Next Generation Sequencing in Forensic Science

A Primer

Kelly M. Elkins, Cynthia B. Zeller

• Publisher: CRC Press
• Year: 2021
• ISBN:
  9781000449259
• Edition: 1
• Topic: Scienze biologiche
• Subtopic: Genetica e genomica

Next Generation Sequencing in Forensic Science: A Primer addresses next generation sequencing (NGS) specific to its application to forensic science. The first part of the book offers a history of human identity approaches, including VNTR, RFLP, STR, and SNP DNA typing. It discusses the history of sequencing for human DNA typing, including Sanger sequencing, SNaPshot, pyrosequencing, and principles of next generation sequencing. The chapters present an overview of the forensically focused AmpliSeq, ForenSeq, Precision ID, PowerSeq, and QIAseq panels for human DNA typing using autosomal, Y and X chromosome STRs and SNPs using the MiSeq FGx and Ion Torrent System. The authors outline the steps included in DNA extraction and DNA quantitation that are performed prior to preparing libraries with the NGS kits.

The second half of the book details the implementation of ForenSeq and Precision ID to amplify and tag targets to create the library, enrich targets to attach indexes and adaptors, perform library purification and normalization, pool the libraries, and load samples to the cartridge to perform the sequencing on the instrument. Coverage addresses the operation of the MiSeq FGx and Ion Chef, including creating a sample list, executing wash steps, performing NGS, understanding the run feedback files from the instrument, and troubleshooting. ForenSeq and Precision ID panel data analysis are explained, including how to analyze and interpret NGS data and output graphs and charts. The book concludes with mitochondrial DNA (mtDNA) sequencing and SNPs analysis, including the issue of heteroplasmy. The final chapters review forensic applications of microbial DNA, NGS in body fluid analysis, and challenges and considerations for future applications.

FEATURES

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• Focuses on human identification using traditional and NGS DNA typing methods targeting short tandem repeats (STRs)

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• Applies the technology and its application to law enforcement investigations and identity and ancestry single nucleotide polymorphisms (SNPs) for investigational leads, mass disaster, and ancestry cases

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• Presents the underlying principles of NGS in a clear, easy-to-understand format for practitioners and students studying DNA in forensic programs

This is the first book to prepare practitioners to utilize and implement this new technology in their lab for casework, highlighting early applications of how NGS results have been used in court. The book can be utilized for upper-level undergraduate and graduate students taking courses focused on NGS concepts. Readers are expected to have a basic understanding of molecular and cellular biology and DNA typing.

1 History of DNA-Based Human Identification in Forensic Science

DOI: 10.4324/9781003196464-1

1.1 Introduction

Forensic biology is the application of serology and DNA typing methods for human, wildlife, pet, and plant identification using bone, teeth, hair, body fluids, and plant materials to help solve a crime. All of these materials, indeed all cellular material with the exception of red blood cells, contain deoxyribonucleic acid (DNA), the chemical whose double-stranded helical structure was elucidated in 1953 (Watson and Crick 1953, Franklin and Gosling 1953, Wilkins et al. 1953). Advances in forensic biology have resulted in tremendous capabilities for human identification and have reduced the reliance on and need for eyewitness accounts of crimes. A brief history of some of the notable advances in forensic biology is listed in Table 1.1.

1.2 Application of DNA Sequencing to Human DNA

The human genome sequence was reported in 2001 (International Human Genome Consortium 2001, Venter et al. 2001) and is comprised of DNA housed in the nucleus. This huge advance built on principles and technology that led to the sequencing of the bacteriophage phi X174 in 1977 (Sanger et al. 1977) and mitochondrial organelle chromosome in 1981 (Anderson et al. 1981). Whereas the human mitochondrial chromosome is circular in structure and is comprised of 16,569 base pairs (bp), the nuclear, or autosomal, genome consists of 3.2 billion bp packaged in twenty-two pairs of linear chromosomes supercoiled on histone proteins (Anderson et al. 1981, International Human Genome Consortium 2001, Venter et al. 2001). An additional set of chromosomes, X and Y, are sex chromosomes that make the total number of chromosomes in humans forty-six. Females have two X chromosomes, while males are characterized by having an X and a Y chromosome. Most of this book is focused on autosomal DNA typing and chapter 7 is focused on mitochondrial DNA typing. Over several years, the sequences of bacteria that have been used as foodborne pathogens and bioterror agents and others found in and on the human body have also been sequenced and used to solve questions in forensic cases; this is the focus of Chapter 8.

