Development of a robust Oxford Nanopore workflow for mitochondrial DNA methylation analysis in reproductive ageing and mitochondrial dysfunction — ASN Events
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  3. Development of a robust Oxford Nanopore workflow for mitochondrial DNA methylation analysis in reproductive ageing and mitochondrial dysfunction

Development of a robust Oxford Nanopore workflow for mitochondrial DNA methylation analysis in reproductive ageing and mitochondrial dysfunction (#485)

Minoli Perera 1 , Mark Corbett 1 2 , Yasmyn Winstanley 1 , Rebecca Robker 1
  1. Robinson Research Institute, School of Pharmacy and Biomedical Science, Adelaide University, Adelaide, South Australia, Australia
  2. School of Medicine, Adelaide University, Adelaide, South Australia, Australia

Background: Mitochondrial DNA (mtDNA) methylation has been implicated as a regulatory mechanism in ageing and cellular function, but its role in reproduction and mitochondrial dysfunction remains unclear. Here, we used Oxford Nanopore sequencing (ONT) to characterize mtDNA methylation in mouse models of reproductive ageing and mitochondrial dysfunction.

Methods: Heart tissue from young (2 months) and reproductively aged (12 months) mice (n=4) and heart and liver tissues from POLG mutator mice (Polg D257A mutant and wild-type; n=4) were analyzed using ONT sequencing on a PromethION 2 Solo platform with adaptive sampling to enrich the mtDNA. Methylation analysis followed the NanoMethViz pipeline, with modifications including primary-alignment filtering and a ≥1000× coverage threshold to minimize Nuclear Mitochondrial DNA segments and retain high-confidence methylation calls.

Results: Age-associated mtDNA methylation levels were low across the mitochondrial genome (methylation probability 0.04–0.08) and exhibited regional rather than global differences. Increased methylation was observed in the COX3, ND4, and D-loop regions of mtDNA from aged mouse hearts. Multidimensional scaling (MDS) analysis indicated subtle age-associated differences, with greater variability among aged samples. Pipeline robustness was demonstrated by no significant associations between methylation frequency and sequencing depth after ≥100× coverage filter application.  Analysis of nuclear genes Srd5a2 and Ptk7 confirmed the ability of the workflow to detect known age-associated differences in methylation patterns and substantially higher methylation levels than those observed in mtDNA. Liver from mutant POLG mice had lower methylation levels across most regions than wild-type mice, whereas the opposite pattern was observed in the heart. MDS plot supported tissue-specific differences, with liver and heart occupying different regions.

Conclusion: Together, these findings establish a robust Oxford Nanopore workflow for mtDNA methylation analysis and demonstrate that mtDNA methylation occurs at low levels with subtle, region-specific changes associated with reproductive ageing and mitochondrial dysfunction.