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

Daniela Cuenca,Michelle Halsing,Jessica Battaglia,Sandra Sheehan

doi:10.3390/genes11111303

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.

Highlights

Mitochondrial DNA has several distinctions from autosomal DNA that have proven to be beneficial in forensic analysis
The success rate (full single source hypervariable regions I and II (HVI/HVII) for Sanger and control region for massively parallel sequencing (MPS) result) for these challenging samples increased from 32.3% with Sanger sequencing to 74.2% with the Precision ID assay
Another limitation of hair is the total amount of mtDNA that can be extracted from the shaft

Summary

Introduction

Mitochondrial (mt) DNA has several distinctions from autosomal DNA that have proven to be beneficial in forensic analysis. Being the powerhouse of the cell, there are hundreds to thousands of mitochondria in each cell [1,2]. This makes mitochondrial DNA analysis more sensitive than the analysis of nuclear DNA (which only has two copies per diploid cell) and increases the probability of successful PCR amplification [3]. It can be used to establish lineage with close or distant kinship. The forensic community has historically limited mtDNA analysis to the relatively small (~700 bases) hypervariable regions I and II (HVI and HVII)

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Conclusion