Damage patterns observed in mtDNA control region MPS data for a range of template concentrations and when using different amplification approaches.

Massively parallel sequencing (MPS) of mitochondrial (mt) DNA allows practitioners the ability to fully resolve heteroplasmic sites. In forensic DNA analysis, identifying heteroplasmy (a naturally occurring mixture of two mtDNA profiles) can provide additional mtDNA profile information which can lead to an increase in the discrimination potential of an mtDNA match between an evidentiary sample and reference source. Forensic samples such as hair and skeletal remains, especially older, more compromised samples, can often exhibit DNA damage. Because both damage and heteroplasmy can manifest as a mixture of two nucleotides, it is important to differentiate between the two conditions when interpreting mtDNA MPS data. In this study, DNA damage was applied under controlled conditions to samples containing a range of template concentrations, including some with identified heteroplasmy. Damage was applied via storage in water at room temperature on samples diluted before or after storage to mimic low template scenarios. Damage was assessed with respect to the following areas: mtDNA quantification and degradation ratios, MPS read depth, MPS profile results, overall damage rates, and the interpretation of heteroplasmy. Datasets were generated to assess and compare two different amplification and library preparation strategies: the Promega PowerSeq™ CRM Nested System kit and a 1.16 kb target amplicon of the entire mtDNA control region followed by a Nextera® XT library preparation. The results of this study provide an evaluation of the Promega 10-plex MPS procedure as an improved process to mitigate the impact of mtDNA damage on low template samples. Some of the negative effects of damage observed in this study were a decrease in mtDNA yield by 20-30% and lower quality MPS sequencing results. These effects were observed more frequently when samples were diluted prior to inducing damage, illustrating that low template samples are more susceptible to damage. The findings of this study will assist forensic laboratories in differentiating between damage and heteroplasmy, which is essential when developing robust mtDNA MPS interpretation guidelines such as setting appropriate reporting thresholds.