{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/339512"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/339512","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"A sequencing-based analyisis of epigenetic modifications of the mitochondrial genome","abstract":"Methylation on CpG residues is one of the most important epigenetic modifications of nuclear DNA, where it regulates gene expression. Methylation of mitochondrial DNA (mtDNA) has been studied using whole genome bisulfite sequencing (WGBS), but recent evidence has uncovered major technical issues, which introduce a potential bias during methylation quantification. In this study, we first validate the technical concerns with WGBS using publicly available datasets. Then we develop and assess the accuracy of a protocol for variant-specific methylation identification using long-read based technology Oxford Nanopore Sequencing. Our approach circumvents mtDNA-specific confounders, while enriching for native full-length molecules over nuclear DNA. Variant calling analysis against Illumina deep re-sequencing showed that all expected mtDNA variants can be reliably identified. By using simulated data sets, we were able to determine that the mtDNA methylation levels identified were likely false positives introduced by the technique. This observation was consistent across the multiple human primary and cancer cell lines and human tissues analysed in this study. We therefore conclude that CpG methylation is not an epigenetic modification occurring 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","abstract_html":"Methylation on CpG residues is one of the most important epigenetic modifications of nuclear DNA, where it regulates gene expression. Methylation of mitochondrial DNA (mtDNA) has been studied using whole genome bisulfite sequencing (WGBS), but recent evidence has uncovered major technical issues, which introduce a potential bias during methylation quantification. In this study, we first validate the technical concerns with WGBS using publicly available datasets. Then we develop and assess the accuracy of a protocol for variant-specific methylation identification using long-read based technology Oxford Nanopore Sequencing. Our approach circumvents mtDNA-specific confounders, while enriching for native full-length molecules over nuclear DNA. Variant calling analysis against Illumina deep re-sequencing showed that all expected mtDNA variants can be reliably identified. By using simulated data sets, we were able to determine that the mtDNA methylation levels identified were likely false positives introduced by the technique. This observation was consistent across the multiple human primary and cancer cell lines and human tissues analysed in this study. We therefore conclude that CpG methylation is not an epigenetic modification occurring 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","abstract_has_math":false,"creators":["Bicci, Iacopo"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Chinnery, Patrick"],"committee_chairs":[],"committee_members":[],"year":2022,"date_issued":"2022-05-20","date_published":"2022-05-20","updated_at":"2026-07-22T22:23:56Z","subjects":["Methylation","Nanopore Sequencing","Mitochondrial DNA"],"languages":["eng"],"rights":[],"rights_urls":["https://creativecommons.org/licenses/by-nc-sa/4.0/"],"identifier_entries":[{"key":"dc:creator.authoridentifier","label":"Author Identifier","values":["0000000169943857"],"render_values":[{"text":"0000-0001-6994-3857","href":"https://orcid.org/0000-0001-6994-3857","code":true}]}]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.86926","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Chinnery, Patrick"]},{"key":"dc:creator","label":"Author","values":["Bicci, Iacopo"]},{"key":"dc:creator.authoridentifier","label":"Author Identifier","values":["0000000169943857"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2022-05-20"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/339512"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Methylation","Nanopore Sequencing","Mitochondrial DNA"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://creativecommons.org/licenses/by-nc-sa/4.0/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["10.17863/CAM.86926"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://www.repository.cam.ac.uk/bitstreams/4ceb3aa5-392b-4d1e-ada7-291e28ec6d65/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Methylation on CpG residues is one of the most important epigenetic modifications of nuclear DNA, where it regulates gene expression. Methylation of mitochondrial DNA (mtDNA) has been studied using whole genome bisulfite sequencing (WGBS), but recent evidence has uncovered major technical issues, which introduce a potential bias during methylation quantification. In this study, we first validate the technical concerns with WGBS using publicly available datasets. Then we develop and assess the accuracy of a protocol for variant-specific methylation identification using long-read based technology Oxford Nanopore Sequencing. Our approach circumvents mtDNA-specific confounders, while enriching for native full-length molecules over nuclear DNA. Variant calling analysis against Illumina deep re-sequencing showed that all expected mtDNA variants can be reliably identified. By using simulated data sets, we were able to determine that the mtDNA methylation levels identified were likely false positives introduced by the technique. This observation was consistent across the multiple human primary and cancer cell lines and human tissues analysed in this study. We therefore conclude that CpG methylation is not an epigenetic modification occurring 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"]},{"key":"dc:format.checksum.md5","label":"Dc Format Checksum Md5","values":["1942d1db03f48c25820f7cb44d64a911"]},{"key":"dc:title","label":"Title","values":["A sequencing-based analyisis of epigenetic modifications of the mitochondrial genome"]}]}],"canonical_facts":{"dc:contributor.advisor":["Chinnery, Patrick"],"dc:creator":["Bicci, Iacopo"],"dc:creator.authoridentifier":["0000000169943857"],"dc:date.issued":["2022-05-20"],"dc:description.abstract":["Methylation on CpG residues is one of the most important epigenetic modifications of nuclear DNA, where it regulates gene expression. Methylation of mitochondrial DNA (mtDNA) has been studied using whole genome bisulfite sequencing (WGBS), but recent evidence has uncovered major technical issues, which introduce a potential bias during methylation quantification. In this study, we first validate the technical concerns with WGBS using publicly available datasets. Then we develop and assess the accuracy of a protocol for variant-specific methylation identification using long-read based technology Oxford Nanopore Sequencing. Our approach circumvents mtDNA-specific confounders, while enriching for native full-length molecules over nuclear DNA. Variant calling analysis against Illumina deep re-sequencing showed that all expected mtDNA variants can be reliably identified. By using simulated data sets, we were able to determine that the mtDNA methylation levels identified were likely false positives introduced by the technique. This observation was consistent across the multiple human primary and cancer cell lines and human tissues analysed in this study. We therefore conclude that CpG methylation is not an epigenetic modification occurring 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:format.checksum.md5":["1942d1db03f48c25820f7cb44d64a911"],"dc:identifier.doi":["10.17863/CAM.86926"],"dc:identifier.uri":["https://www.repository.cam.ac.uk/bitstreams/4ceb3aa5-392b-4d1e-ada7-291e28ec6d65/download"],"dc:language":["eng"],"dc:publisher.institution":["University of Cambridge"],"dc:relation.isreferencedby.uri":["https://www.repository.cam.ac.uk/handle/1810/339512"],"dc:rights":["https://creativecommons.org/licenses/by-nc-sa/4.0/"],"dc:subject":["Methylation","Nanopore Sequencing","Mitochondrial DNA"],"dc:title":["A sequencing-based analyisis of epigenetic modifications of the mitochondrial genome"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-22T22:23:56Z"}