{"doi":"10.1371/journal.pone.0192722","title":"Technical adequacy of bisulfite sequencing and pyrosequencing for detection of mitochondrial DNA methylation: Sources and avoidance of false-positive detection","abstract":null,"journal":"PLOS ONE","year":2018,"id":588960,"datarank":2.567656738686582,"base_score":4.189654742026425,"endowment":4.189654742026425,"self_citation_contribution":0.6284482113039639,"citation_network_contribution":1.939208527382618,"self_endowment_contribution":0.6284482113039639,"citer_contribution":1.939208527382618,"corpus_percentile":null,"corpus_rank":null,"citation_count":65,"citer_count":60,"citers_with_citation_signal":56,"citers_with_endowment":56,"datacite_reuse_total":2,"is_dataset":false,"is_dataset_confidence":null,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":null,"fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":161964,"name":"Matthew Poulin","orcid":null,"position":1,"is_corresponding":false},{"id":1191855,"name":"Liying Yan","orcid":"0000-0001-9572-9440","position":2,"is_corresponding":false},{"id":481672,"name":"Toshi Shioda","orcid":"0000-0002-9434-7835","position":3,"is_corresponding":false},{"id":24524,"name":"Chie Owa","orcid":null,"position":0,"is_corresponding":false}],"reference_count":0,"raw_metadata":{"has_enrichment":true,"resolved":true,"title":"Technical adequacy of bisulfite sequencing and pyrosequencing for detection of mitochondrial DNA methylation: Sources and avoidance of false-positive detection","abstract":"The existence of cytosine methylation in mammalian mitochondrial DNA (mtDNA) is a controversial subject. Because detection of DNA methylation depends on resistance of 5'-modified cytosines to bisulfite-catalyzed conversion to uracil, examined parameters that affect technical adequacy of mtDNA methylation analysis. Negative control amplicons (NCAs) devoid of cytosine methylation were amplified to cover the entire human or mouse mtDNA by long-range PCR. When the pyrosequencing template amplicons were gel-purified after bisulfite conversion, bisulfite pyrosequencing of NCAs did not detect significant levels of bisulfite-resistant cytosines (brCs) at ND1 (7 CpG sites) or CYTB (8 CpG sites) genes (CI95 = 0%-0.94%); without gel-purification, significant false-positive brCs were detected from NCAs (CI95 = 4.2%-6.8%). Bisulfite pyrosequencing of highly purified, linearized mtDNA isolated from human iPS cells or mouse liver detected significant brCs (~30%) in human ND1 gene when the sequencing primer was not selective in bisulfite-converted and unconverted templates. However, repeated experiments using a sequencing primer selective in bisulfite-converted templates almost completely (< 0.8%) suppressed brC detection, supporting the false-positive nature of brCs detected using the non-selective primer. Bisulfite-seq deep sequencing of linearized, gel-purified human mtDNA detected 9.4%-14.8% brCs for 9 CpG sites in ND1 gene. However, because all these brCs were associated with adjacent non-CpG brCs showing the same degrees of bisulfite resistance, DNA methylation in this mtDNA-encoded gene was not confirmed. Without linearization, data generated by bisulfite pyrosequencing or deep sequencing of purified mtDNA templates did not pass the quality control criteria. Shotgun bisulfite sequencing of human mtDNA detected extremely low levels of CpG methylation (<0.65%) over non-CpG methylation (<0.55%). Taken together, our study demonstrates that adequacy of mtDNA methylation analysis using methods dependent on bisulfite conversion needs to be established for each experiment, taking effects of incomplete bisulfite conversion and template impurity or topology into consideration.","is_dataset_classified":null,"base_score":4.189654742026425,"endowment":4.189654742026425,"datacite_reuse_total":2,"file_count":0,"downloads":0,"views":0,"has_version_chain":false,"is_dataset":false,"is_oa":false,"pmid":"29420656","pmcid":"PMC5805350","openalex_id":"https://openalex.org/W2786549807","authors":[],"funders":[{"funder_name":"National Institutes of Health","grant_id":"R01ES023316","title":null},{"funder_name":"National Institutes of Health","grant_id":"R21ES024861","title":null},{"funder_name":"National Institutes of Health","grant_id":"2R01ES023316-06","title":"Transgenerational obesity