{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T19:25:45Z","timestamp":1791487545061,"version":"4.3.4"},"reference-count":54,"publisher":"Public Library of Science (PLoS)","issue":"8","license":[{"start":{"date-parts":[[2025,8,5]],"date-time":"2025-08-05T00:00:00Z","timestamp":1754352000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100010724","name":"Health Systems Research Institute","doi-asserted-by":"crossref","award":["67-095"],"award-info":[{"award-number":["67-095"]}],"id":[{"id":"10.13039\/501100010724","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:p>DNA methylation, a key epigenetic modification, regulates gene expression and diverse cellular functions. Bisulfite sequencing (BS) remains the gold standard for methylation detection, while PacBio HiFi sequencing enables direct detection without chemical conversion. Although both technologies are increasingly used, few studies have directly compared their concordance, particularly in clinically relevant settings such as Down syndrome (DS). We performed a comparative analysis of DNA methylation profiles using whole-genome bisulfite sequencing (WGBS) and PacBio high-fidelity (HiFi) whole-genome sequencing (WGS) in a pair of monozygotic twins with DS. WGBS data were processed with two pipelines, wg-blimp and Bismark, while HiFi WGS data were analyzed using pb-CpG-tools. Our analysis focused on four key aspects: CpG site detection, genomic distribution of methylated CpGs (mCs), average methylation levels, and inter-platform concordance. HiFi WGS detected a greater number of mCs\u2014particularly in repetitive elements and regions with low WGBS coverage\u2014while WGBS reported higher average methylation levels than HiFi WGS. Both platforms exhibited methylation patterns consistent with known biological principles, such as low methylation in CpG islands, and the relative methylation patterns across genomic features were largely concordant. Pearson correlation coefficients indicated strong agreement between platforms (r\u2009\u2248\u20090.8), with higher concordance in GC-rich regions and at increased sequencing depths. Depth-matched comparisons and site-level down-sampling revealed that methylation concordance improves with increasing coverage, with stronger agreement observed beyond 20\u2009\u00d7\u2009. Our findings support the reliability of HiFi WGS for methylation detection and highlight its advantages in regions that are challenging for bisulfite-based methods. This study demonstrates that HiFi WGS can serve as a robust alternative for genome-wide methylation profiling.<\/jats:p>","DOI":"10.1371\/journal.pone.0329593","type":"journal-article","created":{"date-parts":[[2025,8,5]],"date-time":"2025-08-05T17:24:26Z","timestamp":1754414666000},"page":"e0329593","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":3,"title":["A comparison of DNA methylation detection between HiFi sequencing and whole genome bisulfite sequencing in monozygotic twins with Down syndrome"],"prefix":"10.1371","volume":"20","author":[{"given":"Kanyanee","family":"Promsawan","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chalurmpon","family":"Srichomthong","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3228-3351","authenticated-orcid":true,"given":"Monnat","family":"Pongpanich","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1856-0589","authenticated-orcid":true,"given":"Vorasuk","family":"Shotelersuk","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2025,8,5]]},"reference":[{"issue":"1","key":"pone.0329593.ref001","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1038\/npp.2012.112","article-title":"DNA methylation and its basic function","volume":"38","author":"LD Moore","year":"2013","journal-title":"Neuropsychopharmacology"},{"key":"pone.0329593.ref002","first-page":"127","article-title":"Chapter 2-1 - The Role of DNA Methylation in Gene Regulation.","volume-title":"Toxicoepigenetics","author":"PA Bommarito","year":"2019"},{"issue":"8","key":"pone.0329593.ref003","doi-asserted-by":"crossref","first-page":"597","DOI":"10.1038\/nrg1655","article-title":"DNA methylation and human disease","volume":"6","author":"KD