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