Long-read sequencing enables high-accuracy mitochondrial heteroplasmy detection in Parkinson's disease

Low-frequency heteroplasmic mitochondrial DNA (mtDNA) variants are associated with aging and neurological diseases, including Parkinson's disease (PD). Targeted deep mtDNA sequencing using PacBio HiFi long reads has the potential to resolve heteroplasmy across the full mitochondrial genome with high accuracy.

To validate Vega PacBio sequencing for detecting mtDNA heteroplasmy, we analyzed four predefined mixtures of two mtDNA haplotypes. We generated a single long-range PCR amplicon covering the entire mitochondrial genome. These amplicons were mixed at predefined ratios (minor mixture haplotype component: 5%, 2%, 1%, and 0.1%). Variant calling was performed using Mutserve2, and accuracy was assessed by calculating the F₁ score from comparisons between expected and detected variants. Full-length mtDNA PacBio sequencing was applied to explore heteroplasmy across fibroblast passages derived from five LRRK2 p.Gly2019Ser variant carriers (n = 3 affected with PD and n = 2 unaffected carriers). Changes in mtDNA heteroplasmy level and variant load were assessed longitudinally using a linear mixed model as a proof-of-principle.

The single-amplicon approach enabled full-length haplotype resolution without amplification bias associated with overlapping PCR strategies. The F₁ score of the predefined mixtures was 1.0 for heteroplasmy levels between 5% and 1% and remained high (0.91) at 0.1%. At the specifically lowered calling threshold applied to the 0.1% mixture, n = 10/62 additional variants discordant with the Illumina reference were observed, but sensitivity remained very high at 1.00 in that mixture. Detected minor variants closely matched expected heteroplasmy levels, with average variant levels of 0.057 (5%), 0.022 (2%), 0.011 (1%), and 0.001 (0.1%). Across twelve fibroblast passages, we observed fewer mtDNA heteroplasmic variants. In this small proof-of-principle experiment, heteroplasmic variant load was higher in affected (n = 3) than in unaffected (n = 2) LRRK2 variant carriers and with older age. Notably, we observed distinct patterns of heteroplasmic variants that either increased or decreased in heteroplasmy level across passages.

PacBio HiFi sequencing, combined with a single-amplicon strategy, enables accurate full-length mtDNA heteroplasmy detection and longitudinal analysis, providing a valuable tool for studying mitochondrial variation and dynamics in disease.