Since existing software has not allowed for effective alignment of mitochondrial (mt)DNA sequence data generated using a massively parallel sequencing (MPS) approach combined with the ability to perform a detailed assessment of the data, the reported project developed and evaluated a custom software solution, GeneMarker HTS), to address these limitations.
GeneMarker HTS also provides a user-friendly interface for forensic practitioners and others interested in mtDNA analysis of MPS data. GeneMarker HTS generates an exportable consensus mtDNA sequence that produces phylogenetically correct SNP and INDEL calls using a customizable motif-based alignment algorithm. Sequence data from 500 individuals with various alignment asymmetries and levels of heteroplasmy were used to assess the software. Accuracy in producing mtDNA heliotypes, the ability to correctly identify low-level heteroplasmic sequence variants, and the user-based features of the software were evaluated. Analyzed sequences yielded correct mtDNA heliotypes, and heteroplasmic variants were properly identified with minimal manual interpretation. The software offers numerous user-defined parameters for filtering the data that address the interests of researchers and practitioners, and it provides multiple options for viewing and navigating through the data. (Publisher abstract modified)