As submitted by the proposer:
While current techniques and technologies provide for reliable reporting of mitochondrial (mt) DNA profiles (haplotypes), the methods in place today do not effectively identify mtDNA mixtures, including those associated with heteroplasmic variants (mixtures of mtDNA sequence within an individual). Even when heteroplasmy is observed at high levels, the information is typically not included in forensic investigations, reducing the weight of the evidence. This impediment will be overcome with the use of a next generation sequencing (NGS) approach, as the ability to identify, report, and leverage the discrimination potential of heteroplasmy is now available. However, given the current level of detection for low-level heteroplasmic variants (~1%), it is quite possible that the effects of DNA damage will require a modification to the reporting guidelines being developed. Therefore, the goals of this research are to 1) assess the impact of DNA damage on mtDNA sequencing results generated on the Illumina MiSeq, 2) explore whether repair systems can fix the damage, and 3) assess the impact of DNA damage on the interpretation process so that low-level heteroplasmic variants can be reliably reported in forensic investigations. We will do so through both a passive and an active approach to damaging DNA, comparing the results to NGS data generated from pristine samples. In addition, we will assess the effects of damage on low-level template quantities and on mock evidence samples in an attempt to mimic what the forensic practitioner will encounter. Following the development of an NGS approach for the mtDNA genome on the MiSeq platform from Illumina, our initial studies illustrated that low-level heteroplasmy exists in a large percentage of the general population. In addition, we revealed the existence of differing ratios of heteroplasmic variants within maternal lineages, allowing for the distinct identification of maternal relatives. Combined, these findings offer great promise for enhancing the value of mtDNA analysis in forensic casework. We are currently engaged in a study to measure the rate of mtDNA heteroplasmy on a per sample and per nucleotide basis, to evaluate the transmission of heteroplasmic events between maternal relatives and different tissue types, and to develop best practices regarding the reporting of mtDNA heteroplasmy (NIJ Award No. 2014-DN-BX-K022). However, in order to effectively implement an NGS approach, it will be important to understand the effects of DNA damage on mtDNA sequencing results.
This project contains a research and/or development component, as defined in applicable law.