Enhanced mitochondrial genome analysis: bioinformatic and long-read sequencing advances and their diagnostic implications.

Introduction: Primary mitochondrial diseases (PMDs) comprise a large and heterogeneous group of genetic diseases that result from pathogenic variants in either nuclear DNA (nDNA) or mitochondrial DNA (mtDNA). Widespread adoption of next-generation sequencing (NGS) has improved the efficiency and accuracy of mtDNA diagnoses; however, several challenges remain.

Areas covered: In this review, we briefly summarize the current state of the art in molecular diagnostics for mtDNA and consider the implications of improved whole genome sequencing (WGS), bioinformatic techniques, and the adoption of long-read sequencing, for PMD diagnostics.

Expert opinion: We anticipate that the application of PCR-free WGS from blood DNA will increase in diagnostic laboratories, while for adults with myopathic presentations, WGS from muscle DNA may become more widespread. Improved bioinformatic strategies will enhance WGS data interrogation, with more accurate delineation of mtDNA and NUMTs (nuclear mitochondrial DNA segments) in WGS data, superior coverage uniformity, indirect measurement of mtDNA copy number, and more accurate interpretation of heteroplasmic large-scale rearrangements (LSRs). Separately, the adoption of diagnostic long-read sequencing could offer greater resolution of complex LSRs and the opportunity to phase heteroplasmic variants.