skip to content

MRC Mitochondrial Biology Unit


The results of a research study carried out by Iacopo Bicci and Claudia Calabrese in Professor Patrick Chinnery’s laboratory have been published in Nucleic Acids Research

Cytosine methylation is an epigenetic modification of nuclear DNA (nDNA) that can regulate gene expression during development and throughout life, but the presence of CpG methylation on the mitochondrial genome (mtDNA) has been a matter of debate and is an important issue to resolve given the pivotal role of mtDNA in cellular metabolism.

Methylation of mitochondrial DNA (mtDNA) has been studied using whole genome bisulfite sequencing (WGBS), but recent evidence has uncovered technical issues which introduce a potential bias during methylation quantification. In this research study, Drs Bicci, Calabrese and colleagues validate the technical concerns of WGBS, and developed and assessed the accuracy of a new protocol for mtDNA nucleotide variant-specific methylation using single-molecule Oxford Nanopore Sequencing (ONS). This approach circumvents confounders by enriching for full-length molecules over nuclear DNA. Variant calling analysis against showed that 99.5% of homoplasmic mtDNA variants can be reliably identified providing there is adequate sequencing depth.  This shows that some of the mtDNA methylation signal detected by ONS is due to sequence-specific false positives introduced by the technique. The residual signal was observed across several human primary and cancer cell lines and multiple human tissues,but was always below the error threshold modelled using negative controls.

The research team concluded that there is no evidence for CpG methylation in human mtDNA, thus resolving previous controversies.

Additionally, they developed a reliable protocol to study epigenetic modifications of mtDNA at single-molecule and single-base resolution, with potential applications beyond CpG methylation.

Publication reference:

Bicci I, Calabrese C, Golder ZJ, Gomez-Duran A, Chinnery PF. (2021) Single-molecule mitochondrial DNA sequencing shows no evidence of CpG methylation in human cells and tissues. Nucleic Acids Res. 2021 Nov 29:gkab1179.

doi: 10.1093/nar/gkab1179.