from difficult forensic material, but it also laid the groundwork for the many generations of DNA-based typing procedures that are now employed in forensic laboratories. Contemporary biology has given scientists strong tools for reforming the past of the Earth and its residents, including humans. Over the last 25 years, experts have endorsed the multiregional continuity hypothesis (Thorne and Wolpoff 1992) that modern humans left Africa around 350,000 years ago as a single developing species,

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Homo sapiens, and expanded over the world (Schlebusch and Jakobsson 2018). The formation of recognized regional morphologies in the continents of Africa, Europe, and Asia is justified by some skeletal traits that originated and lasted for differing periods in distinct geographical regions. The “recent out of Africa” approach (Wilson and Cann 1992), on the other hand, claimed that since people began to radiate out of Africa, there have been emergent patterns. This scenario similarly claims that H. sapiens first appeared in Africa some 100,000 years ago and quickly spread over the world, displacing other species. During the previous one and a half decade, molecular evidence from populations from many ethnic groups around the world has made a significant contribution to the discussion. Rapid discoveries in DNA sequencing, on the other hand, have opened up a whole new window into the past, showing that human history is more complicated than previously thought. In fact, recent DNA investigations have revealed that humans have migrated and intermingled far more than previously assumed – particularly over the last 10,000 years. In this chapter, it is aimed to shed some light on the crucial associations of DNA profiling in human migrations around the world.

DNA as a Palimpsest of Human Migration When Alec Jeffreys identified considerable individual variation between persons in certain areas of DNA using the variable number of tandem repeats (VNTRs) and restriction fragment length polymorphism (RFLP) analysis in 1985, he coined the term “DNA profiling” as a means of positive identification. While DNA testing was initially used to determine paternity, its use in solving human identification became clear when DNA examination positively identified the skeletal remains of Josef Mengele (Jeffreys et al. 1992). The capacity to examine creatures’ genetic material has been critical to this progress. Today, scientists can extract DNA from microbes, animals, plants, and humans, including their fossils. We can sequence DNA and obtain crucial information about the evolutionary history from the sequences. To date, a substantial amount of human DNA sequence data has been acquired. These have been used to analyze our variety and how individuals from different geographical places or cultural groupings are genetically connected. The Human Genome Project, which began in 1990 and ended in 2003, accompanied a new era of forensic DNA assessment. The Human Genome Project sequence aided in the discovery of a wide range of short tandem repeat (STR) sequences. PCR-based technique by Dr. Kary Mullis aided in rapid amplification of these polymorphic genetic markers, allowing population studies to examine their genetic variation. By the mid-1990s, DNA profiling effectively integrated high sensitivity provided by PCR-based technologies, higher discrimination power than previously achievable, and increased throughput provided by simultaneous amplification of many STR markers in a single multiplexed reaction. Since then, the field has seen a steady increase in the number of STR markers and marker classes used. In human migration research, nuclear DNA, mitochondrial DNA (mtDNA), and Y chromosome DNA profiling have been utilized, and each type of DNA contains

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Table 1 Analytical timeline of milestones in human evolutionary genomics. A great number of research have used genetic data to gain key insights into human history; those with the most clout in terms of the data or data analyses presented are highlighted (Nielsen et al., 2017; Orlando et al., 2021) Year 2010

2011 2012 2014

2015

2016 2017 2018 2019 2020

Analytical timeline of milestones in human evolutionary genomics First ancient human genome First draft Neanderthal genome First draft Denisovan genome 1000 genomes project phase I data First aboriginal Australian sequenced from 90-year-old tuft of hair Neolithic Europeans sequenced 12.6-kyr-old Clovis individual sequenced 23-kyr-old Malta individual sequenced 45-kyr-old Ust’ishim individual from Siberia sequenced 38-kyr-old upper Paleolithic European genome 4 L-kyr-old European individual with recent Neanderthal introgression sequenced Large population genome studies of bronze age Europeans and Asians Large genomic studies of ancient and modern native Americans, polo-Eskimo people, and the Invit Large population genomic studies of Eurosians and Australians First automated capture of ancient DNA Tree-based selection scans kinship inference, READ Ancient metagenomic profiling (HOPS) High-accuracy phylogenetic assignation of metagenomic data Paleofeces host species identifier, coproHD

different genetic information (Holobinko 2012). Only nuclear DNA represents both the parental genetic materials as one copy is inherited from both the parents, while mtDNA and Y chromosome are nonrecombinant. MtDNA is maternally inherited and Y chromosome paternally, and hence both are traceable to a single female and male ancestral type, respectively (Cox and Hammer 2010). The unique identification abilities of each one are enhanced by the difference in copies per cell between nuclear DNA (2 copies) and mtDNA (more than 1000), which enables positive identification of the individual while mtDNA substantiates family relationships. Ahead of the technology horizon, forensic applications of next-generation sequencing (NGS) technologies have also been significantly improved to provide useful information on an individual’s kinship, ancestry, and even phenotype. Analytical milestones of DNA profiling in human migration and evolution are given in Table 1.

General Markers Used in DNA Profiling Short Tandem Repeat (STR) Markers Microsatellites, which include simple sequence repeats, enlarged simple tandem repeats, and STRs are a type of genetic markers that are particularly valuable in DNA profiling. These markers are made up of 2–9 base pair DNA motifs that are

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tandemly repeated 5–50 times. The human genome contains thousands of STRs loci (Collins et al. 2003), and they are distinguished for their high mutation rates caused by polymerase slippage. As a result, these markers are remarkably polymorphic, with varying numbers of tandem repeat units almost in every population. STRs have been the prime genetic marker that is used to individualize evidentiary material for DNA profiling due to their small size and highly variable nature. This large number of STR loci gives forensic laboratories a lot of options when it comes to incorporating them into commercial kits. Separating various size fragments, usually by capillary electrophoresis, and detecting uorescently tagged products are required for analyzing amplified STRs. Profiles created from evidence can then be compared to reference samples to determine whether they match, exclude, or are inconclusive. The most useful STR loci (e.g., European Standard Set: FGA, D2S441, TH01, VWA, D1S1656, D10S1248, D12S391, D8S1179, D18S51, D3S1358, D21S11, and D22S1045), the UK core loci (FGA, D2S1338, D18S51, D3S1358, D8S1179, VWA, D16S539, TH01, D19S433, and D21S11), the German core loci (FGA, TH01, SE33, D8S1179, VWA, D18S51, D3S1358, and D21S11), and the Interpol Standard Set (FGA, TH01, VWA, D3S1358, D8S1179, D18S51, and D21S11) (McKiernan and Danielson 2017), and those that are commonly employed in the field, are those that have enough individual variability to allow an inferred linkage of an evidential DNA profile to a specific individual of interest. These STR loci are thought to have a high discrimination power, especially when many STR loci are used. In most cases, the best loci for human identification are those with the most variance in tandem repeats across human population groupings. One key drawback of utilizing STRs as genetic markers is that they are subject to various degrees of stuttering (Walsh et al. 1996). Stutter is an amplification artifact that occurs as a result of strand slippage during the PCR process. As a result, the DNA polymerase fails to duplicate one or more repeating units or copies the same repeating unit several times. The former produces an amplification product that is one or more repeat units smaller than the genuine genomic allele, whereas the latter produces a product that is typically one repeat larger than the genuine genomic allele (Walsh et al. 1996). STRs, which comprise a four-base-pair core sequence motif (tetranucleotide repeats), are associated with low-stuttering rates and have thus become the most extensively used markers for human identity (Edwards et al. 1991).

Mitochondrial DNA (mtDNA) Despite accounting for a small portion of an organism’s total genome size, mtDNA has become one of the most common markers of genetic variation in animals during the last three decades (Galtier et al. 2009). It is a circular, double-stranded molecule of 16,569 bp size that contains 2 rRNA genes, 22 tRNA genes, and 13 structural genes that code for mitochondrial respiratory chain subunits (DiMauro and Schon 2003). The mutation rate in mitochondrial genomes is several times above in nuclear sequences (Saccone et al. 2000). Many distinct mtDNA variations are detected in one individual as a result of such a high rate of mutation events. The mitochondrial genome has several properties that make it an appealing subject for DNA profiling.

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The circular structure of mtDNA and its subcellular confinement within the mitochondrion contribute to its stability. In many tissues, this results in a large mtDNA copy number per cell (i.e., hundreds to thousands of copies of the mtDNA genome per cell) rather than only one copy of the diploid nuclear genome per cell (McKiernan and Danielson 2017). Allen et al. (1998) and Robin and Wong (1988) found that this feature makes it easier to analyze extremely degraded and/or little amounts of initial material. Human mtDNA (both males and females) is entirely acquired through the mother, therefore an individual’s mtDNA haplotype is a direct re ection of the up to 100,000 copies of the mitochondrial genome, present in the oocyte at conception, and has few key conserved coding regions (Galtier et al. 2009). As a result, the mtDNA haplotype is the female lineage’s counterpart to the Y-STR haplotype. The examination of mtDNA is a viable option in circumstances when analysis of biological material is problematic (e.g., badly decomposed or skeletonized remains) (Loreille et al. 2010) or nuclear DNA cannot be recovered (Budowle et al. 2003) due to the much higher number of mtDNA copies per cell. The possibility of generating an mtDNA profile in these circumstances makes it a particularly useful technique for historical investigations (e.g., Romanov children (Coble et al. 2009)), and modern forensic cases as mtDNA can be compared to any maternal relative if no immediate relatives are available for body identification or a nuclear DNA comparison (Decorte 2010). Investigations on DNAs of mitochondria have aided in the development of hypotheses for significant human migration events. For example, analysis of mtDNA in combination with archaeological and climatological data revealed that prehistoric migrations and demographic expansions were linked with paleoclimate (Forster 2004). A number of key evolutionary findings have come from studies of mtDNA. Most crucially, the first study of worldwide human mtDNA sequence variation led to the adoption of the out-of-Africa theory, which proposes that modern humans began in Africa and spread outward from there (Vigilant et al. 1991). The out-of-Africa scenario is supported by phylogenetic trees of human mtDNA sequences, which have a root in Africa. L1, L2, and L3 were the first haplogroups to emerge from Africa. In Europe and Asia, haplogroup L3 gave rise to haplogroups M and N (Fig. 1). Haplogroup N spawned haplogroups H, I, J, N1b, T, U, V, W, and X, all of which can still be found in Europe today. In Asia, haplogroup M gave birth to haplogroups A, B, C, D, F, and G. Native Americans also have haplogroups A, B, C, and D (Fig. 1). mtDNA sequences are often aligned using the Cambridge reference sequence, which is the first fully sequenced mtDNA genome and is derived from European haplogroup H. (Nesheva 2014).

Y Chromosome Marker The nonrecombining region of the Y chromosome has increasingly become an important tool for studying human evolutionary relationships. With only roughly

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29-42 KYA

12-15 KYA

X ACD B

AB

X W,T,J,K

CD 4050KYA

L3 L2 L1 L1a

B ACD

N M 100200K YA

3060KYA

G

59-69KYA

L1a

Fig. 1 Geographical distribution of human mtDNA haplotypes (letters in map). The image depicts the origin of anatomically modern humans and possible routes of human migrations “out of Africa” (Witas and Zawicki, 2004)

60 million base pairs of sequence, the Y chromosome is the smallest of the human chromosomes. Y chromosome DNA, like conventional STR markers, is found in the nucleus; nevertheless, there are some major distinctions between markers on the Y chromosome and autosomal STR loci (Kayser 2003). To begin with, the Y chromosome is inherited paternally, and nearly all of the DNA in the Y chromosome is nonrecombinant, with the exception of the Y chromosome’s most distal parts (Skaletsky et al. 2003). As a result, a Y haplotype is normally identical among all male relatives of a given paternal family lineage (unless in rare cases of meiotic mutation). The Y chromosome’s single nucleotide polymorphisms (SNPs) make these markers show human genetic variation. Based on the studies of global populations (Jobling and Tyler-Smith 2003), we now have a lot of information about the regional origins of Y-SNPs. This makes Y chromosome markers relevant in human identity and paternity cases, but it drastically reduces the power of discriminating obtainable when compared to autosomal STR loci. SNP haplogroups can be used directly to evaluate mixing among distinct populations without resorting to more complex admixture models (Bertorelle and Excoffier 1998) due to the high geographic specificity of Y-SNPs (Jobling and Tyler-Smith 2003). The current Y chromosome genotype-naming system has identified 20 primary haplogroups, designated A through T (Karafet et al. 2008). The genetic variations observed in the Y chromosome of Native American populations were

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connected with the dispersal of Siberian groups demonstrating the presence of two separate populations in the New World (growth of Asian populations) (Bianchi et al. 1998).

