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dc.contributor.authorBicci, Iacopo
dc.contributor.authorCalabrese, Claudia
dc.contributor.authorGolder, Zoe J
dc.contributor.authorGomez-Duran, Aurora
dc.contributor.authorChinnery, Patrick
dc.date.accessioned2021-11-10T00:31:04Z
dc.date.available2021-11-10T00:31:04Z
dc.date.issued2021-12-16
dc.identifier.issn0305-1048
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/330529
dc.description.abstractMethylation 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.publisherOxford University Press (OUP)
dc.rightsAll rights reserved
dc.rights.urihttp://www.rioxx.net/licenses/all-rights-reserved
dc.titleSingle-molecule mitochondrial DNA sequencing shows no evidence of CpG methylation in human cells and tissues.
dc.typeArticle
prism.publicationNameNucleic Acids Res
dc.identifier.doi10.17863/CAM.77972
dcterms.dateAccepted2021-11-18
rioxxterms.versionofrecord10.1093/nar/gkab1179
rioxxterms.versionAM
rioxxterms.licenseref.urihttp://www.rioxx.net/licenses/all-rights-reserved
rioxxterms.licenseref.startdate2021-11-05
dc.contributor.orcidChinnery, Patrick [0000-0002-7065-6617]
dc.identifier.eissn1362-4962
rioxxterms.typeJournal Article/Review
pubs.funder-project-idCambridge University Hospitals NHS Foundation Trust (CUH) (146281)
pubs.funder-project-idWellcome Trust (212219/Z/18/Z)
pubs.funder-project-idMRC (MR/S035699/1)
pubs.funder-project-idNational Institute for Health Research (NIHRDH-IS-BRC-1215-20014)
cam.issuedOnline2021-11-29
cam.orpheus.successTue Feb 01 19:02:13 GMT 2022 - Embargo updated
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rioxxterms.freetoread.startdate2021-11-29


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