Human Mitochondrial Control Region and mtGenome: Design and Forensic Validation of NGS Multiplexes, Sequencing and Analytical Software

Abstract

Forensic mitochondrial DNA (mtDNA) analysis conducted using next-generation sequencing (NGS), also known as massively parallel sequencing (MPS), as compared to Sanger-type sequencing brings modern advantages, such as deep coverage per base (herein referred to as read depth per base pair (bp)), simultaneous sequencing of multiple samples (libraries) and increased operational efficiencies. This report describes the design and developmental validation, according to forensic quality assurance standards, of end-to-end workflows for two multiplexes, comprised of ForenSeq mtDNA control region and mtDNA whole-genome kits the MiSeq FGxTM instrument and ForenSeq universal analysis software (UAS) 2.0/2.1. Polymerase chain reaction (PCR) enrichment and a tiled amplicon approach target small, overlapping amplicons (60–150 bp and 60–209 bp for the control region and mtGenome, respectively). The system provides convenient access to data files that can be used outside of the UAS if desired. Studies assessed a range of environmental and situational variables, including but not limited to buccal samples, rootless hairs, dental and skeletal remains, concordance of control region typing between the two multiplexes and as compared to orthogonal data, assorted sensitivity studies, two-person DNA mixtures and PCR-based performance testing. Limitations of the system and implementation considerations are discussed. Data indicated that the two mtDNA multiplexes, MiSeq FGx and ForenSeq software, meet or exceed forensic DNA quality assurance (QA) guidelines with robust, reproducible performance on samples of various quantities and qualities.

The Value of Whole-Genome Sequencing for Mitochondrial DNA Population Studies: Strategies and Criteria for Extracting High-Quality Mitogenome Haplotypes

“…However, RtN! and other software that implement similar approaches (e.g., the Converge Forensic Analysis Software from Thermo Fisher Scientific [28] and Verogen's Universal Analysis Software [27]) were developed for small amplicon enrichment data rather than WGS data. The assessment of phasing is more straightforward for enrichment methods that utilize small amplicons since the whole amplicon is sequenced in each read.…”

“…Ring et al [23] utilized alternative mapping strategies, including increased stringency, competitive mapping to the human reference genome (i.e., nuclear and mitochondrial genomes), and stringent alignment to the sample consensus sequence to prevent the mapping of NUMT reads based on the dissimilarity between the NUMT and mtDNA sequences. Other approaches to minimize NUMT reads in mtDNA mappings include filtering the sequences found in a catalog of known NUMTs, which is applied by the Verogen Universal Analysis Software [27] and the Remove the NUMTs! (RtN!)…”

The FORCE Panel: An All-in-One SNP Marker Set for Confirming Investigative Genetic Genealogy Leads and for General Forensic Applications

“…Therefore, mtDNA best serves degraded DNA cases that can be bolstered by other types of evidence, DNA, or otherwise. Recent methods for mitogenome massively parallel sequencing (MPS) have been applied to degraded DNA samples and validated for forensic use, including mini-amplicon PCR [ 7 , 8 ], primer extension capture (PEC) [ 9 ], and hybridization capture [ 10 ]. Such methods are very sensitive and capable of providing authentic mtDNA data from the most challenging of forensic specimens.…”

Mitochondrial DNA in forensic use

“…MPS technology offers cost-effective sequencing of the whole mitochondrial genome (mitogenome) with the provision of increased discrimination and phylogenetic resolution along with an assessment of data quality. Several groups have validated the different methodologies [9][10][11]. Missing person identification frequently makes use of mtDNA analysis, often because of the degraded nature of the remains, with significantly increased success rates when using MPS compared with Sanger sequencing [12].…”