The Human Migration: Origins in Africa The oldest evidence for anatomically modern humans comes from Ethiopian fossils dating from 150,000 to 190,000 years (150–190 kyr) ago (McDougall et al. 2005) (Fig. 2). Beyond Africa, anatomically modern humans have been found in the Middle East as early as 100 kyr ago (Grün et al. 2005), and in southern China as early as 80 kyr ago (Liu et al. 2015). Neanderthals, who vanished from the fossil record about 40 kyr ago (Higham et al. 2014), have been discovered as far back as 400 kyr in Eurasia. Initial studies of genomic diversity revealed that Africans have the highest levels of diversity of any living population (Rosenberg et al. 2002). A study of microsatellite DNA variation in more than 3000 Africans discovered 14 ancestral population clusters that corroborated the evidence that the root of the human mtDNA phylogenetic tree is in Africa revealing the extensive population substructure. These findings were substantially validated by genome-wide SNP genotyping studies (Busby et al. 2016). These and other research findings suggest that African

Paleo-Eskimo expansion 4-5 kyr ago Inuit expansion 3-4 kyr ago Sintashta expansion 2.5-3.5 kyr ago Neolithic expansion 10 kyr ago

Yamnaya expansion 4.5 kyr ago PCS

IP

CA

Out of Africa 55-65 kyr ago

Migration of click-language speakers 50 kyr ago

People of America across Bernigia 15-23 kyr ago

FC

Northern-southern 13 kyr ago

Alternative route to America Paleolithic Eurasian 45-55 kyr ago

Migration into Sahul 47.5-55 kyr ago

Polynesian expansion 3-5 kyr ago

Possible precolumbian contact

Fig. 2 Major human migrations around the planet as deduced from genomic data. The migration routes utilized to colonize the Americas, for example, are still a source of debate. Genomic data has a limited precision when it comes to determining migratory trajectories since future population movements may obfuscate the spatial patterns that can be discerned from the genomic data. Dashed lines show proposed migratory paths that are still up for debate. CA stands for Central Anatolia; FC is for Fertile Crescent; IP stands for Iberian Peninsula; and PCS stands for Pontic-Caspian Steppe (Nielsen et al., 2017)

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populations have remained vast and divided throughout their evolutionary history, with the deepest divisions between human populations occurring in Sub-Saharan Africa. There is also evidence of historical and recent migration events, as well as substantial hybridization, across Sub-Saharan Africa. Other significant migration events include the migration of Pastoralist populations from southern Sudan to eastern and central Africa about 7000 years ago and the migration of Agropastoralists from Ethiopia to Kenya and Tanzania about 5000 years ago. The genetic lineages of click-language-speaking San people of southern Africa capture the deepest divide between human populations, with their separation estimated to have happened roughly 160–110 kyr ago (Veeramah et al. 2012), according to whole-genome sequencing and SNP array data. However, linguistic studies and genetic markers with uniparental inheritance suggest that click-language-speaking hunter-gatherer populations may have originated in eastern Africa and then migrated to southern Africa in the last 50 kyr (Tishkoff et al. 2009), or that they may have originated in eastern Africa and then migrated to southern Africa. Other huntergatherer communities that speak click languages, such as the Hadza and Sandawe, live in Tanzania; however, they have low-genomic similarity with the San people of southern Africa (Schlebusch et al. 2012). The exact origin of anatomically modern humans in Africa is unknown, owing to a lack of fossil and archaeological data in the continent’s tropical parts. However, given the possibilities for migration and admixture across the continent, a multiregional origin of modern humans in Africa (Stringer 2003) is still feasible, in which modern traits arose in a fragmented fashion in different places connected by gene ow. In Africa, there is evidence of anatomically modern humans admixing with archaic populations (Hammer et al. 2011; Hsieh et al. 2016). Because the environment of the samples from which DNA is extracted, particularly the local climatic conditions, is not conducive to the preservation of genetic material, the characterization of genomes from individuals who lived in Africa more than 10 years ago is difficult. However, statistical analysis of whole-genome sequencing data from geographically diverse hunter-gatherer populations reveals archaic human lineages that underwent introgression (the exchange of genetic material through interbreeding) and diverged from modern human lineages as recently as 35 kyr ago (Hsieh et al. 2016). As a result, the degree of archaic hybridization in Africa is a contentious issue, with numerous continuing initiatives aimed at resolving it.

Out of Africa and the Conjunction with Neanderthals The exodus of anatomically modern humans from Africa, a significant event in human evolutionary history, left a profound imprint on non-African genetic variation, including lower levels of diversity and higher levels of linkage disequilibrium. The quantity, geographic origin, migratory routes, and timing of significant dispersals, however, remain unknown. For example, evidence suggests that modern humans originated in eastern, central, and southern Africa (Fig. 2). Three studies

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(Pagani et al. 2016) use new, high-quality whole-genome sequencing data from individuals from more than 270 places around the world to help answer some of these problems. They also suggest that a single out-of-Africa dispersal occurred, with all non-African people deriving from the same ancestral population that migrated out of Africa, maybe with modest genetic contributions from an earlier modern human migration wave into Oceania. Furthermore, as modern people left Africa, they may have split into two waves of dispersal. One wave, as proposed by Rasmussen et al. (2011), led to the establishment of Australasia and New Guinea, whereas the other contributed to the lineage of today’s mainland Eurasians. However, the precise migration routes in the early diversification of people outside of Africa are still a source of debate and investigation. It is now known that all non-African people’s ancestors came into contact with Neanderthals and were admixed with them (Prüfer et al. 2014). All non-Africans investigated so far have roughly 2% Neanderthal ancestry (Vernot and Akey 2015), implying that admixture took place shortly after the dispersal of anatomically modern humans from Africa, which is compatible with a single-dispersal out-ofAfrica hypothesis (Fig. 2). The date of hybridization has been estimated to be around 50–65 kyr ago (Sankararaman et al. 2014), based on linkage disequilibrium characteristics, and knowledge of the time of admixture with Neanderthals helps to constrain estimates of when the ultimately successful out-of-Africa migration occurred. Estimates of the percentage of Neanderthal ancestry found in modern human genomes (Vernot et al. 2014) suggest a more complicated history of interaction amid Neanderthals and modern humans. East Asians have about 20% more Neanderthal sequences than Europeans, which could be due to natural selection. After the population split from Europeans, there was more admixture in the progenitors of today’s East Asians (Vernot et al. 2014; Vernot and Akey 2015; Kim and Lohmueller 2015), or the dilution of Neanderthal ancestry in Europeans due to hybridization with populations from other continents. SNP genotyping of an early modern person from Romania who lived around 40,000 years ago offered more evidence that introgression happened at multiple times and locations throughout Eurasia (Fu et al. 2015), despite the fact that the individual did not contribute detectable ancestry to modern populations. Recent studies have shown a more complicated mixing history than previously anticipated, and that our understanding of admixture models is currently uid and that additional demographic models are compatible with the observed.

The Peopling of Europe Three or more genetic components are likely to make up European populations, some of which arrived in Europe at different times (Haak et al. 2015). As early as 43,000 years ago, the earliest anatomically modern humans lived in Europe, because there is evidence of turnover in the genetic composition of Europeans before the Last

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Glacial Maximum, possibly in relation to climate oscillations (Fu et al. 2016). After the Last Glacial Maximum, some 11 kyr ago, a new way of life based on animal husbandry, agriculture, and sedentarism that is known as the Neolithic lifestyle began to emerge in numerous Fertile Crescent subregions (Asouti et al. 2013). Ancient DNA analysis revealed that this community of farmers spread from Central Anatolia to Europe, but that other Fertile Crescent locations contributed just a small amount of genetic material to the early European farmers (Günther and Jakobsson 2016). They arrived in the Iberian Peninsula around 7000 years ago, and in Britain and Scandinavia approximately 6000 years ago. The widespread migration of groups of farmers and the absorption of local hunter-gatherers (Günther et al. 2015), as indicated by genomic data from Neolithic human remains, drove this process, revealing that the Neolithic way of life spread over Europe through people mobility rather than as a concept or a culture. Although archaeological data suggests that the health of Neolithic farmers was sometimes poor, as there were ample signs of malnutrition and caries, the Neolithic lifestyle helped to increase the size of populations, as seen in estimates of effective population sizes generated from genomic data (Skoglund et al. 2014). During the late Neolithic and early Bronze Age periods, another wave of migration into Europe occurred, introducing the third European genetic component. Herders from the Pontic-Caspian steppe belonging to the Yamnaya civilization migrated to central Europe around 4500 years ago (Haak et al. 2015). The herders were descended from a number of hunter-gatherer tribes from (today) Russia and the Caucasus (Jones et al. 2015). Although some linguistics experts think that Indo-European languages were already spoken by Neolithic farmers (Bouckaert et al. 2012), this movement was likely linked to conquests and technological advancements such as horseback riding and may have spread Indo-European languages to Europe. Clearly, the late Neolithic and Bronze Ages were active periods that resulted in the spread of steppe herders’ genetic material across western and northern Europe (Haak et al. 2015; Allentoft et al. 2015). The contributions of hunter-gatherers to the decolonization of Europe after the Last Glacial Maximum, the migration of Neolithic farmers from Anatolia to Europe in the late-Neolithic period, and the Bronze Age migration to Europe from the east are re ected in the three main genetic components of modern-day European populations. These elements can account for a large portion of the genetic diversity found in today’s Europe. For example, in southern European communities such as the Sardinian people (Lazaridis et al. 2014), the Neolithic genetic component appears to be most prominent. Geographical variety in modern-day Europeans is closely connected with genetic variation (Lao et al. 2008), and there is a gradient of decreasing variety as one moves further north. Despite the fact that the major components of genetic variants were introduced into Europe in different waves, successive processes of gene ow constrained by geography have sculpted the current genetic landscape. In many times of Prehistoric Europe, culture and lifestyle were thus more important factors of genomic divergence and similarity than geography.

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The Colonization of Asia and Oceania The majority of evidence suggests that Asia was colonized in at least two waves. Although additional evidence implies that there was just one dispersal event (Mallick et al. 2016), one wave contained the ancestors of Australians and Papuans, and the other contained other ancestors of East Asians, with admixing between the two (Rasmussen et al. 2011). The specifics of how Asia was colonized are, however, mostly obscure. The genomes of two early modern humans from Asia have been sequenced. The first genome came from a person from the Malta-Buret culture of southern central Siberia who lived about 24,000 years ago (Raghavan et al. 2014), and it shows a strong genetic affinity for both western Eurasians and Native Americans, but a weaker affinity for East Asians and Siberians, implying that there was a very different geographic distribution of genetic signatures during the Upper Paleolithic period in campaigns. When differences in Denisovan admixture are taken into account, the second genome, which comes from a person who lived in the Ust’-Ishim region of western Siberia (Fu et al. 2014) about 45 years ago, displays virtually equal genetic kinship with western Eurasians, East Asians, and Aboriginal Australians. This, combined with evidence from the Kostenki 14 individual’s (36–38-kyr-old) genome from European Russia (Seguin-Orlando et al. 2014), shows a close kinship to contemporary western Eurasians, but not East Asians, and points to a 36–45-kyrold separation between East Asians and western Eurasians. Two later population expansions into central Asia from Europe and western Asia resulted in mixing with and locally displacing Malta-like hunter-gatherers, according to a study that comprised low-coverage sequencing of the genomes of 101 ancient humans from across Bronze Age Eurasia (Allentoft et al. 2015). The first occurrence was a migration of Yamnaya herders into Asia about 5000 years ago, which coincided with the arrival of the first humans in Asia. Similar assertions have been made based on linguistic evidence and lithic technology, as well as the arrival of tamed animals like the dingo (Pugach et al. 2013). However, the only comprehensive population genomic study on Aboriginal Australians and Papuans to date (Malaspinas et al. 2016) finds evidence for only one founding event in Sahul, followed by a genetic divergence between the Papuan and Aboriginal Australian ancestral populations and further genetic diversification in the Aboriginal Australian population, which could have coincided with environmental changes such as desertification. As a result, Aboriginal Australians seemed to have been isolated until recently. Polynesians, who are spread across a triangle of islands in the South Pacific bounded to the east by Rapa Nui (also known as Easter Island), represent an expansion into Oceania of individuals with mixed Melanesian and East Asian ancestry, according to a study (Wollstein et al. 2010) of genome-wide SNP data from modern people in Oceania. After the initial Polynesian spread, Melanesian ancestry was added to the existing East Asian ancestry (Skoglund et al. 2016). It is still debatable if Polynesians reached the Americas and mated with Native Americans during their eastward march, which stopped approximately a thousand years ago.

