Forensic Genetic Approaches for Identification of Human Skeletal Remains: Challenges, Best Practices, and Emerging Technologies 9780128157664, 0128157666

Table of contents :
Cover
Title Page
Copyright
Dedication
In Memory of: Paula C. Brumit, DDS, D-ABFO (1956–2021)
Contributors
Author’s biographies
Editor biography
Preface
Acknowledgments
Missing persons and unidentified human remains: The world’s silent mass disaster
Introduction
Forensic genetic investigations of human skeletal remains: Challenges and considerations
Why so many unidentified human remains?
Natural disasters
Epidemics and pandemics—Mass graves and communal burials
Oppressive regimes, forced disappearances, genocide, and war conflicts
Cemetery mismanagement and funeral malpractice
Overburial—Eastern Cemetery (Louisville, Kentucky)
Unauthorized services (selling of body parts)—The New York Bone Snatchers
Improper disposal of human remains—The Tri-State Crematory Scandal (Georgia)
Organizations and laboratories with specialization in missing persons and unidentified human remains investigations
International Commission on Missing Persons (ICMP)
Armed Forces DNA Identification Laboratory (AFDIL)
Forensic Anthropology Foundation of Guatemala (FAFG)
Argentine Forensic Anthropology Team—Equipo Argentino de Antropologia Forense (EAAF)
Gene Codes Forensics and the Mass Fatality Identification System (M-FISys)
University of North Texas Center for Human Identification (UNTCHI)
National Missing and Unidentified Persons System (NamUs)
The Doe Network—International Center for Unidentified and Missing Persons
Identifinders International LLC
Institute for Human Identification—Lincoln Memorial University (College of Dental Medicine)
Summary
References
Challenges in forensic genetic investigations of decomposed or skeletonized human remains: Environmental exposure, DNA degr ...
Introduction
DNA structure and susceptibility to damage
Inherent instability of DNA and endogenous contributing factors to damage
Environmental (exogenous) influences on DNA degradation
Molecular biology of DNA damage: Types of lesions and causes
Abasic (AP) sites: Depurination and depyrimidination
Strand breakage: Single-strand breaks (SSBs) and double-strand breaks (DSBs)
Crosslinking of DNA bases: Interstrand and intrastrand crosslinks
DNA base modifications: Deamination, oxidation, and alkylation
Deamination
Oxidation
Alkylation
Inhibitors
Endogenous inhibitors
Exogenous (environmental) inhibitors
Exogenous (laboratory-introduced) inhibitors
Low copy number (LCN) DNA
Summary
Acknowledgments and graphic design credits
References
Facilities design and workflow considerations for processing unidentified human skeletal remains
Introduction
Laboratory infrastructure
Laboratory layout
General considerations
Separation of pre-PCR and post-PCR areas
Ventilation
Airlocks and pressure
Pre-PCR areas
DNA-free rooms
Case administration and sample storage room
Reagent preparation room (QA/QC area)
Sampling room
Molecular biology pre-PCR laboratories
High quality/high quantity DNA (HQHQ) pre-PCR room
Low quality/low quantity DNA (LQLQ) pre-PCR rooms
Equipment and reagents
Dedicated personal protective equipment (PPE)
Lab coats/coveralls and sleeves
Gloves
Masks
Gowning protocol
Instruments and equipment
Equipment necessary in a post-PCR lab
Equipment necessary in all pre-PCR rooms
Laminar flow hood
Crosslinkers
Fume hood
Area for skeletal remains sample preparation
Area for degraded DNA extraction and amplification set-up
Commercial reagents and consumables
Anticontamination measures and best practices
Sodium hypochlorite
Irradiation of consumables and buffers with UVC
Other decontamination options
Staff training and laboratory management
Acknowledgments
Disclaimer
References
Further reading
Location, recovery, and excavation of human remains for forensic testing
Introduction
The scene
Discovery
Excavation procedures
Recovery and transport
Packaging
Surface remains recovery
Conclusions
References
Skeletal microstructure, bone diagenesis, optimal sample selection, and pre-processing preparation techniques for DNA testi ...