Robertson","year":"2005","journal-title":"Nat Rev Genet"},{"issue":"4","key":"pone.0329593.ref004","article-title":"DNA methylation: an epigenetic mark of cellular memory","volume":"49","author":"M Kim","year":"2017","journal-title":"Exp Mol Med"},{"key":"pone.0329593.ref005","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1007\/978-1-61779-316-5_2","article-title":"DNA methylation detection: bisulfite genomic sequencing analysis","volume":"791","author":"Y Li","year":"2011","journal-title":"Methods Mol Biol"},{"issue":"1","key":"pone.0329593.ref006","doi-asserted-by":"crossref","first-page":"3438","DOI":"10.1038\/s41467-021-23778-6","article-title":"Systematic benchmarking of tools for CpG methylation detection from nanopore sequencing","volume":"12","author":"ZW-S Yuen","year":"2021","journal-title":"Nat Commun"},{"issue":"6","key":"pone.0329593.ref007","doi-asserted-by":"crossref","first-page":"461","DOI":"10.1038\/nmeth.1459","article-title":"Direct detection of DNA methylation during single-molecule, real-time sequencing","volume":"7","author":"BA Flusberg","year":"2010","journal-title":"Nat Methods"},{"key":"pone.0329593.ref008","unstructured":"Methylation Detection with PacBio HiFi Sequencing. 2021 [cited 2023 July 27]. https:\/\/www.pacb.com\/videos\/methylation-detection-with-pacbio-hifi-sequencing\/"},{"issue":"1","key":"pone.0329593.ref009","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1186\/s13059-024-03207-9","article-title":"A comparison of methods for detecting DNA methylation from long-read sequencing of human genomes","volume":"25","author":"BD Sigurpalsdottir","year":"2024","journal-title":"Genome Biol"},{"issue":"1","key":"pone.0329593.ref010","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1186\/s12929-015-0138-y","article-title":"Down syndrome: an insight of the disease","volume":"22","author":"A Asim","year":"2015","journal-title":"J Biomed Sci"},{"issue":"8","key":"pone.0329593.ref011","doi-asserted-by":"crossref","DOI":"10.3390\/genes12081115","article-title":"DNA Methylation Alterations in Blood Cells of Toddlers with Down Syndrome","volume":"12","author":"OY Naumova","year":"2021","journal-title":"Genes (Basel)"},{"issue":"10","key":"pone.0329593.ref012","doi-asserted-by":"crossref","first-page":"172","DOI":"10.1186\/gb-2012-13-10-172","article-title":"DNA methylation studies using twins: what are they telling us?","volume":"13","author":"JT Bell","year":"2012","journal-title":"Genome Biol"},{"issue":"7","key":"pone.0329593.ref013","doi-asserted-by":"crossref","first-page":"60","DOI":"10.1186\/s13073-014-0060-z","article-title":"Epigenetics of discordant monozygotic twins: implications for disease","volume":"6","author":"JE Castillo-Fernandez","year":"2014","journal-title":"Genome Med"},{"issue":"6","key":"pone.0329593.ref014","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0100191","article-title":"Whole genome and exome sequencing of monozygotic twins with trisomy 21, discordant for a congenital heart defect and epilepsy","volume":"9","author":"P Chaiyasap","year":"2014","journal-title":"PLoS One"},{"issue":"1","key":"pone.0329593.ref015","doi-asserted-by":"crossref","first-page":"169","DOI":"10.1186\/s12859-020-3470-5","article-title":"wg-blimp: an end-to-end analysis pipeline for whole genome bisulfite sequencing data","volume":"21","author":"M W\u00f6ste","year":"2020","journal-title":"BMC Bioinformatics"},{"key":"pone.0329593.ref016","unstructured":"Bwa-meth: Fast and accurate alignment of BS-Seq reads. n.d. [cited 2022 July 14]. https:\/\/github.com\/brentp\/bwa-meth"},{"key":"pone.0329593.ref017","unstructured":"picard: a set of command line tools for manipulating high-throughput sequencing data. n.d. [cited 14 July 2022]. https:\/\/github.com\/broadinstitute\/picard"},{"key":"pone.0329593.ref018","unstructured":"FastQC: A quality control tool for high throughput sequence data. n.d. [cited 14 July 2022]. https:\/\/www.bioinformatics.babraham.ac.uk\/projects\/fastqc\/"},{"issue":"2","key":"pone.0329593.ref019","doi-asserted-by":"crossref","first-page":"292","DOI":"10.1093\/bioinformatics\/btv566","article-title":"Qualimap 