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Epigenome Editing pp 311–349Cite as

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

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1767))

Abstract

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.

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Acknowledgments

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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Author notes

  1. Magali Kernaleguen and Christian Daviaud have contributed equally and should be considered as joint first authors.

Authors and Affiliations

  1. Laboratory for Epigenetics and Environment, Centre National de Recherche en Génomique Humaine, CEA-Institut de Biologie Francois Jacob, Evry, France

    Magali Kernaleguen, Christian Daviaud, Yimin Shen, Jean-François Deleuze, Florence Mauger & Jörg Tost

  2. Laboratory for Bio-analysis, Centre National de Recherche en Génomique Humaine, CEA-Institut de Biologie Francois Jacob, Evry, France

    Eric Bonnet

  3. Laboratory for Bioinformatics, Fondation Jean Dausset – CEPH, Paris, France

    Victor Renault & Jean-François Deleuze

Authors
  1. Magali Kernaleguen
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  2. Christian Daviaud
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  3. Yimin Shen
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  4. Eric Bonnet
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  5. Victor Renault
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  6. Jean-François Deleuze
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  7. Florence Mauger
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  8. Jörg Tost
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Corresponding author

Correspondence to Jörg Tost .

Editor information

Editors and Affiliations

  1. Department of Biochemistry, Institute of Biochemistry and Technical Biochemistry, Stuttgart, Germany

    Albert Jeltsch

  2. Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands

    Marianne G. Rots

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Kernaleguen, M. et al. (2018). Whole-Genome Bisulfite Sequencing for the Analysis of Genome-Wide DNA Methylation and Hydroxymethylation Patterns at Single-Nucleotide Resolution. In: Jeltsch, A., Rots, M. (eds) Epigenome Editing. Methods in Molecular Biology, vol 1767. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7774-1_18

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  • DOI: https://doi.org/10.1007/978-1-4939-7774-1_18

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7773-4

  • Online ISBN: 978-1-4939-7774-1

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