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This concept has been supported by a genetic examination of ancient chicken remains from South America (Storey et al. 2007), but it has also been questioned (Gongora et al. 2008). Genome sequencing of human remains discovered in Brazil circa 1650, which predates the reported transportation of Polynesian slaves to South America (Malaspinas et al. 2014), reveals that the individuals are closely linked to modern Polynesians. These findings could corroborate the theory of early interaction between Polynesians and Native Americans, but they might also be the consequence of people being transported by Europeans. The findings of a genome-wide investigation on modern-day inhabitants of Easter Island (MorenoMayar et al. 2014), which offered statistical support for Native American claims, are much more convincing. The only way to settle the argument is to find evidence of Polynesian and Native American hybridization in human remains that predate colonization of the Americas.

Populating Americas The earliest well-characterized archaeological assemblage in the Americas, the Clovis complex (approximately 12.6–13 kyr ago), dates to about 15–14 kyr ago (Jenkins et al. 2012), and widespread colonization of the Americas appeared with the advent of the Clovis complex (approximately 12.6–13 kyr ago) (Fig. 2). However, until about 13,000 years ago, much of North America was covered in ice, making it difficult for humans to migrate from Beringia (today northern Siberia and northwestern North America) to the southern sections of the Americas. After the ice melted, a 1500 km ice-free inner passage emerged. According to metagenomic investigations of lake cores from Canada, this corridor first became biologically viable around 12.6 kyr ago, which makes it an unlikely early route for the southward migration of pre-Clovis and Clovis groups of people, although a study of bison is in disagreement (Heintzman et al. 2016). It is unclear how and when the first Americans crossed the Pleistocene ice sheets into southern North America, or whether the pre-Clovis and Clovis populations were part of the same migration. However, the most feasible scenario appears to be a movement toward the south along North America’s west coast that happened more than 14,000 years ago and was possibly followed by southerly or northerly backmigrations into the interior. It has been proposed that early Americans were not direct ancestors of contemporary Native Americans but rather were related to Australia-Melanesians, Polynesians, the Ainu people of Japan, or Europeans who were later replaced or assimilated by ancestors of Native Americans from Siberia (Owsley and Jantz 2014), based on cranial morphology and lithic analysis. Several genetic investigations, on the other hand, have generally dismissed these ideas. The earliest and only Clovis-associated human genome from the Americas (discovered in Montana, USA) belonged to an individual who lived around 12.6 million years ago. According to studies, the Clovis group from which the genome was derived was directly ancestral to many modern Native Americans. Similarly, the genome sequence of the 9.5-kyr-old Kennewick

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Man skeleton discovered in the state of Washington in the United States (Rasmussen et al. 2015), which had been thought to be closely related to the Ainu and Polynesians based on cranial morphology, determined that he was most closely related to contemporary Native Americans. Furthermore, communities previously thought to represent relics of an early migration into the Americas and closely related to Australo-Melanesians have been discovered to be genetically connected to modern Native Americans (Raghavan et al. 2015). Based on whole-genome sequencing, estimates of the timing of divergence between Siberians and Native Americans suggest the creation of the Native American gene pool as early as 23 kyr ago (Raghavan et al. 2015), supporting the early arrival of Native American ancestors in the Americas. When the recognized dates for the earliest archaeological sites in the Americas are taken into account, Native American ancestors could have lived in isolation in Siberia or Beringia until roughly 8 kyr ago, after splitting from their Siberian forebears, before traveling eastward into the Americas. Despite the fact that modern Siberians are Native Americans’ closest relatives outside of the Americas, genomic sequencing of a 24-kyr-old Malta skeleton reveals that Native Americans are descended from a combination of Malta-related populations as well as one or more unknown East-Asian lineages. Because the Clovis-associated genome and modern Native Americans have similar levels of the Malta genetic signature (14–38%), the mixing event occurred over 12.6 million years ago. However, it is unknown whether it occurred within or outside of the Americas. Genomic data have been utilized to locate a basal divide in Native Americans that can be dated to around 14,000–13,000 years ago. The southern branch is made up of people who speak Amerindian languages, whereas the northern branch is made up of people who speak Athabascan languages as well as other languages such as Cree and Algonquin. Divergence estimates based on wholegenome sequencing data reveal that both groups diverged from Siberians at the same time, meaning that both Amerindian and Athabascan populations had a single founding event followed by gene ow from Asia (Raghavan et al. 2015). The exact location of the split between the two Native American branches, whether in Siberia or to the north or south of the American ice sheets, is still a matter of contention, which will require the investigation of more ancient genomes. Similarly, it is unclear whether the Australo-Melanesian signal detected in some current Brazilian Native Americans is due to later gene ow (Raghavan et al. 2015) or a previously undiscovered early founding population. This genetic signature has yet to be discovered in the genomes of ancient humans from the Americas. The Inuit of the American Arctic is said to have come from a different migration than other Native Americans (Reich et al. 2012; Gilbert et al. 2008). However, it has long been debated whether the earliest people to occupy the Arctic, the now-extinct PaleoEskimo civilization, who first emerged in the Americas some 5000 years ago, were the progenitors of today’s Inuit or a separate founder group from Siberia. DNA analysis of a 4-kyr-old tuft of hair from Greenland revealed that the group the

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individual belonged to traveled from Siberia to the North American Arctic independently of Native American and Inuit migrations. The tribe then thrived in the Arctic for almost 4000 years, renewing its subsistence techniques and technologies before being displaced by the Inuit around 700 years ago.

The Denisovans When the first modern humans began to appear on the continent, at least one other form of archaic human – the enigmatic Denisovans – thrived in Eurasia alongside the Neanderthals (Fig. 2). Denisovans are only known from the genome sequences of a finger bone and three teeth recovered in the Denisova Cave in Siberia (2015; Stringer and Barnes 2015); therefore, nothing is known about their morphology and distribution. They are most closely related to Neanderthals, with a genetic differential similar to the deepest splits between modern humans (Prüfer et al. 2014) but a divergence time estimated to be between 200 and 400 kyr. Denisovans have a number of unique characteristics, for example, they may contain genetic material (obtained through hybridization) from people linked to earlier human species, potentially Homo erectus. Denisovans are thought to be the eastern or southern end of a spectrum of archaic humans who lived in Eurasia (and possibly beyond), while Neanderthals are thought to be the western end. Denisovans interbred with anatomically modern humans in the same way that Neanderthals did. Some groups of people, such as Melanesians in Oceania, can trace 3–6% of their genome back to a Denisovan-like ancestor. Denisovan genetic DNA is found in 0.1–0.3% of continental southeast Asians (Skoglund et al. 2011). On introgression, both Neanderthals and Denisovans’ genomes were subjected to genetic selection. Because there is a scarcity of introgressed DNA in functional sections of the genome, most selection in humans appears to be directed against it. In addition, extensive genomic areas devoid of both Neanderthal and Denisovan sequences have been discovered (Vernot et al. 2016), indicating that detrimental sequences were rapidly eliminated. Some introgressed DNA, on the other hand, may have aided human adaption to the local environment, such as Tibetan people’s adaptation to high altitudes (Huerta-Sánchez et al. 2014). Two punctuated and very precise instances for hybridization between anatomically modern humans and archaic humans were hypothesized by studies of the first trustworthy genomic data from archaic humans (Reich et al. 2010). Since then, such hybridization is far more widespread, with several episodes occurring in both directions across distinct groups of contemporary and archaic humans (Vernot et al. 2015; Huerta-Sánchez et al. 2014). As the progenitors of current Australasians traveled across the continent, it is unclear whether the Denisovan introgression into Melanesians and Australians took place in Australasia or Asia. If this had happened in Asia, today’s Asians would be primarily descended from other populations who arrived during the subsequent centuries. If that had happened in Asia, the majority of

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today’s Asians would be descended from other groups who arrived during succeeding waves of migration. Similarly, it is unclear whether East Asians’ Denisovan admixture is the consequence of the same mixing event or events that affected Australasians. The investigation of ancient human genomes has advanced significantly in recent years, with studies of single genomes to population-genomic studies including several ancient individuals. Despite the fact that this has supplied a plethora of information about human evolutionary genomics, the science is still in its early stages. Continued attempts to sequence and analyze the genomes of current and ancient humans, with an emphasis on underrepresented areas of the globe, will aid us in forming a more complete picture of the events that have shaped contemporary humans’ cultural and genetic variety.

Future Directions DNA genetic markers will play an important role in human migration and evolution in the future with rapid advancements in technology and data analysis. Wholegenome sequencing will become more affordable and efficient. The most difficult task for scientists will be to analyze massive data sets generated by massive sequencing technologies. As a result, more emphasis can be placed on clarifying the cultural and environmental in uences on gene expression. The future application of genetic markers (DNA fingerprints) is wide open, and study over the next decade will lead to a better understanding of our species’ origins and evolution. It is unclear how far back in time ancient DNA studies will go, but these new methodologies will provide anthropologists with a refined story of human history, unraveling the complexities of human migration, admixture, and the successful and unsuccessful ways in which Hominin genomes were selected by their environment. Research into still-unanswered concerns about human evolution and migration would be one of the work’s future areas. A better understanding of evolution could lead to novel and creative therapeutic options for many diseases that are currently incurable and untreatable.

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Index

A Academy Standards Board (ASB), 1119 Accreditation, 1088, 1089, 1093–1095, 1104 Accu ID software, 238 Acidic pH, 1075 Acid washing, 759 Activity level, 802 Acute GvHD, 651 Acute lymphatic leukemia (ALL) chimerism, 666–668 diagnosis, 665 hematopoiesis, 666 leukemic blasts cells, 665 lymphocyte and granulocyte populations, 666 symptoms, 665 Acute myeloid leukemia (AML) clinical signs, 669 cytogenetic abnormalities, 668 diagnosis, 669 locus, 670 MIX3, 670 myeloblast, 668 phases, 669 samples, 670 symptoms, 667, 669 treatment, 669 Adenosine triphosphate (ATP), 829 Administrative Assistance and Cooperation System for Food Fraud (AAC-FF system), 891 Affymetrix chips, 679 Affymetrix resequencing array technology, 237 Allele(s), 156, 1046 counting method, 1114 dropout, 1078 sharing, 309–311 Alpha thalassemia, 682, 684

AluQuant™ system, 776 Ameloblasts, 757 Amelogenin (AMEL), 618 Amelogenin marker, 178, 179 American Academy of Forensic Sciences (AAFS), 1119 American Board of Forensic Anthropology (ABFA), 376 American Donor Registry, 671 American Society for Histocompatibility and Immunogenetics (ASHI), 652 American Society of Crime Laboratory Directors Laboratory Accreditation Board (ASCLAD-LAB), 1120 American Type Culture Collection (ATCC), 1111 7-Amino Actinomycin D (7-AAD), 651 AmpFISTR ®, 564 AmpFLSTR Identifiler™ Direct, 165 AmpFLSTR Identifiler™ Plus, 165 AmpFlSTR MiniFiler PCR Amplification Kit, 1117 AmpFlSTR YfilerTM PCR Amplification Kit, 195 Ampliconic sequences, 439 Amplification efficiency curve, 792 Amplification refractory mutation screening (ARMS), 683 Amplification-refractory mutation system (ARMS) PCR, 634 Amplified fragment length polymorphism (AFLP), 842, 861 Amylase, 1072 α-Amylase, 571 Anaemia di Fanconi, 650 Anal swab, 571 Analysis software, 1109 Analyst bias, 1113

© Springer Nature Singapore Pte Ltd. 2022 H. R. Dash et al. (eds.), Handbook of DNA Profiling, https://doi.org/10.1007/978-981-16-4318-7

1185

1186 Analytical traceability, 897 Ancestry informative markers (AIM), 39, 240, 390 Ancient DNA (aDNA), 757, 758, 763, 788–789 ANDE 6C system, 989 Anderson sequence, 433 Androgenic alopecia (AGA), 1137 Aneuploidy, 677 Angelman syndrome, 681–682 Animal breeds, 892 Animal Tubulin-Based-Polymorphism (aTBP), 900 ANSI/ASB Standard 020, 1119 ANSI/ASB Standard 040, 1119 ANSI National Accreditation Board (ANAB), 1088 Antenatal testing, 638 Anti-counterfeiting technologies, 892 Antley Bixler syndrome (ABS), 700 Apert syndrome, 695 Appeal, 20 Applied Biosystems 3500xL Genetic Analyzer, 1091 Applied Biosystems RapidHIT™ ID System, 1102 Argonautes, 124 Array CGH, 678 Association fallacy, 817, 818 Association of Southeast Asian Nations (ASEAN), 260, 264, 273, 274, 278, 280, 292 Automated electrospray ionization mass spectrometry method, 247 Automated extraction, 719–722 Automated fingerprint identification technology, 341 Automate express, 613 Autosomal chromosomal, 154 Autosomal DNA quantification, 785–786 Autosomal DNA typing, 565 Autosomal InDels, 1046 Autosomal markers, 616 Autosomal short tandem repeat (STR) analysis, 571 Autosomal SNPs, 400 Autosomal STR markers criteria, 155 nomenclature, 156 types, 156, 157 Autosomal STR multiplex kits, 160 cases, 161 components, 161 discrimination powers, 161 variants, 162, 163

