Massively Parallel Mitogenome Sequencing: Building a Strong Foundation for the Interpretation of MPS MtDNA

Project Abstract: Massively Parallel Mitogenome Sequencing: Building a Strong Foundation for the Interpretation of MPS MtDNA     Mitochondrial DNA (mtDNA) is a lineage marker that holds a significant role in forensic casework. Even today, with the implementation of high-density single nucleotide polymorphism (SNP) detection and genetic genealogy, there are limitations to the use of autosomal DNA markers that can be overcome with mtDNA. MtDNA is of high copy number, being beneficial for low quality/quantity DNA analysis. Additionally, as a haploid genome, mtDNA can be sequenced in very small fragments, enabling DNA profiling from degraded samples lacking intact template for short tandem repeat (STR) typing. Another benefit of mtDNA is that it can be traced unilaterally over many generational events. Therefore, mtDNA can extend the reach of genetic genealogy beyond that of autosomal SNPs to include all maternal relatives.  Finally, massively parallel sequencing (MPS) technologies offer versatile and efficient means to mtDNA profiling.     Forensic casework has historically focused on the non-coding mitochondrial control region (CR) due to a combination of technological and ethical reasons.  Today, mitochondrial genome (mitogenome) MPS is feasible and maximizes the genetic information that can be gleaned from the mtDNA locus. Furthermore, recent studies have shown that pathogenic variants can be filtered from mitogenome haplotypes, thereby allaying the privacy concerns in sequencing the coding region portion. The primary hurdles to the incorporation of mitogenomes in U.S. forensics are associated with the statistics and the haplotype matching / exclusion criteria. First, there is an insufficiency of population frequency data that are needed to evaluate the weight of the mtDNA evidence. Second, exclusion criteria utilized for familial mtDNA comparisons are lacking for the entire mitogenome locus. As family reference DNA is often required in missing persons cases, there is a need to characterize the expected number of differences between haplotypes of maternal relatives.     Further, laboratories previously unfamiliar with mtDNA may now be inclined to implement mtDNA analysis with the commercial availability of MPS panels. While this democratizes mtDNA analysis in new and exciting ways for the forensic science community, cross-platform interpretation guidelines must be developed to ensure haplotyping integrity amongst forensic mtDNA practitioners using different methodologies.  Each of these techniques can be disparately complicated by nuclear mtDNA segments (NUMTs), sequencing error, and/or phantom mutations among other workflow-specific issues. Misinterpretation of the mitogenome MPS data can then lead to erroneous conclusions, which may ultimately impact forensic cases. Thus, it is critical that MPS mtDNA interpretation is standardized within the field.     The proposed project aims to address and level the implementation challenges of MPS mitogenome analysis for the forensic community. First, 3,000 haplotypes from U.S. and global populations will be developed to increase publicly available mitogenome reference data. Second, the mtDNA substitution rate will be characterized using 500 maternally related pairs (1000 total samples) up to six generations removed. This family study will help to refine exclusion criteria for mitogenome data comparisons between maternal relatives. Lastly, commercially available mitogenome MPS kits will be compared to understand methodological bias and ultimately develop standards for cross-platform data generation. The results of this project will be essential to the adoption of MPS mtDNA throughout the forensic community.