Table 1.1 Brief History of Some Notable Advances in Forensic Biology

Year	Advance
1953	Rosalind Franklin records X-ray autoradiographs of crystallized DNA fibers and deduces basic features including that the structure was helical with the phosphates on the outside and its basic dimensions of DNA strands and publishes with Raymond Gosling in Nature
1953	James Watson and Francis Crick solve three-dimensional structure of DNA from Franklin’s X-ray crystallography data and publish it in Nature
1977	Frederick Sanger invents a method for DNA sequencing
1981	Anderson and team sequence human mitochondrial chromosome
1983	Kary Mullis invents polymerase chain reaction method as reported in Science
1985	Alec Jeffreys develops the first multi-locus DNA typing method and publishes it in Nature
1986	Human leukocyte antigen HLA-DQα multi-allelic locus was published for forensic DNA typing and used in the first US criminal case
1986	First application of Jeffrey’s method to rape and murder cases in Leicestershire, England
1990	FBI establishes the Combined DNA Index System (CODIS) to allow for national DNA comparisons
1994	Promega introduces first commercial three loci STR typing kit targeting CSF1PO, TPOX, and TH01, named “CTT” using the first letter of each locus
1996	In Tennessee v. Ware, mitochondrial DNA typing was admitted for the first time in a US court.
1997	Applied Biosystems introduces the three-dye AmpFlSTR^® Profiler Plus^® PCR Amplification Kit for typing nine STRs and Amelogenin
1998	Applied Biosystems introduces the three-dye AmpFlSTR^® COfiler^® PCR Amplification Kit for typing six STRs and Amelogenin
2000	Promega introduces PowerPlex™ 16 System, the first commercial STR typing kit that targeted all thirteen CODIS loci as well as the ENFSI loci, Interpol loci, and GITAD loci in one PCR reaction
2001	Applied Biosystems introduces five-dye AmpFLSTR™ Identifiler™ PCR Amplification Kit targeting sixteen STR loci
2014	ThermoFisher introduces GlobalFiler™, the first twenty-four-plex, six-dye STR kit
2014	Promega introduces the PowerPlex^® Fusion 24-locus STR DNA typing system
2016	Promega introduces PowerSeq™ 46GY kit
2017	Qiagen introduces the Investigator 24plex GO! and DS kits that target the CODIS and ESS loci
2018	Applied Biosystems introduces Precision ID NGS library prep kit
2017	Verogen introduces NGS ForenSeq™ library prep kit for forensic applications
2019	Precision ID and ForenSeq™ data approved for inclusion in CODIS
2019	First application of NGS in a Dutch criminal case.

1.3 History of DNA Typing

Analysis of the DNA sequence and repeat polymorphisms is referred to as DNA typing, or DNA profiling. DNA typing is used to determine the origin of forensic evidence and is applied to criminal, paternity, and missing persons cases. When forensic DNA typing was pioneered and adopted in the 1980s, relatively small segments of the genome were probed and used to differentiate the origin of samples (Gill et al. 1985). Even without sequencing the entire human genome, these DNA typing methods were shown to provide high statistical confidence that a stain or sample can be assigned as originating from a specific individual (Butler 2005). However, as sequencing capabilities and tools have improved, they are now being applied to forensic cases. DNA sequencing can overcome limitations of the now standard DNA typing methods. For example, monozygotic twins and body fluids can now be differentiated genetically. In addition, new, so-called next generation sequencing (NGS) methods can be used to determine the biogeographical ancestry and phenotype characteristics including eye color, skin tone, and hair color from fragments of human remains and trace body fluid or fingerprint sources.

It has been estimated that 30% of the human genome is comprised of repeated segments. The origins of forensic DNA typing began with restriction fragment length polymorphisms (RFLPs). The RFLP targets were sites with a variable number of tandem repeats (VNTRs). These sites are also known as minisatellite sequences and are comprised of long repeats of ten to one hundred nucleotide bases consisting of thousands of bases in total (Butler 2005). The first RFLP DNA test was developed by Sir Alec Jeffreys in 1984 and published in the journal Nature in 1985 (Gill et al. 1985). The test involved analysis of patterns from multiple RFLP loci. In RFLP, restriction enzymes were used to cut the DNA repeat reg...