Index Autosomal STRs, 399–401 Autosomal STR typing, 386 Avuncular Index (AI), 404–405

B Baecchi’s staining, 204 Bahrain, 593, 594 Barcode DNA high-resolution melting (BAR-HRM), 859, 863 Barcode of Life Data Systems (BOLD), 844, 846, 927 Basic Local Alignment Search Tool (BLASTing), 791 Bayesian allele frequency approach, 212 Bayes’ theorem, 803 BeadChip method, 332 Bench-top automated systems, 613 Bertillon system, 1126 Bespoke validation support solution, 1095 Beta thalassemia, 683 Beverages ontology, 897 Bio-agents, 942 Biobanks, 267, 268, 270, 272, 292 Biofilms, 505, 508 Bioforensics, 508 Biofouling, 509 Bio-geographical origin of individual, 437 Bio-geographic ancestry (BGA), 39, 533 Bioinformatics, 905, 906 Biological contamination, 7 Biological evidence, 71, 72 chain of custody, 82 cutting method, 77 DNA content, 72–74 double swabbing method, 76, 77 dried blood stains, 80 hair, 82 liquid blood, 79 picking method, 79 scrapping method, 76 sexual assault, 80, 81 soft organs, 81 tape lifting method, 78 teeth and bones, 81 touch DNA samples, 82 Biological exhibit material, 13 Biological macro-trace, 86 Biological matter, 960 Bio-stain identification, 86 approaches for, 89–93 efficiency of technological tools for detection of latent biological traces, 91 forensic lights, 86

Index Luminol, 88 protocols for use of detection technologies, 91 substrate color, 90 synergistic screening, 91 techniques for detection of latent biological traces evidence, 86 textile substrates, 91 Wood’s lamp, 88 Biosurveillance, 507 Bioterrorism, 142, 936–937 Biotin, 131 Blood, DNA biological sources components, 53 extraction protocol of DNA from, 54 fingerprinting technique, 56 pre-analytical factors, 53 scraping/tape-lifting, 53 storage conditions, 53 typing, 53 Blood group testing, 122–123 Bloodstain pattern analysis, 13 BM transplantation, 647 Body uid(s), 510 Body uid identification challenges, 323 DNA-compatible cell-specific identification, 333–334 DNA methylation profiling method, 329–333 epigenetics, 327–329 methods for, 324 miRNAs profiling, 326–327 mRNA profiling, 324–326 BOLD Identification System (IDS), 844, 848 Bone and Tooth Lysate Tube, 612 Bone DNA Extraction Kit, 765 Bone marrow (BM) transplant, 646 Bone Marrow Donor Registry (BMDR), 649 Bone matrix, 757 Bone/tooth pulverization, 385 Botanical evidence, 841, 919 Botanical origin, 899 British Forensic Science Service, 179 Bryophytes, 842 C Cambridge reference sequence (CRS), 433, 490–491 Canadian National DNA Database, 1025 Cancer, 505, 512 Capillary electrophoresis, 30, 129–131, 359, 410, 526, 531, 535, 536, 578, 967, 1001, 1046, 1091, 1112

1187 data analysis similarities, 36 vs. next-generation sequencing, 33–36 similarities with NGS/MPS methodology, 35 Carborundum discs, 611 6-carboxy-4’, 5’-dichloro-2’, 7’-dimethoxyuorescein (JOE), 166 Carboxy-tetramethylrhodamine (TMR), 166 Carboxy-X-rhodamine (CXR), 166 Cardiac genetic disorders, 697–699 Case documentation, 1110 Cell free fetal DNA, 639 Cellular phone, 22 Cementum, 607 Center for International Blood and Marrow Transplant Research (CIBMTR), 671 Certified and sworn in experts for biological stains, 572 Cetryl trimethylammonium bromide (CTAB) method, 922–924 Chediak Higashi syndrome, 693 Chelex, 736–737 extraction methos, 716–717 Chemiluminescent probe, 126 Chemoradiotherapy, 647 Chimeras, 647 Chimeric status, 648 Chimerism, 647, 648, 656, 659, 666, 672 ChimerMarker™, 646, 654, 655 ChimerPanel, 656, 658, 666 Chloroplast (cp) genome, 844 Chromosomal abnormalities, 632, 635 Chromosomal disorders, DNA testing, 677–680 Chromosomal microarray, 638, 677 Chromosomal translocations, 668 Chromosomes, 154 Chronic GvHD, 651 Ciclosporina (CSA), 651, 652 Cinta Senese genotypes, 905 City Police Department (KCPD), 971 Cognitive challenges, 1157 Cold cases, 512 Cold cases, DNA analysis, 365 arrest of Nebraska sex offender in 1983 slaying of UNO student, 366–367 Boston Strangler case, 365–366 Jeffrey Gafoor case, 367–368 murder and sexual assault of girl in 1988, 366 unsolved female homicide cases, 1990–1999, 367 Collection, biological samples, 74 Combined aspartic acid racemization, 389 Combined Avuncular Index (CAI), 405

1188 Combined DNA Index System (CODIS), 182, 343, 363, 984, 985, 1020, 1022, 1024–1028, 1031, 1127 autosomal STR markers, 159, 160 categories, 158 definition, 158 DNA database, 1089 Combined Paternity Index (CPI), 402, 404, 405 Combined probability of inclusion (CPI), 1003 Comparative and reconstructive identification, 607 Complementary metal-oxide-semiconductor (CMOS) sensor, 782 Complete chimerism (CM), 647 Congenital adrenal hyperplasia (CAH), 685–686 Congenital hypothyroidism, 696 Consanguineous marriages, 546 homozygous loci, 549, 551 inbreeding, 546, 547, 549 technical updates, 548 Consortium for the Barcode of Life (CBOL), 843, 844, 927 Contamination, 1155–1156 Convention on International Trade in Endangered Species (CITES), 841 Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), 824 Convicted Offender DNA Databasing Laboratories, 1108 Convicted Offender Index (COI), 1025 Cord blood banks, 646, 650 Costello syndrome, 698 Council for International Organizations of Medical Sciences (CIOMS), 267, 268, 273, 292 Council on Health Research for Development (COHRED), 283, 284 Counting method, 435 CQ inter-assay reproducibility, 654 CQ Intra-assay reproducibility, 654 Craniosynostosis syndrome, 695 Cranium, 390 Cri-du-Chat syndrome, 705 Crime, 1020, 1022–1027, 1030–1035 Crime scene, 11 analysis, 1154 examiner, 9 revisit, 21 Crime Scene Index (CSI), 1025 Criminalistics, 376 Cross-contamination, 381

Index Cross-forensic investigative leads, 20 Crouzon syndrome, 695 Cryogenic Grinding technique, 610 C.S.I. effect, 568 Cutting out technique, 965 Cystic fibrosis, 684–685 Cytochemistry analysis, 665 Cytochrome b (Cyt b), 828–829 Cytochrome c oxidase 1 (COI), 843, 899 Cytochrome oxidase I (COI), 829 CytoScan Optima, 679

D Dabney extraction method, 386 Data acquisition process, 654 Data analysis, 1095 Databases, 343 Data interpretation, 1113 Data protection, 263–264 Declaration of Helsinki, 267, 268, 270, 273 Declaration of Taipei, 267, 273, 292 Decomposed/skeletonized bodies, DNA extraction, 741–743 biological samples, 743–744 organic method, 745–746 PrepFiler ® BTA Forensic DNA Extraction Kit, 748–749 QIAamp DNA Investigator Kit protocol, 746–748 solid-phase, 746 Deconvolution, 657, 660 Defence hypothesis (Hd), 801, 804, 816 Defendant’s fallacy, 811, 812 Defense hypothesis (Hd), 1004, 1114 Degradation/inhibition models, 312 Degraded DNA, 1073 degradation factors, 1075–1077 mechanisms, 1074 Demineralization, 385 Dental DNA sampling, 610 Dental identification, 606 Dental papila, 757 Dental pulp, 619 Dentin, 756, 757 Denys Drash syndrome, 700 Deoxyribonucleic acid (DNA), 11, 154, 179, 341, 1109 amplification, 652, 1112 analysis methods, 1094 analyst factors, 1114 collection, 1109 contamination, 381, 382

Index damage, 379–381 database, 347, 351 degradation, 381 evidence, 346 extraction, 608, 611, 612, 1110, 1111 extraction methods, 385, 386, 1091 fingerprinting (see DNA fingerprinting) forensics, 342 humans, 342 isolation, 615 laboratory, 1089–1091, 1093 microarray assays, 617 phenotyping, 348 profiling, 343, 347, 607, 608, 646 profiling methods, 387 quantitation, 1111 quantity, 616 structure, 342 testing, 376, 378 testing process, 1110 typing methods, 608 Developmental validation, 299, 1090, 1102 DFC, 594 40 ,6-Diamidino-2-phenylindole (DAPI), 678 Dideoxynucleotidetriphospahtes, 925 Dietary factors, 893 Differential extraction, 722 Di-George syndrome, 632, 681 Digital polymerase chain reaction (dPCR), 792–793, 898 Dihydrocaffeic acid (DHCA), 893 Diploid organism, 1129 Direct analysis in real time-high resolution mass spectrometry (DART-MS), 881 Direct cutting, 596 Directional focused fragmentation charge (DFFC), 594, 598 Directionally focused charges, 598 Direct PCR, 722–723 Direct phenotyping, 349 Direct-to-consumer testing (DTC), 260 Direct-to-DNA Y-screening, 204 Disaster victim identification (DVI), 207, 377, 576, 580, 582, 586, 983–984 Disorders of sex development, 699–700 Dithiothreitol (DTT), 611, 763 DNA Advisory Board (DAB), 615, 1108, 1120 DNA barcodes, 862 DNA Barcoding Consortium, 844 DNA-barcoding techniques, 899 DNA-based identification system, 844

1189 DNA based prediction eye color, 1132–1133 hair, 1134–1135 skin, 1135 DNA based tests, 629 DNA-based tracking, 906 DNA-compatible cell-specific identification, 333–334 DNA contamination, 1079–1081 sources, 1080–1081 DNA conviction, 8 DNA Criminal Intelligence Database (DCID), 1021 DNA Data Bank of Japan, 830 DNA databases, 6, 18, 547, 551, 556 ethical and legal standards, 1028, 1034 national, 1021, 1028 DNA databasing, 521 DNA evidence, 20, 560–561, 800 activity level, 802 association fallacy, 817, 818 “consistent with” approach, 817 “could have come/originated from” approach, 816 “could not be excluded” approach, 817 defendant’s fallacy, 811, 812 identical twins, 818, 819 initial assessment, 801, 802 logic of evidential proof, 805, 806 numerical conversion error, 814, 815 offence level, 802 probability of another match error, 813, 814 probative value, 803, 805 prosecutor’s fallacy, 806, 811 relative frequency of occurrence, 815, 816 source level, 802 sublevel (sub-source level), 802 uniqueness fallacy, 812, 813 verbal scale, 818 DNA extraction, 577, 579–580, 582, 905 automated extraction, 719–722 challenges, 724–728 Chelex extraction methods, 716–717 contamination, 726–727 decomposed/skeletonized bodies, 741–749 differential extraction, 722 direct PCR, 722–723 DNA quantity and quality, 724–726 forensic sample, 713–714 fresh bodies, 734–741 history, 712 inhibitors, 727–728 magnetic beads extraction, 716–718

1190 DNA extraction (cont.) methodologies, 714–722 organic extraction, 715–716 protocols, 1079 spin column extraction, 719–720 storage conditions, 723 DNA findings, 25 DNA fingerprinting, 341, 343, 398, 399, 401, 576, 842, 998, 1166, 1180 collect DNA from suspects, 344 evidence, 343 technique, 70, 895 DNA fragments, 70 DNA identification act, 158, 1127 DNA intelligence, 38 DNA isolation, human remains, 762 Bone DNA Extraction Kit, 765 Cells and Tissue DNA Isolation Kit, 766 Chelex ® resin, 763 Crime Prep Adem-Kits for casework, 766 EZ1 DNA Investigator Kit, 766 incorporation of manual pretreatment protocols to commercial kits, 766–767 manual DNA isolation, 762–764 manual purification of DNA extracts, 763–764 organic extraction, 763 PrepFiler™ BTA Forensic DNA Extraction Kit, 764–765 QIAamp DNA Investigator Kit, 765–766 total demineralization, 762–763 DNA metabarcoding, 850 DNA methylation profiling method, 329–333 DNA microarray, 245 DNA migrant identification databases biological samples, 1060–1061 building, updating and managing genetic databases, 1061 power of identification, 1059–1060 robust, reliable, universal technology, 1059 victim’s data with relatives’ data, 1060 DNA mini-barcoding, 899 DNA mutation, 903 DNA profiles, 12, 15, 305–307, 314, 551, 554, 556, 961, 970 DNA profiling, 70, 71, 520, 521, 523, 526, 529, 534, 535, 576, 580, 582, 586, 597, 626, 1127 and capillary electrophoresis, 578 chemical analysis, 595 collection method, 596, 597 combination kits, 528, 529 DFC, 598, 599

