Mitochondrial Sequencing of Missing Persons DNA Casework by Implementing Thermo Fisher’s Precision ID mtDNA Whole Genome Assay

Abstract

The advent of massively parallel sequencing (MPS) in the past decade has opened the doors to mitochondrial whole-genome sequencing. Mitochondrial (mt) DNA is used in forensics due to its high copy number per cell and maternal mode of inheritance. Consequently, we have implemented the Thermo Fisher Precision ID mtDNA Whole Genome panel coupled with the Ion Chef™ and Ion S5™ for MPS analysis in the California Department of Justice, Missing Persons DNA Program. Thirty-one mostly challenging samples (degraded, inhibited, low template, or mixed) were evaluated for this study. The majority of these samples generated single source full or partial genome sequences with MPS, providing information in cases where previously there was none. The quantitative and sensitive nature of MPS analysis was beneficial, but also led to detection of low-level contaminants. In addition, we found Precision ID to be more susceptible to inhibition than our legacy Sanger assay. Overall, the success rate (full single source hypervariable regions I and II (HVI/HVII) for Sanger and control region for MPS result) for these challenging samples increased from 32.3% with Sanger sequencing to 74.2% with the Precision ID assay. Considering the increase in success rate, the simple workflow and the higher discriminating potential of whole genome data, the Precision ID platform is a significant improvement for the CA Department of Justice Missing Persons DNA Program.

Interpreting NUMTs in forensic genetics: Seeing the forest for the trees

“…Yet, the main reason for picking up NUMTs in MPS-based studies is that most applications target hundreds of mini amplicons/ mtDNA fragments using primers homologous to both mtDNA and NUMTs. Mini amplicon workflows are relatively simple to perform (e.g., [50]) and automate (e. g., [49]) in the laboratory; they are also increasing the success rate of mtDNA analysis in even heavily degraded mtDNA that is typically present in old and/or environmentally challenged specimens [39,44,[51][52][53]. However, they are also more likely to enrich NUMT fragments than long-range mtDNA workflows do [42,48,49,54].…”

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

“…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].…”