2: advanced multi-sample quality control for high-throughput sequencing data","volume":"32","author":"K Okonechnikov","year":"2016","journal-title":"Bioinformatics"},{"key":"pone.0329593.ref020","unstructured":"MethylDackel: A universal methylation extractor for BS-seq experiments. n.d. [cited 14 July 2022]. https:\/\/github.com\/dpryan79\/MethylDackel"},{"issue":"11","key":"pone.0329593.ref021","doi-asserted-by":"crossref","first-page":"1571","DOI":"10.1093\/bioinformatics\/btr167","article-title":"Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications","volume":"27","author":"F Krueger","year":"2011","journal-title":"Bioinformatics"},{"key":"pone.0329593.ref022","unstructured":"Zymo-Seq WGBS Library Kit: Bisulfite library preparation in one tube. 2023. https:\/\/www.zymoresearch.com\/products\/zymo-seq-wgbs-library-kit"},{"key":"pone.0329593.ref023","year":"2023"},{"key":"pone.0329593.ref024","unstructured":"pb-CpG-tools: Collection of tools for the analysis of CpG data. n.d. [cited 15 Sep 2023]. https:\/\/github.com\/PacificBiosciences\/pb-CpG-tools"},{"key":"pone.0329593.ref025","unstructured":"CSS: Generate Highly Accurate Single-Molecule Consensus Reads. n.d. [cited 1 Sep 2023]. https:\/\/ccs.how\/"},{"key":"pone.0329593.ref026","unstructured":"LongQC: a tool for the data quality control of the PacBio and ONT long reads. n.d. [cited 4 Apr 2023]. https:\/\/github.com\/yfukasawa\/LongQC"},{"key":"pone.0329593.ref027","unstructured":"Jasmine: Predict 5mC in PacBio HiFi reads. n.d. [updated 1 Sep 2023]. https:\/\/github.com\/PacificBiosciences\/jasmine"},{"key":"pone.0329593.ref028","unstructured":"pbmm2: A minimap2 SMRT wrapper for PacBio data. n.d. [cited 2 Sep 2023]. https:\/\/github.com\/PacificBiosciences\/pbmm2"},{"issue":"16","key":"pone.0329593.ref029","doi-asserted-by":"crossref","first-page":"2078","DOI":"10.1093\/bioinformatics\/btp352","article-title":"The Sequence Alignment\/Map format and SAMtools","volume":"25","author":"H Li","year":"2009","journal-title":"Bioinformatics"},{"issue":"8","key":"pone.0329593.ref030","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pcbi.1003118","article-title":"Software for computing and annotating genomic ranges","volume":"9","author":"M Lawrence","year":"2013","journal-title":"PLoS Comput Biol"},{"key":"pone.0329593.ref031","doi-asserted-by":"crossref","DOI":"10.1007\/978-0-387-98141-3","volume-title":"ggplot2: Elegant Graphics for Data Analysis","author":"H Wickham","year":"2009"},{"issue":"2","key":"pone.0329593.ref032","doi-asserted-by":"crossref","first-page":"289","DOI":"10.1007\/s00438-020-01729-y","article-title":"Uncovering patterns of the evolution of genomic sequence entropy and complexity","volume":"296","author":"RP Sim\u00f5es","year":"2021","journal-title":"Mol Genet Genomics"},{"key":"pone.0329593.ref033","doi-asserted-by":"crossref","first-page":"341","DOI":"10.1016\/B978-0-12-809633-8.20111-8","article-title":"Epigenetics: Analysis of Cytosine Modifications at Single Base Resolution.","volume-title":"Encyclopedia of Bioinformatics and Computational Biology","author":"M Prater","year":"2019"},{"key":"pone.0329593.ref034","doi-asserted-by":"crossref","first-page":"618955","DOI":"10.3389\/fgene.2021.618955","article-title":"Bisulfite-Converted DNA Quantity Evaluation: A Multiplex Quantitative Real-Time PCR System for Evaluation of Bisulfite Conversion","volume":"12","author":"SR Hong","year":"2021","journal-title":"Front Genet"},{"key":"pone.0329593.ref035","article-title":"Epigenetic conservation at gene regulatory elements revealed by non-methylated DNA profiling in seven vertebrates","volume":"2","author":"HK Long","year":"2013","journal-title":"Elife"},{"issue":"5","key":"pone.0329593.ref036","doi-asserted-by":"crossref","first-page":"543","DOI":"10.1016\/j.celrep.2012.03.013","article-title":"Differential GC content between exons and introns establishes distinct strategies of splice-site recognition","volume":"1","author":"M Amit","year":"2012","journal-title":"Cell Rep"},{"issue":"10","key":"pone.0329593.ref037","doi-asserted-by":"crossref","DOI":"10.1186\/gb-2002-3-10-reports0058","article-title":"Evolution