Index DFFC, 598 EFP, 598 forensic DNA intelligence, 597–599 forensic investigative team, 591, 592 forensics, 531, 533, 534 human genotyping, 523 human identification, 528 IEDs, 597–599, 602 intelligence forensics DNA, 590, 591 mDNA, 1169, 1170 RDX-C4, 593, 599, 602 single nucleotide polymorphisms, 524 STRs, 520, 1169 SWGDAM, 526 technical updates, 594 terrorism, 590 X-STR, 524 Y chromosome DNA, 1171 DNA-Prokids program, 1062 DNA-ProORGAN Program, 1063–1064 DNA purification protocol, 895 DNA quantification, 774, 775 human-and higher primate-specific methods, 776 total genomic methods, 775–776 DNA reference, 22 DNA repositories, 830 DNA sequencing, 850 DNA source identifier (DSI)-SemenTM, 332 DNA testing, 13 DNA typing, 1058–1061 DNA variation, 12 DNeasy PowerClean Pro Cleanup Kit, 1073 Dot blot technique, 967 Double-donor chimerism analysis, 658, 670 Double swabbing method, 77 Dremel, 610 Duchenne muscular dystrophy, 688–689 Duplication syndromes, 682 Dynamic criminology, 115

E Eelectrophoresis, 127 Ejaculation, 571 Electrokinetic injection, 1113, 1114 Electropherogram, 180, 551, 554, 1047 Electrophoresis, 988 Enamel, 607, 757 Endocrine disorders, 696–697 Engraftment, 648, 652 Environmental DNA (eDNA), 859, 879, 881 Environmental microbiology, 508–509

Index Enzymatic predigestion, 759 Enzyme proteinase K, 737 Enzymes, 1111 Epigenetics, 327–329 Erroneous forensic DNA findings, 25 Error of the transposed conditional, 806 Estrogen receptor 1, 334 Ethanol washing, 758–759 Ethical and legal standards, forensic DNA databases, 1028, 1029 crime scene DNA profile, 1032 errors and improper utilization of police resource, 1033, 1034 familial looking, research use and counterpsychological oppression, 1031, 1032 human rights and racial contemplations, 1029, 1030 privacy (lack of safeguard), 1031 Ethical review board (ERB), 264 Ethical review committee (ERC), 264 Ethylenediaminetetraacetic acid (EDTA), 385, 759, 1073 European Federation for Immunogenetics (EFI), 652 European Molecular Biology Laboratories (EMBL), 830 European Network of Forensic Science Institutes (ENFSI), 164, 1021 European Observatory on Health Systems and Policies, 262 European Standard Set (ESS), 182 European standard set of loci (ESSL), 169 Evaluation, 1091 Evidential value, 9, 10, 13, 14, 23 Evolutionary biology, 514 Exhibit integrity, 7 Exhibit material, 13, 21 Exoneration of convicted individuals, DNA analysis Gary Dotson’s case, 368–369 Kirk Bloodsworth’s case, 369 Michael Shirley’s case, 369–370 post-conviction DNA testing, 369–370 ExoSAP, 246 Explosively formed penetrator/projectiles (EFP), 594, 598 EZ1 DNA Investigator Kit, 766

F False match, 809 False positive error, 809 False positive fallacy, 809, 810

1191 Familial DNA searches (FDS), 18, 341, 345–347, 352 Familial DNA search methodology, 1118 Familial searching, 210–211 Family Tree DNA (FTDNA), 211 Fast mutating Y-STRs, 190 FBI Laboratory, 616 Fecal matter, DNA biological sources bacteria and viruses, 63 extraction protocols, 63 isolation, 63 Federal Bureau of Investigation (FBI), 1020, 1108 Federal database of genome information (FDBGI), 1023 Federal quality standards, 1108 Fetus, 626, 640, 641 Fichier National Automatisé des Empreintes Génétiques, 1022 Filled pasta, 898 Fire scene, genetic traces in, 99–102 biological/genetic damage from fire, 110 biological traces evidence, 101–102 denaturation of biological traces on textile substrates, 111 direct investigations, 102 DNA evidence, 105–112 genetic electropherogram, 112 heat denaturation process, 106 imprints on ceramic material, 104 indirect investigations, 102 mitochondrial DNA, 107–109 nuclear DNA, 107 papillary traces, 105 technique for papillary print identification, 103 First-degree assault, 344 First generation multiplex (FGM), 161 Fixation index, 252 Fluorescein (FL), 166 Fluorescence detection, 155 Fluorescence resonance energy transfer (FRET), 241 Fluorescent assays, 776 Fluorescent dye, 967 Fluorescent in situ hybridization (FISH), 629–632, 669 Food fraud, 892, 896 Food intake, 893 Food security, 891, 895 Food supply chain, 891 Food traceability, 841 ForenSeq™ DNA Signature Prep, 444

1192 ForenSeqTM kit, 510 Forensic analysis, 1159 Forensic anthropology (FA) bones and teeth, 378 classical approach, 378 definition, 376, 377 identification process, 377 skeletal elements, 383, 384 Forensic biology, 510–513 Forensic botany, 912, 915–916 and DNA barcoding, 843–851 Forensic cases, 1040 Forensic caseworks, 181 Forensic DNA, 976, 978, 985, 987, 991, 993 challenge, 1079 database, 15, 16 laboratories, 1088, 1094 testing, 1088 work ow, 1088 Forensic DNA Databank of Malaysia (FDDM), 285, 1027 Forensic DNA databases, in Southeast Asia Brunei Darussalam, 280–281 Cambodia, 281–282 Indonesia, 282–283 Laos, 283–284 Malaysia, 284–286 Myanmar, 286–287 Philippines, 287–288 Singapore, 288–289 Thailand, 289–290 Vietnam, 290–291 Forensic DNA phenotyping (FDP), 38, 98, 348–351, 1128–1131, 1143, 1144 IDentify, 1139 individual freedoms and privacy, 1142 legal and ethical aspects, 1141–1143 legislations, 1140–1141 Parabon NanoLabs, 1140 racial profiling, 1142 right not to know, 1142 slippery slope theory, 1141–1142 SNaPshot, 1139, 1140, 1143 Forensic DNA typing, 166 blood group testing, 122–123 capillary electrophoresis, 129–131 description, 120 forensic protein profiling, 123–125 microbial and animal forensics, 142–146 mitochondrial DNA, 137–140 next generation sequencing, 146–147 principles, 121 RFLP-based testing, 125–126

Index STR typing, 134–137 X-chromosome analysis, 140–142 Y-chromosome DNA testing, 131–134 Forensic examination, 342 Forensic geneology, 1117 Forensic genetic genealogy, 348 case studies, 43 Ekeby man case, 43 ow chart for, 42 GEDmatch, 42 golden state killer, 43 Forensic genetic genealogy dNA analysis (FGGS), 534 Forensic genetics, 155, 193, 735, 750 analysis techniques, 93–98 bio-stain identification, 86–93 DNA polymorphisms, 94 in fire scene, 98–112 forensic DNA phenotyping, 98 justice method, 113–114 low copy number genetic profiles, 97 mitochondrial DNA analysis, 97 next-generation sequencing, 97 polymerase chain reaction, 94 probabilistic genotyping software, 96 qPCR in (see Quantitative polymerase chain reaction (qPCR)) random sampling, 86 SNP analysis, 95 STRs, 95 VNTRs, 95 Y-chromosome DNA testing, 97 Forensic Genetics Policy Initiative, 259 Forensic investigative leads, 17, 18, 20 Forensic laboratories, 608, 1152, 1153, 1157, 1160 Forensic lights, 86 Forensic microbiology, 507 Forensic molecular anthropologists (FMA), 378, 383 Forensic nurses, 571 Forensic odontology, 606 applications, 606 definition, 606 DNA, 608, 609 Forensic palynology, 849, 850, 853, 913 Forensic phenotyping, 344 familial DNA, 345 Forensic practitioners, 377 Forensic protein profiling, 123–125 Forensic science, 376, 1043 DNA typing, 1057–1061 human identification, 30–31

Index next-generation sequencing, 32–33 rapid DNA instruments, 43–45 software tools, 31 Forensic Science Laboratories, 609, 610, 1108–1110, 1113, 1114, 1117, 1119–1121 Forensic Science Service (FSS), 158, 163 Forensic scientists, 1047 Forensic validation categories, 179 parameters, 180, 181 proficiency test, 180 Formal identification parades, 23 Forum for Ethical Review Committees in Thailand (FERCIT), 289 Fragment analysis, 1046 Frank-American-British (FAB), 669 Fresh bodies, DNA extraction, 734–735 biological samples, 735–736 Chelex, 736–737 organic method, 737–738 QIAamp DNA Investigator Kit protocol, 739–741 solid-phase, 739 Whatman FTA ® paper, 737 FTA cards, 613 Full DNA profile, 95 Fusion 6C system advantages, 176 circumstances, 176 6-dye technology, 176 features, 175 loci, 175 primers, 176 STR markers, 177

G Gap-Polymerase chain reaction, 684 Gel-based quantification, 776 GenBank, 830, 846, 848 Gene, 154 Genealogical determination, 1047 Genealogical research, 260 Genealogical studies, 449–450 Gene expression, 520 GeneMapper ® ID-X (GMID-X) ® software, 197 Genetically modified food (GM-food), 893 Genetic analyzer, 166, 170, 173, 176 Genetic data, 261 Genetic diversity, 292 Genetic genealogy, 504

1193 Genetic Investigation of Anthropocentric Traits (GIANT), 390 Genetic marker, 1042 Genetic polymorphisms, 426 Genetic transmission, 227, 229 Genetic variations, 1042 Genodermatoses, 692–694 Genome wide association studies (GWAS), 249 Genomic DNA, 610 Genotype, 649 Geographic identification, 830–832 Geographic origin, 873 Geolocation, 509 Global Biodiversity Information Facility (GBIF), 927 Globalfiler kit, 564 6-dye technologies, 169, 172 enhanced buffer system, 170 features, 169, 170 STR markers, 170, 171 GlobalFiler™ IQC PCR Amplification Kit, 1095 Globalized market, 892 Golden State Killer, 346 Gonosomal InDels, 1046 Good Laboratory Practice, 1088 Governmental subsidies, 1093 Gowers’s sign, 688 Graft versus Host Disease (GvHD), 651 Graft versus Leukemia (GvL) effect, 665 Grapevine products, 904 Griscelli syndrome, 693

H Half-sisters, 469 Haplodiploid markers applications, 226–230 association at the population level, 225–226 genetic relatedness, 227–230 individual profiling, 226 linkage, 225 single locus, 221–223 theoretical and statistical analyses, 221–226 Haplodiploidy, 220, 222, 230 Haplotypes, 649, 1171 HapMap project, 251 Hard binding, 126 Hardy–Weinberg Equilibrium (HWE), 221–223, 999, 1117 Heat denaturation process, 106 Hematoma, 571 Hematopoietic maternal cells, 669

1194 Hematopoietic Stem cells (HSC), 664 Hematopoietic Stem Cell Transplantation (HSCT), 647, 662, 671 Hemopoietic, 648 Herbal dietary supplements (HDS), 900 Hermansky Pudlak syndrome, 694 Heteroplasmy, 140, 427–429, 484, 485, 488, 489, 492, 493, 495, 496 Heterozygosity, 1047 Heterozygote peak imbalance, 1078 Heterozygous, 156 imbalance, 657 Hexaethyleneoxide (HEO), 164 HID Ion GeneStudio™ S5 Prime System, 1101, 1102 Hierarchy of the propositions, 802 High resolution melting (HRM), 899, 901 High-throughput sequencing (HTS), 850 HIrisPlex test system, 389 Homeland Security News Wire, 347 Homicide, 568–570, 842 HOMINGS technique, 506 Homoplasmy, 484 Homozygosity, 546, 547, 551 Homozygous, 156 loci, 551 House robbery, 23 Human DNA analysis Colin Pitchfork case, 359–360 Craig Harman case, 362 identification of 9/11 victims, 362–363 Joseph Castro case, 360–361 O. J. Simpson murder case, 361–362 Tommie Lee Andrews case, 360 Human DNA databases data protection, 263–264 funding agencies, role of, 266 funding and benefit-sharing, 265 genetic research and DNA databasing, ethical elements, 261–265 independence of ethics committees, 264 informed consent, 262–263 multiple stakeholder responsibility, ethical framework for, 265–273 public engagement, 265 RECs, role of, 266–272 researcher, role of, 265–266 research institutions, role of, 266 scientific journals, role of, 267–273 Human genome, 132 Human identification (HID), 385, 386, 520, 525, 535, 608, 610, 616, 617, 619, 732, 733, 744, 750, 1042, 1046, 1047, 1108, 1118