Introduction
Bone composition, microstructure, and diagenesis
Taphonomy and diagenesis: Microscopic destruction of bone
Non-uniform, heterogeneous diagenesis of bone microstructure
Correlation between bone diagenesis and DNA preservation
Microanatomy of a human tooth
Selection of optimal skeletal elements for DNA testing
Weight-bearing long bones
Teeth
Petrous region of the temporal bone (skull)
Foot bones
Additional considerations in selecting skeletal samples for DNA testing
Correlation between skeletal weathering and DNA quality and quantity
Pre-processing of skeletal remains for DNA extraction: Surface-sanding, sectioning, decontamination, and pulverization
Contamination during recovery or exhumation: DNA transfer from direct contact and handling of human remains
Contamination prevention in the laboratory
Quality Assurance (QA) standards and Forensic DNA Grade (ISO 18385) products for identification of human skeletal remains
Sterilization and decontamination of saw blades, tools, and accessories
Mechanical and physical pre-processing: Surface-sanding and sectioning of long bones
Chemical washing of external surfaces of bone cuttings or teeth
Pulverization of sectioned bones and teeth into powder
Summary and conclusions
Acknowledgment
References
Additional reading—Contamination prevention
DNA extraction methods for human skeletal remains
Introduction
Forensic DNA casework—Standard operating procedure (SOP)
Importance of demineralization
DNA extraction in designated low copy number (LCN) area
Preparation for DNA extraction—Sterilization of tubes, equipment, and work surfaces
Importance of polypropylene tubes and barrier (filter) pipette tips
Validated organic DNA extraction method for forensic casework involving skeletonized human remains
Organic DNA extraction from skeletonized human remains (bones or teeth)
Field-deployable “Rapid DNA” technology for disaster victim identification (DVI) and mass fatality incidents
Summary
References
Further reading
Quantitative and qualitative assessment of DNA recovered from human skeletal remains
Overview
DNA quantification methods
Real-time quantitative PCR (qPCR)
SYBR® Green I assays
TaqMan® probe assays
Scorpions® primer assays
Plexor® assays
Fluorescence detection and measurement
Quality indicators
Commercial DNA quantification kits
Thermo Fisher Scientific qPCR kits
Promega Corporation qPCR kits
QIAGEN qPCR kits
InnoGenomics Technologies qPCR kits
Comparison of the latest generation of DNA quantification kits
Custom DNA quantification assays
Mitochondrial DNA (mtDNA) quantification assays
Sex determination DNA quantification assays
A qualitative PCR assay
DNA quantification instruments
Thermo Fisher Scientific real-time PCR systems
Applied Biosystems™ 7500 Real-Time PCR System
Applied Biosystems™ QuantStudio™ 5 Real-Time PCR System
Other real-time PCR systems
Rotor-Gene Q System
Interpretation of results
Assessment of standard curve
Standard curve
Slope
The R 2 value
Y-intercept
Assessment of controls
No template control (NTC)
Internal positive control (IPC)
Assessment of DNA quantity
Assessment of DNA quality
Procedural modifications
Reduced reagent volumes
Removal of PCR inhibitors
Summary
References
Autosomal short tandem repeat (STR) profiling of human skeletal remains
Overview
Short tandem repeats (STRs)
Modern STR multiplexes
Sex-determining markers
STR markers
Discrimination power
Quality indicators
Commercial STR kits
Autosomal STR kits
Thermo Fisher Scientific STR kits
Promega Corporation STR kits
QIAGEN STR kits
Comparison of the latest generation of STR multiplex kits
Direct amplification for genotyping reference samples
Mini short tandem repeat (mini-STR) kits
Thermo Fisher Scientific mini-STR kit
Custom mini-STR kits
Y-chromosome short tandem repeat (Y-STR) kits
X-chromosome short tandem repeat (X-STR) kits
Dual amplification strategies
PCR thermal cycling instruments
Thermo Fisher Scientific PCR thermal cyclers
Applied Biosystems™ GeneAmp® PCR System 9700
Applied Biosystems™ Veriti® 96-Well Thermal Cycler
Applied Biosystems™ ProFlex™ PCR System
Other PCR thermal cyclers
Optimization of STR profiling of skeletal samples
Manufacturer guidelines
DNA input amount
Reaction volume
Cycle number
PCR reagent modifications
Post-PCR purification
High-throughput and Rapid STR profiling for large-scale identification efforts
Automation
Rapid amplification
Rapid DNA systems
Direct PCR
Summary
References
Y-chromosome analysis for unidentified human remains (UHR) investigations
Introduction
Basic structure and inheritance patterns of the Y chromosome
Y-chromosome polymorphisms
Y-chromosome short tandem repeats (Y-STRs)
Y-chromosome single nucleotide polymorphisms (Y-SNPs)
Y-chromosome databases
Y-STR haplotype frequencies and matching: Identical by Descent (IBD) vs. Identical by State (IBS); Rapidly Mutating Y-STRs ...
Predicting biogeographic ancestry of unidentified human remains (UHR)
Y haplogroup prediction tools
Genetic genealogy applications for Y-chromosome DNA
Summary
References
Further reading
Mitochondrial DNA and its use in the forensic analysis of skeletal material
Introduction
Cellular location of mtDNA
Endosymbiont theory and the origins of mtDNA
Structure of mtDNA
Inheritance patterns of mtDNA
Heteroplasmy in the mtDNA genome
The use of mtDNA in forensic casework
Sample processing in the laboratory
mtDNA amplification strategy
Data interpretation and comparison
mtDNA nomenclature
Statistical calculations
Emerging mtDNA methods
Conclusion
Acknowledgments
Disclaimer
References
X-chromosome short tandem repeats (X-STRs): Applications for human remains identification
Introduction
The X chromosome: Discovery and pattern of inheritance
Forensic applications of X-chromosome analysis
Forensically relevant X-chromosome markers, commercial amplification kits, and other multiplex systems available for X-STR ...
