Advancing mitochondrial DNA analysis: Utility of PacBio long-read sequencing technology in accurate and precise mtDNA SNV and CNV variant detection

Background Primary Mitochondrial Diseases (PMD) can arise from pathogenic variants in mitochondrial DNA (mtDNA), including single-nucleotide variants (SNVs), small indels, single large-scale mtDNA deletions (SLSMD), and multiple mtDNA deletions (MMD). Accurate detection of and distinction between MMDs and SLSMDs, along with heteroplasmy quantification, is essential for PMD diagnosis. Clinical mtDNA diagnostic tests utilize targeted Short Read Sequencing (SRS) for variant detection and droplet-digital PCR (ddPCR) for large-scale deletion heteroplasmy quantification. This multi-tiered approach is necessary to address a) the limitations of SRS in detecting deletion heteroplasmy and precise breakpoints (in MMDs) due to the PCR amplification utilized in this assay and b) the inability of ddPCR to detect SNVs. This study evaluates amplification-free PacBio Long Read Sequencing (LRS) for detecting mtDNA SNV and large-scale deletion, and heteroplasmy quantification. Methods 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. Two samples were sequenced twice to assess reproducibility. A bioinformatics pipeline was developed for mtDNA large deletion detection and breakpoint clustering. The SNVs, large deletions, and heteroplasmy levels from LRS data were compared against the standard of care clinical mtDNA testing assay. Results 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 r ²  = 0.98). LRS detected 100% of SNVs with heteroplasmy > 10% previously identified through clinical testing. LRS also enabled us to investigate breakpoint microhomology by detecting precise breakpoints in all deletion-positive samples. Conclusions This study presents PacBio LRS as a promising technique for advancing mtDNA analysis by evaluating its ability to detect SNVs, large-scale mtDNA deletions, quantify heteroplasmy, and precisely locate deletion breakpoints. Our results demonstrate the utility of PacBio LRS as a comprehensive tool for clinical mtDNA testing.