Mitochondrial DNA heteroplasmy patterns in hair shaft samples: Insights from Whole Mitogenome Sequencing using the Precision ID mtDNA Whole Genome Panel

Mitochondrial DNA (mtDNA) is a robust marker in forensic casework, particularly crucial for forensic casework involving samples containing small amounts of nuclear DNA (nuDNA), such as hair shafts. However, the interpretation of mtDNA faces challenges, notably in the reporting of heteroplasmy. In this study, we aim to identify heteroplasmy patterns in hair shaft samples using whole mitogenomes sequenced with massively parallel sequencing and compare these heteroplasmy patterns with those obtained in reference samples. Blood and buccal reference samples from 36 unrelated individuals, alongside 317 hair segments (each measuring 2 cm) were included in this study. DNA extractions were performed using in-house pre-treatment protocols followed by DNA extraction with the EZ-1 extraction robot (Qiagen). Quantification of nuDNA in the reference samples was performed using the Quantifiler Trio DNA quantification Kit (ThermoFisher Scientific, TFS), while mtDNA (and nuDNA if present) in hair samples was quantified using the qPCR SD quants assay (Xavier et al., 2019). All quantification experiments were performed on the 7500 Real-Time PCR system (Applied Biosystems). Hair samples with less than 100 mtDNA copies were up-concentrated and re-quantified. The Precision ID mtDNA Whole Genome Panel and the Ion Torrent Ion Gene Studio S5 sequencing platform with Ion 530 chip (both TFS) were used for library preparation and sequencing. Successful amplification of mtDNA was obtained for all samples, even those with low mtDNA copy numbers. Our study provides insight into the heteroplasmy patterns observed in hair samples, revealing a higher frequency of heteroplasmy events in hair compared to those obtained in reference samples. Furthermore, variations in heteroplasmy patterns exist both within and between hairs from the same individual. These findings advance our understanding of mtDNA dynamics and hold promise to enhance the interpretation and reporting of mtDNA data in forensic contexts.