Investigator Argus X-12 QS (Qiagen Corporation)
Mentype Argus X-8 Kit (Biotype Forensics)
AGCU X19 STR Kit (Jiangsu, China)
Goldeneye DNA ID System 17X Kit (Beijing, China)
X-STR Decaplex: System A and System B (GHEP-ISFG)
12X-Plex System ( Turrina et al., 2007)
11X-Plex System ( Rodrigues et al., 2008)
Two X-chromosomal multiplexes—15 X-STR loci ( Diegoli et al., 2013)
17 X-Plex System ( Prieto-Fernández et al., 2015)
ForenSeq™ DNA Signature Prep Kit (Verogen)
X-STR population data
Conclusions
References
Single nucleotide polymorphisms (SNPs): Ancestry-, phenotype-, and identity-informative SNPs
Introduction
History of SNP use in forensic genetics
SNP typing methodologies
Allele-specific oligonucleotide hybridization
Taqman 5 ′ nuclease assays
Pyrosequencing
Minisequencing (SNaPshot assays)
Massively parallel sequencing
SNP databases for forensic genetic applications
SNPs in forensic DNA mixtures
SNP classes for forensic investigative and intelligence purposes
Identity-informative SNPs
Commercially available kits for iiSNP genotyping
Lineage-informative SNPs
Ancestry-informative SNPs (aiSNPs) for prediction of biogeographic ancestry
Phenotype-informative SNPs for prediction of externally visible characteristics
Eye (iris) color
Hair color
Skin color
Forensic DNA phenotyping as a private forensic service
Microhaplotypes
Conclusion
References
Diallelic Markers: INDELs and INNULs
Introduction
Existing INDEL panels in the literature
Existing INNUL panels in the literature
Limitations of diallelic markers
References
Genotyping and sequencing of DNA recovered from human skeletal remains using capillary electrophoresis (CE)
Overview
Principles of capillary electrophoresis (CE)
Sample injection
Sample separation
Sample detection
Spatial and spectral calibration
CE instrumentation
Thermo Fisher Scientific genetic analyzers
Applied Biosystems™ 31XX Series Genetic Analyzer
Applied Biosystems™ 3500 Series Genetic Analyzer
Promega Corporation genetic analyzers
Spectrum CE System
Spectrum Compact CE System
Short tandem repeat (STR) genotyping
Polymerase chain reaction (PCR)
CE requirements
Polymer type and capillary length
Run modules
Internal size standard
Allelic ladder
Quality thresholds
Analytical thresholds
Stochastic thresholds
Stutter thresholds
Data analysis
Applied Biosystems™ data collection software
Applied Biosystems™ genotyping software
Probabilistic genotyping software
Genotyping considerations for human skeletal remains
Common artifacts
Optimizing electrophoresis parameters
Mitochondrial DNA (mtDNA) sequencing
DNA template preparation
Pcr
Quantification of PCR products
Purification of PCR products
Cycle sequencing
Process
Sequencing chemistries
Thermal cycling conditions
Purification of sequencing products
CE requirements
Data analysis
Applied Biosystems™ data collection software
Applied Biosystems™ sequence analysis software
Other sequence analysis software
Common artifacts observed for skeletal samples
Common artifacts
Alternative sequencing technologies
Limitations of CE
Summary
References
Rapid DNA identification of human skeletal remains
Introduction and historical context of skeletal samples in DNA-based forensic human identification
Overview of the ANDE Rapid DNA Identification System
Selection of bone samples for Rapid DNA Analysis
Bone preprocessing protocols for Rapid DNA Identification
Bone cleaning
Bone fragmentation
Processing intact bone
Processing burned bone
Processing old and degraded bone
Processing demineralized bone and tooth samples in the ANDE Rapid DNA Identification System
Representative results from Rapid DNA processing of degraded bones and teeth
Above-ground exposed and refrigerated remains
Submerged remains
Cremated remains (cremains)
Formalin/formaldehyde-treated remains
The oldest bones processed by Rapid DNA
Developmental validation of Rapid DNA Identification for bones and teeth
Reproducibility
Accuracy and concordance
Peak height and Peak Height Ratios (PHRs)
Resolution and precision
Conclusions
References
Emerging technologies for DNA analysis of challenged samples
Introduction
Massively parallel sequencing
MPS library preparation multiplexes
STR multiplexes for MPS
SNP multiplexes
Indel and microhaplotype multiplexes
Mitochondrial genome multiplexes
MPS platforms
Template preparation methods for MPS
Emulsion PCR
Rolling circle amplification
DNA colony generation using bridge amplification
Single molecule templates
Sequencing approaches
Pyrosequencing
Sequencing-by-synthesis
Ion semiconductor sequencing
Multiplex and platform compatibility
Direct PCR
Rapid DNA testing
Conclusion
References
Further reading
Best practices in the development and effective use of a forensic DNA database for identification of missing persons and un ...
