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Single-molecule mitochondrial DNA sequencing shows no evidence of CpG methylation in human cells and tissues
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...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8682748/ https://www.ncbi.nlm.nih.gov/pubmed/34850165 http://dx.doi.org/10.1093/nar/gkab1179 |
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author | Bicci, Iacopo Calabrese, Claudia Golder, Zoe J Gomez-Duran, Aurora Chinnery, Patrick F |
author_facet | Bicci, Iacopo Calabrese, Claudia Golder, Zoe J Gomez-Duran, Aurora Chinnery, Patrick F |
author_sort | Bicci, Iacopo |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-8682748 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-86827482021-12-20 Single-molecule mitochondrial DNA sequencing shows no evidence of CpG methylation in human cells and tissues Bicci, Iacopo Calabrese, Claudia Golder, Zoe J Gomez-Duran, Aurora Chinnery, Patrick F Nucleic Acids Res Gene regulation, Chromatin and Epigenetics 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. Oxford University Press 2021-11-29 /pmc/articles/PMC8682748/ /pubmed/34850165 http://dx.doi.org/10.1093/nar/gkab1179 Text en © The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research. https://creativecommons.org/licenses/by/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Gene regulation, Chromatin and Epigenetics Bicci, Iacopo Calabrese, Claudia Golder, Zoe J Gomez-Duran, Aurora Chinnery, Patrick F Single-molecule mitochondrial DNA sequencing shows no evidence of CpG methylation in human cells and tissues |
title | Single-molecule mitochondrial DNA sequencing shows no evidence of CpG methylation in human cells and tissues |
title_full | Single-molecule mitochondrial DNA sequencing shows no evidence of CpG methylation in human cells and tissues |
title_fullStr | Single-molecule mitochondrial DNA sequencing shows no evidence of CpG methylation in human cells and tissues |
title_full_unstemmed | Single-molecule mitochondrial DNA sequencing shows no evidence of CpG methylation in human cells and tissues |
title_short | Single-molecule mitochondrial DNA sequencing shows no evidence of CpG methylation in human cells and tissues |
title_sort | single-molecule mitochondrial dna sequencing shows no evidence of cpg methylation in human cells and tissues |
topic | Gene regulation, Chromatin and Epigenetics |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8682748/ https://www.ncbi.nlm.nih.gov/pubmed/34850165 http://dx.doi.org/10.1093/nar/gkab1179 |
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