cam.issuedOnline	2021-11-29
cam.orpheus.counter	2
cam.orpheus.success	Tue Feb 01 19:02:13 GMT 2022 - Embargo updated
dc.contributor.author	Bicci, Iacopo
dc.contributor.author	Calabrese, Claudia
dc.contributor.author	Golder, Zoe J
dc.contributor.author	Gomez-Duran, Aurora
dc.contributor.author	Chinnery, Patrick F
dc.contributor.orcid	Chinnery, Patrick F [0000-0002-7065-6617]
dc.date.accessioned	2021-11-10T00:31:04Z
dc.date.available	2021-11-10T00:31:04Z
dc.date.issued	2021-12-16
dc.description.abstract	Methylation on CpG residues is one of the most important epigenetic modifications of nuclear DNA, regulating gene expression. 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. Here, we validate the technical concerns of WGBS, and develop and assess the accuracy of a new protocol for mtDNA nucleotide variant-specific methylation using single-molecule Oxford Nanopore Sequencing (ONS). Our 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. We show 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. We conclude that there is no evidence for CpG methylation in human mtDNA, thus resolving previous controversies. Additionally, we developed a reliable protocol to study epigenetic modifications of mtDNA at single-molecule and single-base resolution, with potential applications beyond CpG methylation.
dc.identifier.doi	10.17863/CAM.77972
dc.identifier.eissn	1362-4962
dc.identifier.issn	0305-1048
dc.identifier.uri	https://www.repository.cam.ac.uk/handle/1810/330529
dc.language.iso	eng
dc.publisher	Oxford University Press (OUP)
dc.publisher.url	http://dx.doi.org/10.1093/nar/gkab1179
dc.rights	All rights reserved
dc.rights.uri	http://www.rioxx.net/licenses/all-rights-reserved
dc.subject	Cell Line
dc.subject	Cell Line, Tumor
dc.subject	CpG Islands
dc.subject	DNA Methylation
dc.subject	DNA, Mitochondrial
dc.subject	Genetic Variation
dc.subject	Humans
dc.subject	Nanopore Sequencing
dc.subject	Whole Genome Sequencing
dc.title	Single-molecule mitochondrial DNA sequencing shows no evidence of CpG methylation in human cells and tissues.
dc.type	Article
dcterms.dateAccepted	2021-11-18
prism.publicationName	Nucleic Acids Res
pubs.funder-project-id	Cambridge University Hospitals NHS Foundation Trust (CUH) (146281)
pubs.funder-project-id	Wellcome Trust (212219/Z/18/Z)
pubs.funder-project-id	MRC (MR/S035699/1)
pubs.funder-project-id	National Institute for Health and Care Research (IS-BRC-1215-20014)
pubs.funder-project-id	Medical Research Council (MC_UU_00015/7)
rioxxterms.licenseref.startdate	2021-11-05
rioxxterms.licenseref.uri	http://www.rioxx.net/licenses/all-rights-reserved
rioxxterms.type	Journal Article/Review
rioxxterms.version	AM
rioxxterms.versionofrecord	10.1093/nar/gkab1179

Single-molecule mitochondrial DNA sequencing shows no evidence of CpG methylation in human cells and tissues.

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