Evaluation of the ForenSeq mtDNA Whole Genome Kit for massively parallel sequencing of mitochondrial genomes

Abstract

The performance of Verogen’s ForenSeq mtDNA Whole Genome Kit and its possible application in a forensic setting was evaluated. We carried out three sequencing runs that included 1) a dilution series of two different samples (200–6.25 pg input nuclear DNA), 2) casework samples, and 3) hair, blood, and buccal samples from the same individual. For samples described in 1), the method was tested using different reagent volume in the PCR and library building (recommended volume or half-volume). Reference samples were analysed in duplicates, and positive and negative controls were included in every run. All mtDNA profiles were known from previous genotyping with the Precision ID mtDNA Whole Genome Panel or using long range PCR and the MiSeq instrument. The average read depth per sample was > 1200x in all samples, although some regions had read depth 50x. Using half volume of reagents did not compromise the quality of the results and can be a cost-effective alternative for reference samples. For an analytical threshold of 10%, complete concordance was observed between the duplicates and previous results. Heteroplasmy was consistent with prior observations although frequencies varied up to 10% compared to previous results.