Evaluation of GeneMarker® HTS for improved alignment of mtDNA MPS data, haplotype determination, and heteroplasmy assessment.

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 regions of sequence that are typically difficult to align are homopolymeric stretches, isolated patterns of SNPs (single nucleotide polymorphisms), and INDELs (insertions/deletions). A custom software solution, GeneMarker® HTS, was developed and evaluated to address these limitations, and to provide 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 haplotypes, 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 haplotypes, 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 provides multiple options for viewing and navigating through the data.