Minimal detection and low biological fluctuation of mitochondrial CpG methylation at the single-molecule level

Cytosine DNA methylation in the CpG context (5mCpG) is associated with the transcriptional status of nuclear DNA. Due to technical limitations, it has been less clear if mitochondrial DNA (mtDNA) is methylated and whether 5mCpG has a regulatory role in this context. The main aim of this work was to develop and validate a novel tool for determining methylation of mtDNA and to corroborate its existence across different biological contexts. Using long-read nanopore sequencing we found low levels of CpG methylation (with few exceptions) and little variation across biological processes: differentiation, oxidative stress, and cancer. 5mCpG was overall higher in tissues compared to cell lines, with small additional variation between cell lines of different origin. Although we do show several significant changes in all these conditions, 5mCpG is unlikely to play a major role in defining the transcriptional status of mitochondrial genes. ### Competing Interest Statement C.G. and H.H.-V. have received travel and accommodation support to attend conferences for Oxford Nanopore Technology. The authors declare that they have no additional competing interests.