of genomic GC variation","volume":"3","author":"DP Genereux","year":"2002","journal-title":"Genome Biology"},{"issue":"4","key":"pone.0329593.ref038","doi-asserted-by":"crossref","first-page":"973","DOI":"10.1534\/g3.115.025437","article-title":"Mapping of variable DNA methylation across multiple cell types defines a dynamic regulatory landscape of the human genome","volume":"6","author":"J Gu","year":"2016","journal-title":"G3 Genes|Genomes|Genetics"},{"issue":"10","key":"pone.0329593.ref039","doi-asserted-by":"crossref","first-page":"1321","DOI":"10.7150\/ijbs.26605","article-title":"Genome-wide Identification and Characterization of Enhancers Across 10 Human Tissues","volume":"14","author":"L Xiong","year":"2018","journal-title":"Int J Biol Sci"},{"issue":"1","key":"pone.0329593.ref040","doi-asserted-by":"crossref","first-page":"536","DOI":"10.1186\/s12864-017-3934-9","article-title":"Short DNA sequence patterns accurately identify broadly active human enhancers","volume":"18","author":"LL Colbran","year":"2017","journal-title":"BMC Genomics"},{"issue":"5","key":"pone.0329593.ref041","doi-asserted-by":"crossref","first-page":"555","DOI":"10.1016\/j.cell.2004.08.011","article-title":"Chromatin architecture of the human genome: gene-rich domains are enriched in open chromatin fibers","volume":"118","author":"N Gilbert","year":"2004","journal-title":"Cell"},{"issue":"10","key":"pone.0329593.ref042","doi-asserted-by":"crossref","first-page":"1606","DOI":"10.1038\/s41587-023-02057-3","article-title":"Characterization and visualization of tandem repeats at genome scale","volume":"42","author":"E Dolzhenko","year":"2024","journal-title":"Nat Biotechnol"},{"key":"pone.0329593.ref043","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1007\/978-981-13-6037-4_7","article-title":"Bionano Genome Mapping: High-Throughput, Ultra-Long Molecule Genome Analysis System for Precision Genome Assembly and Haploid-Resolved Structural Variation Discovery","volume":"1129","author":"S Bocklandt","year":"2019","journal-title":"Adv Exp Med Biol"},{"issue":"9","key":"pone.0329593.ref044","doi-asserted-by":"crossref","DOI":"10.3390\/ijms23094755","article-title":"On the base composition of transposable elements","volume":"23","author":"S Boissinot","year":"2022","journal-title":"Int J Mol Sci"},{"issue":"6","key":"pone.0329593.ref045","doi-asserted-by":"crossref","first-page":"655","DOI":"10.2217\/epi.12.55","article-title":"CG methylation","volume":"4","author":"C Vinson","year":"2012","journal-title":"Epigenomics"},{"issue":"9","key":"pone.0329593.ref046","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0239196","article-title":"Lower DNA methylation levels in CpG island shores of CR1, CLU, and PICALM in the blood of Japanese Alzheimer\u2019s disease patients","volume":"15","author":"R Mitsumori","year":"2020","journal-title":"PLoS One"},{"issue":"1","key":"pone.0329593.ref047","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1186\/s13148-019-0803-1","article-title":"DNA methylation changes in Down syndrome derived neural iPSCs uncover co-dysregulation of ZNF and HOX3 families of transcription factors","volume":"12","author":"L Laan","year":"2020","journal-title":"Clin Epigenetics"},{"issue":"3","key":"pone.0329593.ref048","doi-asserted-by":"crossref","first-page":"259","DOI":"10.1007\/s13237-021-00367-y","article-title":"DNA methylation and regulation of gene expression: Guardian of our health","volume":"64","author":"GA Dhar","year":"2021","journal-title":"Nucleus (Calcutta)"},{"issue":"4","key":"pone.0329593.ref049","doi-asserted-by":"crossref","first-page":"462","DOI":"10.18632\/oncotarget.497","article-title":"On the presence and role of human gene-body DNA methylation","volume":"3","author":"D Jjingo","year":"2012","journal-title":"Oncotarget"},{"issue":"7","key":"pone.0329593.ref050","doi-asserted-by":"crossref","first-page":"484","DOI":"10.1038\/nrg3230","article-title":"Functions of DNA methylation: islands, start sites, gene bodies and beyond","volume":"13","author":"PA Jones","year":"2012","journal-title":"Nat Rev 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