Whole-Genome Bisulfite Sequencing for the Analysis of Genome-Wide DNA Methylation and Hydroxymethylation Patterns at Single-Nucleotide Resolution

  • Magali Kernaleguen
  • Christian Daviaud
  • Yimin Shen
  • Eric Bonnet
  • Victor Renault
  • Jean-François Deleuze
  • Florence Mauger
  • Jörg TostEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1767)


The analysis of genome-wide epigenomic alterations including DNA methylation and hydroxymethylation has become a subject of intensive research for many biological and disease-associated investigations. Whole-genome bisulfite sequencing (WGBS) using next-generation sequencing technologies is currently considered as the gold standard for a comprehensive and quantitative analysis of DNA methylation throughout the genome. However, bisulfite conversion does not allow distinguishing between cytosine methylation and hydroxymethylation requiring an additional chemical or enzymatic step to identify hydroxymethylated cytosines. Here we provide two detailed protocols based on commercial kits for the preparation of sequencing libraries for the comprehensive whole-genome analysis of DNA methylation and/or hydroxymethylation. If only DNA methylation is of interest, sequencing libraries can be constructed from limited amounts of input DNA by ligation of methylated adaptors to the fragmented DNA prior to bisulfite conversion. For samples with significant levels of hydroxymethylation such as stem cells or brain tissue, we describe the protocol of oxidative bisulfite sequencing (OxBs-seq), which in its current version uses a post-bisulfite adaptor tagging (PBAT) approach. Two methylomes need to be generated: a classic methylome following bisulfite conversion and analyzing both methylated and hydroxymethylated cytosines and a methylome analyzing only methylated cytosines, respectively. We also provide a step-by-step description of the data analysis using publicly available bioinformatic tools. The described protocols have been successfully applied to different human samples and yield robust and reproducible results.


Whole-genome bisulfite sequencing DNA methylation Hydroxymethylation Bisulfite conversion Low input Spike-in Oxidative bisulfite sequencing PBAT Ovation® Methyl-seq TrueMethyl® WholeGenome 



The protocol for hydroxymethylation analysis has been set up in the laboratory of Jörg Tost in the framework of the ANR-BMBF-funded project “Epigenomics of Parkinson’s Disease” (EpiPD, ANR-13-EPIG-0003-05). Further work is supported by grants from the ANR (ANR-13-CESA-0011-05), Aviesan/INSERM (EPlGl2014-18 and EPIG2014-01), INCa (PRT-K14-049), a Sirius research award (UCB Pharma S.A.), a Passerelle research award (Pfizer), iCARE (MSD Avenir), and the institutional budget of the CNRGH.


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Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Magali Kernaleguen
    • 1
  • Christian Daviaud
    • 1
  • Yimin Shen
    • 1
  • Eric Bonnet
    • 2
  • Victor Renault
    • 3
  • Jean-François Deleuze
    • 1
    • 3
  • Florence Mauger
    • 1
  • Jörg Tost
    • 1
    Email author
  1. 1.Laboratory for Epigenetics and Environment, Centre National de Recherche en Génomique HumaineCEA-Institut de Biologie Francois JacobEvryFrance
  2. 2.Laboratory for Bio-analysis, Centre National de Recherche en Génomique HumaineCEA-Institut de Biologie Francois JacobEvryFrance
  3. 3.Laboratory for BioinformaticsFondation Jean Dausset – CEPHParisFrance

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