Comprehensive Detection and Quantification of mtDNA Variants Using PacBio Long-Read Sequencing

Accurate detection of all types of mitochondrial DNA (mtDNA) variants, including single large-scale mtDNA deletions (SLSMDs) and multiple mtDNA deletions (MMDs), along with heteroplasmy quantification, is essential for Primary Mitochondrial Disease (PMD) diagnosis. This study compares amplification-free PacBio Long Read Sequencing (LRS) mtDNA analysis with long-range PCR-based targeted mtDNA sequencing by Short Read Sequencing (SRS) in terms of detection sensitivity and accuracy. Seventeen samples, including SLSMD cases (3 blood, 2 muscle), 8 MMD muscle samples, and 4 deletion-negative controls (2 blood, 2 muscle), were sequenced using the PacBio Sequel II. Our findings demonstrate LRS’s efficacy in detecting SNVs and large mtDNA deletions with precise breakpoints. Deletion heteroplasmy computed from LRS was highly correlated with the ddPCR estimates (Pearson’s r2 = 0.95). LRS detected 100% of SNVs with heteroplasmy > 10% previously identified through clinical testing. Our findings highlight the utility of PacBio LRS as a valuable tool for advanced mtDNA analysis.