Index Human Identification (HID) Professional Services (HPS), 1095 Human Leukocyte Antigen (HLA), 646, 649, 671 Human migration, 1052–1054, 1062–1064 Africa, 1172, 1173 Asia/Oceania, colonization, 1176 contemporary biology, 1166 Denisovans, 1179 DNA, 1166, 1167 Europe, 1174, 1175 populating Americas, 1177, 1178 research, 1180 Human Remains Index (HRI), 1026 Human research ethics committee (HREC), 264 Human subject protection, 292 Human trafficking, 1053, 1055–1057 Humic acid, 1072 Humic substances, 1076 Humidity, 380 Hydrogen peroxide washing, 759 Hydrolysis, 379 Hydrolysis-induced rupture, 1075 Hydroxyapatite mineral matrix, 385 Hypertrophic cardiomyopathy, 697 Hypervariable points I, 139 Hypervariable regions, 483

I Ideal genetic markers, 1040 Identical-by-descent alleles, 227 Identical twins, 818, 819 Identifiler Kit advantages, 163 autosomal STR markers, 163, 164 dyes, 163 emission spectra, 165 IDentify, 1139, 1140, 1143 Identity-testing SNPs, 240 Illegal adoption, 1053, 1058, 1062, 1064 Illumina MiSeq instrument, 507 Immunophenotypic analysis, 665 Inadequate validation, 1094 Inborn errors of metabolism, 689–692 Inconsistencies, 398, 403–405, 408, 409, 412, 418 InDel loci, 1043, 1045 InDel multiplex panel kits, 1046 InDelPlex INDEL Polymorphism Detection Kit, 1044 Individual identification, 833–835 Individualization, 874–875 Individual profiling, 226

Index Infectious disease, 505, 507 In ammatory bowel disease (IBD), 609 Informationals markers, 653 Informed consent, 262–263 Inherited paternal antigens (IPA), 665 Injection syringes, 563 Innocence project, 369, 370 InnoQuant ® H-Dye, 786 Insertion-deletion (InDel), 409, 410, 859, 862 Insertion / deletion markers (Ins/Del), 648 Insertion/deletion (InDel) mutations, 1042 Insertion/deletion (InDel) polymorphism, 1043, 1047 Institutional grants, 1093 Institutional review board (IRB), 264 Internal Lane Standard (ILS), 655 Internal PCR control (IPC), 383 Internal positive control (IPC), 1111 Internal quality control (IQC), 172, 173 Internal transcribed spacer (ITS), 918–919 Internal validation, 299, 1090, 1091, 1102, 1104, 1112 standards and guidelines, 300–304 International Criminal Police Organization, 983 International Genetics of Anthropomorphic Traits, 1138 International research networks, 897 International Society for Forensic Genetics DNA Commission, 383 International Society of Forensic Genetics (ISFG), 827 International Society of Hematotherapy and Graft Engineering (ISHAGE), 650 International standard, 1088 International standards and guidelines, 1093 Interspersed Simple Sequence Repeats (ISSR), 901 Investigative steps, 20 Investigative tool, 348 Investigator ® DIPplex Kit, 1043 Investigator ® Quantiplex Pro Kit, 786 Ion torrent method, 503 Ion Torrent™ PGM ®, 1081 IrisPlex model, 1133 ISO/IEC 17025:2017, 1093 ISO/IEC 17025 standardization, 1121 Isometric alleles, 36 Israel Police DNA Index System (IPDIS), 1021 J Jacquard coefficient, 463 Jacquard’s coefficients partitions, 466 Junior investigators, 23 Junk DNA, 342

1195 K Kansas City Police Department (KCPD), 971 Kerala, India Ockhi cyclone disaster, 581–582 Puttingal temple firework disaster, 579–581 Kinship analysis, 248–249 Kinship analysis and X-chromosome, 464 female-female duo, 464 female-male duo, 465–472 male-male duo, 465 paternal aunt–niece, 472 paternal grandmother–granddaughter, 470 Kinship and Data Analysis Panel (KADAP), 363 Klebsiella pneumoniae infection, 508 Klinefelter syndrome, 699 Kolmogorov-Smirnov’s test, 223

L Laboratories data analysis and interpretation guidelines, 1100 fundamental studies, 1100 infrastructure, 1101 project planning, 1101, 1102 standardised or controlled sample sets, 1100 validation data, 1103 Law enforcement, 1024, 1035 agencies, 259, 1143 Rapid DNA (see Rapid DNA) Legislative response, 350 Length polymorphism, 95 Leukemia, 653 Leukocytes, 654 Life-long prison sentence, 568 Likelihood ratio (LR), 96, 435, 804, 999, 1004, 1007, 1114 approach, 201 Lineage markers, 832 mitochondrial DNA, 424–438 Y-chromosome (see Y chromosome) Lineage SNPs, 240 Liquid blood, 79, 80 LMNA gene mutations, 698, 699 Local DNA index system (LDIS), 1025 Locard’s exchange principle, 144, 561 Locus/loci, 156, 656 Long period grating (LPG), 903 Low copy number (LCN), 6, 1005–1006, 1077 problems with, 1077–1079 Luminol, 88

1196 Lymphohistiocytosis criteria, 661 with deconvolution, 662, 663 immune dysfunction, 661 pathology subtypes, 661 treatment, 662 without deconvolution, 662 M Magnetic-activated cell sorting (MACS), 663 Magnetic beads extraction method, 716–719 Maillard reactions, 1075 Major Histocompatibility Complex (MHC), 648 Male-specific region (MSY), 439 Manual DNA isolation Chelex ® resin, 763 manual purification of DNA extracts, 763–764 organic extraction, 763 total demineralization, 762–763 Markers characteristics, 155 Markov chain Monte Carlo (MCMC), 1008, 1009 Mass disaster, 240 Mass disaster victim identification Ockhi cyclone disaster, Kerala, India, 581–582 Puttingal temple firework disaster, Kerala, India, 579–581 Mass fatality cases, 1043 Massively parallel DNA sequencing, 862 Massively parallel sequencing (MPS), 33, 35, 259, 279, 371, 496, 503, 950–951, 1010–1013, 1076 Mass spectrometry, 247 Match confirmation, 984–985 Matched unrelated donor (MUD), 649 Match probability, 95 Match unrelated donor (MUD), 664 Maternal cell contamination (MCC), 635 Maternal inheritance, 485, 489 Matrilineal inheritance, 427 Matrix metalloproteinase (MMP), 949 Maturase K (matK), 916–917 Maximum likelihood estimation (MLE), 1008 Medical diagnostics, 504 Medical microbiology, 505–508 Medical Research Ethics Committee (MREC), 285 Melissopalynological analyses, 900 Mendelian laws, 1043

Index Mentype ® DIPplex PCR Amplification Kit, 1044 Metabarcoding, 862 Metabolomic profile, 892 Metagenomics, 40, 951 Method-assessment processes, 1091 Methylation PCR and chromosomal microarray test, 680 Methylation-sensitive restriction enzyme-PCR (MSRE-PCR) method, 332 Methylation specific MLPA, 637 Metotrexato (MTX), 651 Microarray techniques, 617 Microbes, 505 Microbial and animal forensics, 142–146 Microbial evidence, 936, 939, 944, 945 Microbial food spoilage, 894 Microbial forensics, 936 analytical techniques, 947 bioterrorism, 936–937 body uids, detection of, 940 detection and identification, 943 development of strain repository, 944 and epidemiology, 940–943 individualistic micro ora for person identification, 938 information and databases, 944 interpretation of results, 951–952 metagenomics, 951 micro ora of soil and water for forensic use, 940 molecular techniques, 947 MPS, 950–951 need for validation, 944 PCR, 948, 949 physical analysis, 947 post-mortem examination, 938–939 quality assurance guidelines, 944 RT-PCR, 948, 949 sample collection, 944–946 SNP array, 949–950 VNTR, 950 Microbiology, 936 Microbiome, 40 Microchimerism, 647, 672 Microdeletion syndromes Prader Willi syndrome, 681–682 22q11.2 deletion syndrome, 681 subtelomeric deletion, 682 Microhaplotypes, 1076 Microsatellite polymorphisms of Y chromosome, 444

Index Microsatellites, 95, 653, 832 Microscopic methods, 389 Migrant smuggling, 1055 Milk fats, 894 MiniFiler kit, 1117 Minimum quantity detectable (MQD), 90 Minisatellites, 95, 653 Minisequencing, 246 Mini short tandem repeats (miniSTRs), 136, 1040, 1076 Mini Y-STR, 191 miRNAs profiling, 326–327 Missing persons and migrations, 1055–1056 Missing Persons Index (MPI), 1026 Mitochondrial DNA (mtDNA), 107–109, 137–140, 342, 424, 525, 528, 610, 827, 1076, 1167 analysis, 97 biogeographical origin of the individual, 437 characteristics, 426–430 degradation resistance, 429 forensic applications, 435–438 genome sequencing, 513 haplogroup diagnosis, 434–435 haplotype determination, 433 heteroplasmy, 427–429 high mutation rate and neutrality, 429 high number of copies, 426 kinship detection, 437 localization, origin and structure, 425–426 low template DNA damples, 436 markers, 400–401 matrilineal inheritance, 427 mitochondrial reference sequence, 433 nomenclature, 431–432 NUMTs, 430 results and interpretation, 432–433 sequencing, 37–38, 1101 single nucleotide polymorphisms and variations, 430–431 statistical evaluation, 435 twins differentiation, 438 variants segregation, 428 Mitochondrial DNA (mtDNA), in human identification age estimation, 487–488 AmtDB, 493 analytical methodologies, 490–491 CE technique vs. NGS, 495 CRS, 490–491

1197 degraded compromised samples, 486–487 EMPOP, 493, 494 geneology tracing, 489–490 HmtDB, 493 human mitochondrial DNA sequence database, 493–494 massive parallel sequencing, 495–496 MITOMAP, 493 MitoVariome, 493 mtDNA sequencing result interpretation, 492–493 nomenclature of mtDNA sequence, 491–492 screening approaches for mtDNA analysis, 494 Mitochondrial genome, 481–483 inheritance of, 484–485 MitoMap project, 514 Mixed Chimerism (MC), 647 Mixed DNA samples LCN, 1005–1006 mixture and complex DNA profiles, 1001–1005 MPS approaches, 1011–1013 probabilistic genotyping, 1006–1010 Mixed gonadal dysgenesis, 699 Mixture and complex DNA profiles, 1001–1005 Mobility modifying, 164 Moderately mutating Y-STRs, 190 Modus Operandi, 18–20, 22 Molecular analysis, 385, 609 Molecular authentication, 896 Molecular beacons (MB), 241–245, 779–780 Molecular biology, 666, 672, 896 Molecular cytogenetics, 626, 629 Molecular genetics, 504, 1040 Molecular marker, 616 Molecular taphonomic processes, 380 Morphological analysis, 665 mRNA, 617 mRNA-profiling, 124 mtDNA typing, 616 Mucosal cells, 334 Multidisciplinary team, 606 MultiLocus Probe (MLP), 998 Multilocus variable number tandem repeat analysis, 950 Multiple endocrine neoplasia, 696 Multiplex human identification genotyping assay, 248

1198 Multiplex ligation-dependent probe amplification (MLPA), 636, 640, 680 applications, 636 methylation specific, 637–638 reaction for deletion/duplication analysis, 637 Murder automated DNA extraction method, 563 autosomal STR DNA typing, 564–565 case history, 561 description and sources of samples, 563 DNA evidence, 560–561 Mutation(s), 400, 401, 403–405, 408–412, 1041 screening, 632–635

N National Accreditation Board for Testing and Calibration Laboratories (NABL), 1088 National Association of Testing Authorities (NATA), 1088 National Center for Biotechnology Information (NCBI), 791 National DNA databases African region, 1021 American region, 1024, 1026 Asia, 1026, 1028 Australasia, 1024 European region, 1021, 1023 Middle East, 1021 National DNA index system (NDIS), 158, 977, 1024, 1127 National DNA Profile Databank (NDPD), 1024 National Ethics Committee for Health Research (NECHR), 281, 284 National Forensic DNA Database of South Africa, 1021 National Forensic Institute (NFI), 1021 National Institute for Criminalistics and Criminology, 1022 National Institute of Standards and Technology (NIST), 1108 National Marrow Donor Program, 671 National Missing Persons DNA Program (NMPDP), 1026 National Police Agency (NPA), 1027 National Research Council of Thailand (NRCT), 289 National System of Judicial Genetic Data, 1023

