AbstractBackgroundThere is growing evidence for the role of DNA methylation in regulating the transcription of mitochondrial genes. This is particularly important in neurodegenerative disorders characterised by mitochondrial dysfunction, including Alzheimer’s disease. However, current methods to create genome‐wide, high‐resolution analysis of the mitochondrial DNA methylome are challenging and lack control standards.MethodHere, we present a new methodological approach to interrogate the mitochondrial DNA methylome at single base resolution using Oxford Nanopore technology and the creation of independent controls. Firstly, we isolate enriched mtDNA from SH‐SY5Y cell lines and post‐mortem brain tissue and use long‐range PCR (LR‐PCR) to generate 0%, 50% and 100% methylation controls. These controls were then validated using pyrosequencing and then run on the Oxford Nanopore (OXNP) long read sequencer. Finally, we used data generated by our controls to train existing methylation calling packages to better call mtDNA methylation in a post‐mortem human brain sample.ResultWe present a new and effective method of profiling mtDNA methylation on native DNA. Using this approach, we show that mitochondrial DNA methylation is relatively low but conserved, with peaks in DNA methylation >5% at several sites.ConclusionWe have annotated patterns of DNA methylation at single base resolution across the mitochondrial genome in human brain, using our new laboratory pipeline and Oxford Nanopore technology to profile native DNA. Looking to the future this method could be utilized to further investigate the role of mitochondrial epigenetic mechanisms in neurodegenerative disorders, such as Alzheimer’s disease.