Assessment of Illumina® Human mtDNA Genome assay: workflow evaluation with development of analysis and interpretation guidelines

Mitochondrial DNA (mtDNA) is a small but significant part of the human genome, whose applicability potential has gradually increased with advent massively parallel sequencing (MPS) technology. Knowledge particular workflow, equipment, and reagents used, along extensive usage negative controls to monitor all preparation steps constitute prerequisites for confident reporting results. In this study, we performed an assessment Illumina® Human mtDNA Genome assay on MiSeq FGx™ instrument. Through analysis several types controls, as well positive established thresholds data interpretation, consisting components: minimum read depth (220 reads), quality score (41), percentage minor allele sufficient (3.0%), interpretation (6.0%), major homoplasmic variant call (97.0%). Based these criteria, defined internal guidelines results obtained by MPS. Our study shows that whole produces repeatable reproducible results, independent analyst, which are also concordant Sanger-type control region, MPS produced NextSeq®. Overall, were successfully applied complete mitochondrial genomes from high-quality samples. The underlying principles proposed methodology definition laboratory may be applicable similar workflows, e.g. targeting good-quality samples in forensic genetics molecular diagnostics.