Index Newborns, 626 Newborn screening, 701–703 premarital/pre-conception/prenatal/ preimplantation genetic screening, 703–705 New generation kits, 167, 168 Nextera XT approach, 512 Next-generation sequencing (NGS), 146–147, 371, 386, 388, 410–411, 495, 617, 638, 640, 642, 649, 785, 894, 1001, 1011, 1012, 1014, 1076, 1081, 1089, 1094, 1101, 1102 applications in DNA profiling, 504–514 ancestry informative markers, 39 benefits over CE, 40–41 vs. CE, 33–36 degraded samples, 39 DNA intelligence, 38 environmental microbiology, 508–509 in evolutionary biology, 514 forensic applications, 35–40 forensic applications of DNA profiling, 504 in forensic biology, 510–513 forensic DNA phenotyping, 38 impact in forensics and diagnostics, 504 markers, amplification, 32–33 medical microbiology, 505–508 mitochondrial DNA sequencing, 37–38 molecular genetics and diagnostics, 504–505 monozygotic twin identification, 39 in plant biology, 513–514 similarities with CE, 35 SOLiD method, 503 STRs and SNP sequencing, 36–37 work ow, 35 Non-coding SNPs, 239 Non-human DNA analysis, 789, 840 Bogan case, 364–365 Snowball case, 363–364 Non-human paternity testing, 835 Non-invasive prenatal testing (NIPT), 639, 704 Non-nucleotide linkers, 164, 170, 173 Non pigmented traits, DNA based inference age, 1137–1138 body height/stature, 1138–1139 face, 1136 hair loss/baldness, 1137 hair structure, 1136–1137 Non-recombinant region, 439 Non-syndromic cardiomyopathy, 698 Non synonymous type SNP, 239 Noonan syndrome, 697

Index No Template Control (NCT), 653 Nuclear DNA, 101, 107, 153 Nuclear magnetic resonance (NMR), 900 Nuclear-mitochondrial DNA fragments (NUMTs), 430 Nucleic acids analysis, 897 Nuclein, 713 Nucleotides, 1111 Number of contributors (NOC), 1120 Numerical conversion error, 814, 815 Nusinersen, 687 Nylons ocked swabs, 596

O Objective probative acquisition investigations, 102 Objective thresholds, 314 Ockhi cyclone disaster, Kerala, India DNA extraction, 582 DNA profiling, 582 postmortem samples, 581 Odontoblasts, 757 Offence level, 802 Offender profiling, 19 Oral uids, DNA biological sources deposition of trace samples, 60 double swab technique, 59 in drowning case, 60 extraction from different surfaces, 59 isolation technique, 59 occurrence, 59 Oral swab, 571 Organic extraction, 715–716 method, 922 Organic method, 737–738, 745–746 Organization of Scientific Area Committees (OSAC), 1108, 1118–1120 Organ trafficking, 1063–1064 Outer enamel epithelium, 757 Ownership, 10 Oxidation, 379 Oxidative DNA damage, 1074

P Palindromes, 440 Parabon NanoLabs, 1140, 1143, 1144 Parentage testing autosomal and lineage markers in, 401–402 Avuncular Index, 404–405 calculation of mutation rate, 404 complementary markers, 409–410

1199 human identification, autosomal STRs in, 401 NGS, 410–411 statistical analysis, of paternity test results, 402–403 STR inconsistencies/mismatches, 403–404 STR mutation, 405–409 types of samples, 399 Partial DNA profile, 95 Paternal aunt–niece, 472 Paternal grandmother–granddaughter, 470 Paternity cases, 1045 Paternity Index (PI), 402 Paternity testing, 207, 248–249, 398, 399, 403, 410, 835 Pathogens, 505 PCR inhibition, 1070 inhibition factors, 1072–1073 mechanism, 1071–1072 Performance check, 1091 Periodontitis, 506 Persistent mixed chimerism (PCM), 647 Personal protective equipment (PPE), 1109 Person of interest (POI), 260, 1003, 1008 pH, 380 Phenol chloroform extraction, 715 Phenol-chloroform/organic DNA extraction, 612, 613 Phenylbutazone, 898 Philadelphia chromosome, 666 Philippine Health Research Ethics Board (PHREB), 287 Phonotypic informative SNP, 241 Photographic documentation, 571 Phylotree, 434 Physical evidence, 6, 7, 9, 11, 22, 71 Physiognomic characteristics, 607 Plant biology, 513–514 Plant DNA Barcoding Project, 846 Plant species identification, DNA barcoding collection of plant samples for DNA isolation, 919–921 CTAB method, 922–924 database, 927–928 forbidden/controlled materials, 913–914 forensic botany, 915–916 ITS, 918–919 matK, 916–917 organic extraction method, 922–923 PCR amplification, 924–926 plant DNA isolation, 921–922 plant genome sequencing, 925–927 poisoning, 914–915

1200 Plant species identification, DNA barcoding (cont.) preservations of samples, 921 protected plants, environment laws, 914 quality and quantity assessment, 923–924 rbcL, 917–918 transferred trace evidence, in physical and sexual assault cases, 912–913 Plexor HY System, 786 Point or gene mutations, 1042 Poisson distribution, 898 Police, 1020, 1022, 1027, 1029–1031, 1033, 1034 Pollen DNA metabarcoding, 850 Pollen morphology, 849 Polymerase chain reaction (PCR), 71, 94, 359, 370, 371, 577, 578, 615, 616, 618, 633, 864, 867, 872, 924–925, 948, 949, 977, 1001, 1090, 1097, 1101, 1103, 1111, 1112, 1114, 1117, 1120, 1166 amplification, 388 based DNA tests, 126–131 based tests, 126–131 cycling, 382 duplication of mtDNA, 139 inhibitors, 382 kits, 388 techniques, 961 Polymerase shift, 1041 Polymorphic systems, 1047 Polymorphisms, 156, 1041–1043, 1045–1047 Polyploidy, 677 Polyvinylpyrrolidone (PVP), 923 Population genetics, 248 Post-conviction DNA testing, 369–370 Posterior odds, 804 Postmortem (PM), 379 Postmortem interval (PMI), 378, 617, 938–939 Post-PCR analyses, 382 Post-PCR products, 1080 Post-transplant chimerism, 655, 671 Posttraumatic stress disorder, 572 Powerplex 16HS system autosomal markers, 167 definition, 165 penta D, 166 penta E, 166 reproducible results, 166 PowerPlex ® Y System, 194 PowerPlex kits, 163 Prader Willi syndrome, 632, 681–682 Precision ID kit, 511 Predentin, 757

Index PredYMaLe, 212 Pregnancies, 1045 Premarital screening, 703 Prenatal diagnosis of genetic disorders, 627 ARMS PCR, 634 case studies, 639–642 for chromosomal testing, 628–629 FISH testing, 629–632 indications for genetic testing, 627 lab methods, 630–631 MLPA (see Multiplex ligation-dependent probe amplification (MLPA)) mutation screening, 632–635 next generation sequencing, 638–639 non-invasive prenatal testing for cytogenetic abnormalities, 639 polymerase chain reaction, 633 pre and post-test genetic counselling, 628 prenatal cytogenetics and limitations, 628–633 quantitative uorescent polymerase chain reaction, 635 RFLP, 634–635 triplet-primed PCR, 635–636 pre-PCR techniques, 1101 PrepFiler ® BTA Forensic DNA Extraction Kit, 748–749, 765 PrepFiler™ BTA Lysis, 611 Preservation, 70, 71 Preserving, 76 biodiversity, 900 Pre-trial preparation, 23 Primary contact, 570 Primers, 342 Prior odds, 803 Privacy, 1029–1031 Probabilistic genotyping (PG), 1006–1010, 1094 Probabilistic genotyping software (PGS), 96, 298–300, 1113, 1114 allele sharing, 309–311 DNA template quantities, 307 number of contributors, 308–309 principles, 304 standards and guidelines governing internal validation, 300–304 transparency of validation data for independent review, 315–316 validation incorporation into standard operating procedures, 313–315 validation workshops and technical merit evaluation by, 303–304 variation in contributor ratio, 307–308

Index Probability of another match error, 813, 814 Probability of exclusion (PE), 402, 404, 1007 Probability of identity (PI), 176 Probability of Paternity, 402 Probative evidence, 9 Progressive cardiac conduction system disease (PCCD), 698 Promega and Applied Biosystem, 161 Promega Powerplex series, 1115, 1116 Promega PowerSeqTM system, 511 Promoter SNPs, 239 Prosecution, 568 Prosecution hypothesis (Hp), 801, 804, 807, 816, 818, 1004, 1114 Prosecutor’s fallacy, DNA evidence, 806, 810, 811 false positive fallacy, 809, 810 source probability error, 807, 808 ultimate issue error, 808, 809 Protected Designations of Origin (PDO), 906 Protected Designation of Origin of Extravergin Olive oil (PDO EVO oil), 899 Protected Geographical Indication (PGI), 906 Protocol drift, 1119 Public engagement, 265 Published guidance documents, 300–303 Puttingal temple firework disaster, Kerala, India DNA extraction, 579–580 DNA profiling, 580 matching of DNA profiles, 580 postmortem samples of victims, 579 success rate of DNA profiling, 581 Pyrimidine dimers, 379 Q 22q11.2 deletion syndrome, 681 QIAamp DNA Investigator Kit protocol, 739–741, 746–748, 765–766 Quality assurance, 13 guidelines, 944 systems, 1088, 1089, 1093, 1094, 1104 Quality Assurance Program, 1108 Quality Assurance Standards (QAS), 1089, 1091, 1108 Quality control, 654, 841, 1109, 1110, 1112, 1119, 1120 Quantifiler™ Trio, 786 Quantitative uorescent polymerase chain reaction (QF-PCR), 635, 679 Quantitative polymerase chain reaction (qPCR), 648, 652, 774, 782, 967 aDNA studies, 788–789

1201 advantages and limitations, 790 assessment of DNA degradation, 785 autosomal DNA quantification, 785–786 background calibration, 782 calibration curve and DNA quantity calculation, 780–782 customization, 783–784 detection of PCR inhibition, 785 dPCR, 792–793 dye spectral calibration, 782 mitochondrial DNA quantification, 787–788 molecular beacons, 779–780 NGS libraries, 785 non-human DNA analysis, 789 optimisation of DNA extraction, 785 primer design, 784 probe design, 784 ROI calibration, 782 sex determination and detection of DNA contaminants/mixtures, 785 SNPs, 790 SYBR ® Green qPCR assays, 777–779 TaqMan probes, 776–777 X and Y chromosome quantification, 787 Quantitative trait loci (QTL) analysis, 513 Quercus geminata, 842 R Racial identifiers, 349 Racial profiling, 349, 350, 352, 1142 Radioactive probe, 126 Radiography, 606 Randomly amplified polymorphic DNA (RAPD), 841, 861, 901 Random match probability (RMP), 252, 805, 999, 1003 Random sampling, 86, 946 Rape, 572 Rapid DNA, 990, 991, 1089, 1094, 1102, 1152, 1153, 1158–1160 booking station, 981 cognitive challenges, 1157 contamination, 1155–1156 cost, 991–992 crime scene, 981–982 DVI, 983–984 ease of use, 979–980 expanded testing capabilities, 992–993 history, 977–978 instruments, 43–45 match confirmation, 984–985 mixture interpretation, removing bias in, 985

1202 Rapid DNA (cont.) portability, 980 privacy, 1156–1157 quality of training, 1159 retesting of swabs, 985–986 satellite DNA laboratories, 982–983 transportability, 1154–1155 turnaround time, 1153–1154 RapidHITTM ID system, 44, 986 Rapidly mutating (RM) Y-STRs, 189–191, 441 RDX-C4, 595, 599, 602, 603 Reagent preparation, 1110 Real-time PCR (RT-PCR), 386, 652, 653, 788–794, 892, 924, 948, 949 Red blood cells (RBC), 735 Reference DNA, 653 Reference marker, 653 Reference population DNA database, 259 Re-interview victims, 21 Relative uorescence units (RFU), 569, 654 Relative frequency of occurrence, 815, 816 Relatives of Missing Persons Index (RMI), 1026 Replicate analysis, 1078 Report writing, 1095 Research ethics committees (REC), 261, 264, 266–267, 274, 284, 286, 292 Research publications, 271 Resolving criminal casework, 26 Restriction fragment length polymorphism (RFLP), 358, 359, 370, 371, 576, 634–635, 999, 1126, 1167 analyses, 716 Restriction fragment length polymorphismbased DNA testing, 125–126 Re-trial in Germany, 569 Reverse C.S.I. effect, 568 Revised CRS (rCRS), 491, 492 Ribulose-1,5-bisphosphate carboxylase/ oxygenase, 917–918 Right to Privacy, 346, 350 Robertsonian translocation, 678, 682 Robust thresholds, 314 Routine laboratory, 573 Royal Canadian Mounted Police (RCMP), 1025 Royal Malaysia Police Forensic Laboratory (RMPFL), 1027

