Long-read sequencing enables high-accuracy mitochondrial heteroplasmy detection in Parkinson’s disease
Discuss this preprint
Start a discussion What are Sciety discussions?Listed in
This article is not in any list yet, why not save it to one of your lists.Abstract
Background
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.
Methods
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 F 1 score from comparisons between expected and detected variants. Full-length mtDNA PacBio sequencing was applied to investigate 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.
Results
The single-amplicon approach enabled full-length haplotype resolution without amplification bias associated with overlapping PCR strategies. The F 1 score of the predefined mixtures was 1.0 for heteroplasmy levels between 5% and 1% and remained high (0.91) at 0.1%. We detected n=10/62 variants discordant with the Illumina reference at the 0.1% mixture, 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 (β=-3.2, p=0.026). Increased heteroplasmic variant load over time was also associated with older age (β=1.50, p=0.001) and PD affection status (β=5.0, p=1.0 × 10 - ⁴) in LRRK2 variant carriers. Notably, we observed distinct patterns of heteroplasmic variants that either increased or decreased in heteroplasmy level across passages.
Conclusion
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.