caused by ancestral exposure to obesogens in utero: changes in germline genomic architecture, roles of gonadal somatic cells, and metabolomic analysis of sexual dimorphism"},{"funder_name":"National Institutes of Health","grant_id":"5R21ES024861-02","title":"Roles of Nuclear Receptors in Generation of Heritable Epimutations in Germ Cells"}],"total_grants":4,"fwci":2.9662,"citation_percentile":0.92368597,"influential_citations":0,"citation_trend":[{"year":2018,"count":4},{"year":2019,"count":8},{"year":2020,"count":12},{"year":2021,"count":12},{"year":2022,"count":10},{"year":2023,"count":9},{"year":2024,"count":4},{"year":2025,"count":5},{"year":2026,"count":1}],"oa_status":"gold","license":"cc-by","oa_locations":[{"url":"https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0192722&type=printable","host_type":"journal"},{"url":"https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0192722&type=printable","host_type":"publisher"},{"url":"http://dx.plos.org/10.1371/journal.pone.0192722","host_type":"publisher"},{"url":"https://doi.org/10.1371/journal.pone.0192722","host_type":"journal"},{"url":"https://pubmed.ncbi.nlm.nih.gov/29420656","host_type":"repository"},{"url":"http://nrs.harvard.edu/urn-3:HUL.InstRepos:35014895","host_type":"repository"},{"url":"https://doaj.org/article/fbe7565012a6440cbe59851a0c2cb9f7","host_type":"repository"},{"url":"https://figshare.com/articles/dataset/Technical_adequacy_of_bisulfite_sequencing_and_pyrosequencing_for_detection_of_mitochondrial_DNA_methylation_Sources_and_avoidance_of_false-positive_detection/5872038","host_type":"repository"},{"url":"https://www.ncbi.nlm.nih.gov/pmc/articles/5805350","host_type":"repository"},{"url":"https://europepmc.org/articles/PMC5805350","host_type":"Europe_PMC"},{"url":"https://europepmc.org/articles/PMC5805350?pdf=render","host_type":"Europe_PMC"},{"url":"http://dx.doi.org/10.1371/journal.pone.0192722","host_type":""},{"url":"https://dx.doi.org/10.1371/journal.pone.0192722","host_type":""},{"url":"https://doi.org/https://doi.org/10.1371/journal.pone.0192722","host_type":""}],"fields_of_study":["Epigenetics and DNA Methylation","RNA modifications and cancer","Cancer-related gene regulation","0301 basic medicine","03 medical and health sciences","Animals","CpG Islands","Cytosine","DNA Methylation","DNA, Mitochondrial","Humans","Mice","Sequence Analysis, DNA","Sulfites"],"mesh_terms":["Animals","Cytosine","DNA, Mitochondrial","Humans","Sulfites","Sequence Analysis, DNA","CpG Islands","DNA Methylation","Mice"],"keywords":["Bisulfite","Pyrosequencing","Bisulfite sequencing","DNA methylation","Illumina Methylation Assay","Methylation","CpG site","Biology","Primer (cosmetics)","Molecular biology","Amplicon","DNA","Mitochondrial DNA","Methylated DNA immunoprecipitation","Cytosine","Gene","Genetics","Polymerase chain reaction","Chemistry","Gene expression","DNA cloning","Gene Sequencing","Artificial Gene Amplification and Extension","Biochemistry","Mice","Sequencing techniques","DNA sequencing","Nucleotides","Organic Compounds","Q","Chemical Reactions","R","Chromatin","Nucleic acids","Physical Sciences","Medicine","Epigenetics","DNA modification","Chromatin modification","Chromosome biology","Research Article","570","Cell biology","Forms of DNA","Science","610","DNA, Mitochondrial","Animals","Humans","Sulfites","Molecular Biology Techniques","Shotgun Sequencing","Biology and life sciences","Organic Chemistry","Chemical Compounds","Sequence Analysis, DNA","Pyrimidines","CpG Islands","Cloning"],"sdg_mappings":[{"sdg_number":0,"sdg_label":"Clean water and sanitation"}],"linked_datasets":[{"doi":"10.6084/m9.figshare.23884430.v1","title":"Additional file 1 of Optimized bisulfite sequencing analysis reveals the lack of 5-methylcytosine in mammalian mitochondrial DNA","publisher":"figshare","resource_type":"JournalArticle"},{"doi":"10.6084/m9.figshare.23884430","title":"Additional file 1 of Optimized bisulfite sequencing analysis reveals the lack of 5-methylcytosine in mammalian mitochondrial DNA","publisher":"figshare","resource_type":"JournalArticle"}],"clinical_trials":[],"software_tools":[],"database_accessions":[],"source":"live","citation_network_status":"fetched"},"created_at":"2026-07-23T09:06:18.649781Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}