Massively Parallel Sequencing as an Operational Forensic Tool: Current and Future Perspectives—Improving Results for Current Applications and Paving the Way for Expanded Capabilities

Abstract

Advances in genomic technologies underpin the practical and powerful application of massively parallel sequencing (MPS) to forensic science. MPS provides the highest resolution of targeted, forensic PCR amplicons, harnessing the full power of genomic technology. Illumina Sequencing By Synthesis (SBS) provides the core sequencing technology of the MiSeq FGxTM Forensic Genomics System and ForenSeq® chemistries, provided exclusively by Verogen (www.verogen.com). MiSeq FGx-generated short tandem repeat (STR) allele calls are fully compatible with current database formats, providing a seamless link between capillary electrophoresis (CE)-based and MPS data, such that laboratories may transit from legacy to contemporary data with minimum disruption to existing training and infrastructure. Unlike CE-based STR typing systems, forensic MPS approaches are not limited to dye channels and size restrictions, as genetic markers may occupy the same “real estate” by length such that 100s of polymorphisms of different categories can be analyzed simultaneously. The ForenSeq DNA Signature Prep Kit is the first commercially developed and validated assay to combine 27 autosomal STRs, 24 Y-STRs, and 7 X-STRs with a dense SNP set in a single test, which can reduce iterative testing, redundant quality assurance and quality control 498procedures, and training programs for increased efficiencies. Targeting the smallest forensic amplicons possible (<150 bases), identity SNP amplicons aid in data recovery from partially degraded DNA samples, phenotypic SNPs enable analysis of visible traits such as hair and eye color and biogeographic ancestry is estimated using a third class of SNPs. These data may arm investigators with actionable genetic intelligence as opposed to CE-based genetic clues which are effectively limited to whether or not DNA originated from a male or female. Because alleles are resolved at the nucleotide level using MPS, more conclusive results can be reached, especially in complex mixtures. Although not as powerful (in statistical terms) as nuclear DNA, mitochondrial DNA (mtDNA) is important for specific forensic scenarios and has been hampered by the limitations of Sanger sequencing on CE platforms. MPS-based mtDNA analysis is quicker, simpler and, for some methods, less expensive than Sanger. MPS also extends mtDNA variant analysis capabilities beyond the control region to the entire mtGenome sequence in a practical way. ForenSeq mtDNA Control Region and ForenSeq mtDNA Whole Genome kits spare laboratories of the need to QC in house (“home brew”) reagent formulations, while ForenSeq software flattens analysis and interpretation time such that onsite bioinformatics expertise is not required. This chapter clarifies some of the misperceptions associated with MPS and highlights milestones that have been reached in the global forensic community. MPS allows exploration of a variety of forensic analyses that have not been in the forensic realm historically. Laboratories that are expert in MPS are future proofing their operations for the “omics” era that encompass the transcriptome, nuclear genome, epigenome, and microbiome. Investigative intelligence that incorporates genetic data, including but not limited to the use of GEDmatch for long-range familial searching, is on the rise and expected to become part of the mandate and day-to-day affairs of crime lab systems. Strategic planning in forensic infrastructure should anticipate advanced workflows that answer more forensic questions than ever before, extending capabilities of the contemporary forensic laboratory to truly exploit the power of “omics.”