Evaluation of PacBio Long-Read and PCR-Based Short-Read Sequencing for Mitochondrial DNA (mtDNA) Variant Detection, with an Emphasis on Detection and Quantification of mtDNA Deletion

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. In total, 17 samples, including 4 SLSMD cases (3 blood, 1 muscle), 9 MMD muscle samples, and 4 deletion-negative controls (1 blood, 3 muscle), were sequenced using the PacBio Sequel IIe. Our findings demonstrate LRS's efficacy in detecting single nucleotide variants (SNVs) and large mtDNA deletions with precise breakpoints. LRS can accurately detect and distinguish SLSMD from MMD, providing deletion heteroplasmy without the need for a second methodology. Deletion heteroplasmy computed from LRS was highly correlated with the Droplet Digital PCR (ddPCR) estimates (Pearson's r2 = 0.95). While LRS can detect SNVs with approximately 5% heteroplasmy, only variants exceeding 10% heteroplasmy can attain 100% sensitivity, specificity, and precision when compared to those previously identified through clinical testing. In conclusion, our findings establish PacBio LRS as a robust tool for comprehensive mtDNA analysis capable of accurately detecting and quantifying heteroplasmic mtDNA variants and complex deletions.