One of the persisting challenges in the forensic DNA analysis field is interpreting mixtures with nuclear genetic markers when the DNA is degraded or present in trace amounts. In these scenarios, utilizing mitochondrial DNA (mtDNA) analysis can be beneficial due to the higher copy number per cell compared to nuclear DNA. However, until the emergence of Next-Generation Sequencing (NGS) with its capability of clonal sequencing, analysis of mtDNA mixtures was very challenging.
We used our custom mtgenome probe capture Next-Generation Sequencing (NGS) system to sequence complex mtDNA mixtures. We report here analysis of contrived mixtures of two contributors in 50:50 and 95:5 ratios as well as three-person mixtures ranging from equal proportions (33:33:33 ratio) to low amounts of the minor contributors, as in a 90:5:5 ratio. Additionally, we have applied this system to the analysis of mtDNA mixtures from forensically relevant samples. Furthermore, by utilizing the massively parallel, clonal aspects of NGS, we can bioinformatically separate and count the individual sequence reads and calculate the proportions of the contributors. For data analysis, we use both variant frequency-based software program GeneMarker ®HTS and phylogenetic-based software program Mixemt to de-convolute the mixtures. Mixemt allows us to assign each sequence read to its originating contributors using phylogenetically informative polymorphisms. GeneMarker®HTS allows us to detect all mutations, including “private” mutations (non-phylogenetically informative polymorphisms) and assign them to individual contributors based on frequency. Using our custom probe capture NGS system and both GeneMarker®HTS and Mixemt software programs, we show that we can interpret complex mixtures of equal proportion contributors, trace amount contributors, and more than two contributors in contrived mixtures as well as challenging forensic specimens.
(Publisher abstract provided.)