Introduction
Upstream context—Sample collection
Upstream context—Antemortem sample collection
Upstream context—Postmortem sample collection
Downstream context—Match confirmation
Context—Conclusion
Relational databases
Data mapping
Database performance
This is not an online search
Expunging records
Specifying capacity
Conclusion
Software and database functionality for direct identification and kinship analysis: The Mass Fatality Identification System ...
Introduction
Representing and directly comparing profiles
Direct matching
Non-STR direct match assays
Quality control (QC) issues to consider in direct match data
Kinship matching
Different formats of kinship searching
Pedigree search
Modifying the family structure in a pedigree search
Additional QC controls in a pedigree search
Kinship hypothesis
Reports
Advanced topics
Complexity of pedigrees
Pedigrees with multiple hypotheses
Secure coordination with collaborators
Anthropological information
Conclusion
Bioinformatic tools for interrogating DNA recovered from human skeletal remains
Introduction
Relevant loci
Short tandem repeats
Autosomal STRs
Y-chromosome STRs
X-chromosome STRs
Single nucleotide polymorphisms
Identity-informative SNPs
Ancestry-informative SNPs
Phenotype-informative SNPs
Mitochondrial DNA
Relevant data file formats
Common file formats
Sequence-specific file formats
Extracting genotype information
Capillary electrophoresis platforms
STR allele calling
Sequencing with CE
CE output
Massively parallel sequencing platforms
STRait Razor
STRinNGS
lobSTR
Variant calling
Bioinformatic tools
Population genetic parameters
Individualization and source attribution
Ancestry inference
Global ancestry estimation
Maternal lineage estimation
Paternal lineage estimation
Phenotype inference
Emerging tools
Summary
References
The emerging discipline of forensic genetic genealogy
Introduction
Genetic genealogy and Y-STR testing
Application of genetic genealogy Y-STR data to forensic casework
Massively parallel sequencing, Y-SNPs, and Y-STRs
Genetic genealogy autosomal SNP testing
Application of genetic genealogy autosomal testing to forensic casework
Feasibility of using GEDmatch for data obtained from degraded DNA
Genetic genealogy autosomal SNP analysis tools
A simple example
Existing challenges
The future
References
Forensic anthropology in a DNA world: How anthropological methods complement DNA-based identification of human remains
Introduction
History of forensic anthropology
Medicolegal significance
Creating a biological profile for unidentified human remains
Sex determination
Ancestry estimation
Age-at-death estimation
Stature estimation
Personal identification using comparative radiography
Comparative radiographic methods
Stable isotope analysis as an investigative tool
Example case study
Conclusion
References
Further reading
Generation of a personal chemical profile from skeletonized human remains
Introduction
Protocol theory and development
Mass spectrometry
Preparation of materials
Solvent selection
Ambient ionization
Protocol
Preparation of skeletal materials
Preparation of tooth materials
Solvent treatment
GC/MS instrument loading
Analysis
Application
Summary
Disclaimer
References
Forensic odontology: Historical perspectives and current applications for identification of human remains
Introduction
History of forensic dental identification of human remains
Scientific rationale for human dentition as a means of identification
Common dental approaches for identification of human remains
Comparison of antemortem (AM) and postmortem (PM) radiographs
Dental implant batch numbers and unique serial numbers on implanted medical devices
Dental age assessment (DAA): Forensic estimation of a decedent’s chronological age
Age assessment techniques for infants, children, and adolescents
Post-adolescence (adult) age assessment methodologies
Biochemical age assessment techniques
Additional distinctive morphological features-of-interest in dental remains
cusp of Carabelli
Shovel-shaped incisors
Hutchinson’s incisors and Mulberry molars
Dental fluorosis
Turner’s hypoplasia
Isolated microdontia (peg lateral incisor)
Overview of dental restorative materials
Gold foil restorations
Gold bridgework
Vulcanite denture teeth
Identification networks for the missing and unidentified
National Crime Information Center (NCIC)
National Missing and Unidentified Persons System (NamUs)
OdontoSearch
References
Further reading
Forensic facial reconstruction of skeletonized and highly decomposed human remains
Introduction
Standard reconstruction methods
Basic working techniques
Building a simple cranial stand/armature
Two-dimensional (2D) reconstruction
Cadaver image reconstruction
Conclusion
Appendix
References
Index
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