Optimized bisulfite sequencing analysis reveals the lack of 5-methylcytosine in mammalian mitochondrial DNA.

DNA methylation is one of the best characterized epigenetic modifications in the mammalian nuclear genome and is known to play a significant role in various biological processes. Nonetheless, the presence of 5-methylcytosine (5mC) in mitochondrial DNA remains controversial, as data ranging from the lack of 5mC to very extensive 5mC have been reported. By conducting comprehensive bioinformatic analyses of both published and our own data, we reveal that previous observations of extensive and strand-biased mtDNA-5mC are likely artifacts due to a combination of factors including inefficient bisulfite conversion, extremely low sequencing reads in the L strand, and interference from nuclear mitochondrial DNA sequences (NUMTs). To reduce false positive mtDNA-5mC signals, we establish an optimized procedure for library preparation and data analysis of bisulfite sequencing. Leveraging our modified workflow, we demonstrate an even distribution of 5mC signals across the mtDNA and an average methylation level ranging from 0.19% to 0.67% in both cell lines and primary cells, which is indistinguishable from the background noise. We have developed a framework for analyzing mtDNA-5mC through bisulfite sequencing, which enables us to present multiple lines of evidence for the lack of extensive 5mC in mammalian mtDNA. We assert that the data available to date do not support the reported presence of mtDNA-5mC.