S Saliva, 571, 608, 1109 Salivary microbiome, 609 Salmonella typhimurium, 507

Index Sample pretreatment, DNA isolation acid washing, 759 combination of techniques, 760 compact bone sampling, inner part of, 760 decontamination, 758–760 enzymatic predigestion, 759 ethanol washing, 758–759 Freezer/Mill ®, 761 hydrogen peroxide washing, 759 manual mortars, 761 milling and sanding, 759 pulverization, 761–762 sodium hypochlorite washing, 758 surface washing, 758 TissueLyser II system, 761 ultraviolet irradiation, 760 Sanger sequencing, 33, 636, 649 analysis, 846 Sange technique, 139 Santa Cruz, 251 Scene of Crime Officers (SOCOs), 1159 Science and technology, 606 Science-based identification method, 344 Scientific communities, 181 Scientific universal-multidisciplinary approach, 376 Scientific Working Group on DNA Analysis Methods (SWGDAM), 180, 300, 521, 793, 985, 1003, 1089, 1091, 1095, 1108, 1119 Scraping technique, 965 Scrapping method, 76 Secondary transfer of DNA, 569 Semen, DNA biological sources extraction protocols, 57 individualization of, 56 Pitchfork Case’ of Leicestershire (1988), 56 sexual assault cases, 56 Sentencing, 22 Sequence polymorphism, 94 Sequence specific oligonucleotides (SSO), 649 Sequence specific primers (SSP), 649 Sequencing by hybridization (SBH), 246 Serial investigation, 23 Serial offender casework, 23 Serial rape/serial murder, 19 Serological probes, 608 Sex markers, 616 Sexual assault, 571–573 Shared Allele Imbalance (SAI), 657, 660 Short-fatty acids (SFCAs), 894

Index Short tandem repeats (STRs), 71, 155, 156, 239, 240, 247, 248, 370, 398, 399, 411, 412, 418, 520, 564, 576, 580, 582, 616, 646, 653, 671, 775, 826, 834–835, 862, 977, 1000–1002, 1004–1007, 1011, 1012, 1090, 1092, 1093, 1127, 1154, 1167 analysis, 1046 anomalous band patterns, 409 autosomal STRs, 399–400 capillary electrophoresis and DNA profiling, 578 DNA pre-processing, extraction and quantification, 577–578 kits, 1117 loci, 1040, 1046 marker systems, 1117 number, 1046 original form, 1115 PCR amplification of STR Loci, 578 primer binding site mutations, 405–408 profile, 569 sample collection, storage and transport, 577 strand slippage mutations, 408 STR typing methodology, 402 systems, 1047 technology, 342 trends, 1117, 1118 UPD, 408–409 Y chromosomal STRs, 400 Short tandem markers, 6 Simple sequence repeats (SSR), 861, 901 Single donor chimerism analysis, 656, 657, 659, 662 Single gene disorders, DNA testing adult onset/late onset diseases, 700–701 alpha thalassemia, 684 beta thalassemia, 683 cardiac disorders, 697–699 congenital adrenal hyperplasia, 685–686 cystic fibrosis, 684–685 disorders of sex development, 699–700 Duchenne muscular dystrophy, 688–689 endocrine disorders, 696–697 genodermatoses, 692–694 inborn errors of metabolism, 689–692 skeletal dysplasia, 694–696 spinal muscular atrophy, 686–687 Single locus probe (SLP), 999

1203 Single nucleotide polymorphism (SNP), 237, 400, 409, 495, 524, 616, 789–790, 826, 833, 862, 874, 879, 899, 1001, 1011, 1041, 1043, 1129, 1130, 1132–1136, 1138, 1139 advantages of, 237–239 ancestral informative markers, 240 array, 679, 949–950 autosomal SNPs, 400 constraints of, 250 dbSNP, 250–251 DNA microarray, 245 ensemble, 251 forensic applications, 247–250 HapMap project, 251 human identification from skeletal remains, 247–248 identity-testing, 240 information databases, 250–251 lineage, 240 markers, 239–240 mass spectrometry method, 247 molecular beacons, 241–245 paternity testing and kinship analysis, 248–249 phenotypic information of missing suspect, 249–250 phonotypic informative, 241 Santa Cruz, 251 SNaPshot multiplex method, 246 vs. STR, 238 techniques for analysis of, 241–252 Sinofiler, 1117 Sisterhood probability, 466 Skeletal biological profile age at death, 388, 389 biogeographical ancestry, 390 craniofacial features, 391 height, 390 pigmentation, 389 sex identification, 387, 388 Skeletal dysplasia, 694–696 Skin akes, 572 Slippery slope theory, 1141–1142 Slot blot, 776 Slowly mutating Y-STRs, 190 SNaPshot, 1139, 1140, 1143 multiplex method, 246 Sodium dodecyl sulphate (SDS), 386, 737, 763 Sodium hypochlorite washing, 758 Sodium lauryl sulphate (SLS), 386 Soil composition, 381 Solar radiation, 1075

1204 Solid-phase DNA extraction decomposed/skeletonized bodies, 746 fresh bodies, 739 Somatic chromosomes, 1045 Somatic/gonosomal chromosome mutations, 1042 Source level, 802 Source probability error, 807, 808 Species specific primers, 828 Spinal muscular atrophy, 686–687, 703 Spin columns extraction, 719 Standard curve, 792 Standardized validation process, 1088 Standardization, 654 Standard operating procedure (SOP), 312–315, 1091 Stepwise mutation model (SMM), 403, 444 Stochastic detection threshold, 1077 Storage conditions, 723 Strategic Initiative for Developing Capacity in Ethical Review (SIDCER), 279 STRBase website, 1116 STR markers, 141–142 STRmix™, 1009, 1010 STR typing, 134–137 Stutters, 1078 Sublevel, 802 Subtelomeric deletion, 682 Sugar-phosphate structure, 1108 Surface abrasion, 1080 Surface washing, 758 Swabbing, 965 Swab technique, 964, 965 Sweat chloride tests, 685 Sweat, DNA biological sources chemicals on DNA profiling, 62 concentration of DNA, 62 importance of, 61 sample collection, 61 shedding rate, 61 substrate bearing sweat stains, 61 Touch DNA, 60 Transfer DNA, 60 SYBR ® Green qPCR assays, 777–779 Syndromic cardiomyopathy, 698 Synonymous type SNP, 239 System vulnerability, 264 T Tailored techniques, 895 Tape-lifting, 596, 963 method, 78 technique, 965

Index TaqMan ® PCR assay, 904 TaqMan probes, 776–777 Targeted sampling, 946 Taxonomic identification, 870–873 Tay Sachs disease, 704 Teachback training, 1095 Technical Working Group on DNA Analysis Methods (TWGDAM), 1024 Technical Working Groups, 1108 Test-on-arrest, 344 Tetranucleotide repeats, 134 Tetranucleotide STRs, 144 Texas Forensic Science Commission, 1094 β-Thalassemia donor and recipient, 658, 659 groups, 658 hemoglobin, 658 Thanatophoric dysplasia (TD) type I, 695 The Mummy, 618 Thermal cycling process, 1112 Thermo Fisher Scientific company, 169, 1095 ThermoFisher, scientific human identification kit, 163, 172 Tiling strategy, 246 Time resolved uoro-immunoassay, 684 TissueLyser II system, 761 Tooth anatomical parts, 607 DNA extraction, 614, 615 formation, 757 samples, 609 vertical and horizontal sectioning, 611 Total Number Cells (TNC), 650 Touch DNA, 1077 biological matter, 960 evidentiary value, 967–970 examination, 966 extraction, 966 history, 962 isolation, 967 samples, 9 sampling techniques, 970 techniques, 963–965 Traceability, 896 Traditional Chinese Medicine, 824 Traditional DNA methods data interpretation, 989 electrophoresis, 988 extraction, 987 PCR amplification, 988 quantification, 988 sample collection, 987 troubleshooting, 990

Index Traditional Specialty Guaranteed (TSG), 906 Transportability, 1154–1155 Transportation, biological evidences, 70 Trengglalek shipwreck, 583 Triplet primed PCR (TP-PCR), 635–636, 702 Trypsin-Giemsa stain, 678 Tuberous sclerosis, 692 Tumoral necrosis factor (TNF), 651

U Ultimate issue error, 808, 809 Ultraviolet irradiation, 760 Ultraviolet radiation, 1075 Ultraviolet (UV) spectrophotometry, 775 Umbilical cord blood (UBC), 646, 650, 664, 671 Uniparental disomy (UPD), 408–409 Uniqueness fallacy, 812, 813 United Nations Conference on Trade and Development, 264

V Vaginal secretion, DNA biological sources contamination of genetic material, 58 cross-contamination, 58 sexual assault, 58 Vaginal swab sample, 571, 1045 Validation approaches, 1092 confidence and quality, 1093 context, 1092 definition, 1089 experimental design, 1094 interlocus balance, 1097, 1099 laboratories, 1093 laboratory’s purposes, 1090 peak height ratios, 1097–1099 repeatability of peak heights, 1097, 1099, 1100 standardized scope, 1095–1097 transformation, 1094 types, 1090 Validation and Verification of Quantitative and Qualitative Test Methods, 1094 Variable number of short tandem repeats (VNTRs), 1115 Variable number of tandem repeats (VNTRs), 95, 120, 576, 648, 653, 950, 1040, 1127, 1167 Velocardiofacial syndrome, 681 Vendor’s validation, 1102

1205 Verifiler kit advantages, 173 6-dye technology, 172, 173, 175 features, 172 optimization, 172 primers, 173 STR makers, 174 Victim assistance act, 572 Visual recognition, 606

W Weight of DNA evidence, 804 Western-diet, 894 Whatman FTA ® paper, 737 White blood cells (WBC), 651, 664, 735 Whole exome sequencing (WES), 495 Whole genome sequencing (WGS), 495 Whole-genome shotgun sequencing (WGSS), 949 Wildlife DNA profiling and forensic relevance COI, 829 control region (D-loop), 829 Cyt b, 829 DNA repositories, 830 DNA sequencing, 827 geographic origin, identification of, 830–833 individual identification, 833–835 lineage markers, 832 microsatellites, 832 non-human paternity testing, 835 SNPs, 833 species of origin, identification of, 826–830 species specific primers, 828 species testing, 827–828 Wines DNA fingerprints (WDF), 901–903 Wolf Hirschhorn syndrome, 682 Wood’s lamp, 88

X X-chromosome, 230, 618, 1045, 1046 analysis, 140–142 evolution, 456–458 and forensic analysis of low template DNA, 475 genealogies, 472–473 identical-by-descendants, 461 identical-by-state alleles, 461 incest cases, 473–475 and kinship analysis (see Kinship analysis and X-chromosome)

1206 X-chromosome (cont.) likelihood ratios, 463 markers and usefulness, 459–460 population data, 460–461 statistical approach, 461–472 structural characteristics, 458–459 X-Chromosome short-tandem repeats (X-STRs), 524 Xeroderma pigmentosum, 694 X-InDels, 459, 475 X-linked polymorphism analysis, 1045 X-SNP, 460 X-STRs, 459, 460, 475

Y Y chromosome, 618, 1045 ancestry, 1118 applications of, 448–451 biogeography ancestry and migrations human, 450–451 forensic analysis, 448 forensic use, 203 genealogical studies, 449–450 markers, 583, 584 origin of, 438 paternity testing and historical investigations, 448 polymorphisms of, 441–448 sex determination, 205–207 sexual assault, 203–205

Index STRs, 400 structure, 186, 438–440 variations and polymorphisms, 187 Y-single nucleotide polymorphisms, 193, 447–448 Y STRs, 441–446 Y chromosome DNA, 1171 testing, 97, 131–134 Y chromosome haplotype reference database (YHRD), 446 Y chromosome-related polymorphism analysis, 1045 Yfiler Plus kit ®, 195 Y short tandem repeats (Y-STRs), 188–189, 571 analysis, 523 disaster victim identification, 207 factual mismatches/exclusions, 199–200 forensic familial searching, 210–211 haplogroup prediction, 211–212 haplotype databases, 209–210 historical investigations, 208–209 inheritance pattern of, 191 interpretation of the results, 197–198 kits, 193–197 match, 200–202 mini, 191 mixture interpretations, 202–203 paternity testing, 207 rapidly mutating, 189–191 Y-STR Haplotype Reference Database, 134