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Whole-Genome Bisulfite Sequencing Using the Ovation® Ultralow Methyl-Seq Protocol

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DNA Methylation Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1708))

Abstract

The analysis of genome-wide epigenomic alterations including DNA methylation has become a subject of intensive research for many complex diseases. Whole-genome bisulfite sequencing (WGBS) using next-generation sequencing technologies can be considered the gold standard for a comprehensive and quantitative analysis of cytosine methylation throughout the genome. Several approaches including tagmentation- and post bisulfite adaptor tagging (PBAT)-based WGBS have been devised. Here, we provide a detailed protocol based on a commercial kit for the preparation of libraries for WGBS from limited amounts of input DNA (50–100 ng) using the classical approach of WGBS by ligation of methylated adaptors to the fragmented DNA prior to bisulfite conversion. The converted library is then amplified with an optimal number of PCR cycles to ensure high sequence diversity and low duplicate rates. Spike-in of unmethylated DNA allows for the precise estimation of bisulfite conversion rates. We also provide a step-by-step description of the data analysis using publicly available bioinformatic tools. The described protocol has been successfully applied to different human samples as well as DNA extracted from plant tissues and yields robust and reproducible results.

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Acknowledgments

We thank Benjamin G Schroeder (NuGEN, San Carlos, CA) for help with setting up the technology in the laboratory and Doug Amorese (NuGEN, San Carlos, CA) and Steven McGinn (CNRGH) for the critical reading of the manuscript. Work in the laboratory of Jörg Tost is supported by grants from the ANR (ANR-13-EPIG-0003-05 and ANR-13-CESA-0011-05), Aviesan/INSERM (EPlGl2014-18 and EPIG2014-01), INCa (PRT-K14-049) and the joint CEA-EDF-IRSN program (CP-PHE-102).

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Authors and Affiliations

  1. Laboratory for Epigenetics and Environment, Centre National de Recherche en Génomique Humaine, CEA-Institut de Biologie Francois Jacob, Bâtiment G2, 2 rue Gaston Crémieux, 91000, Evry, France

    Christian Daviaud, Florence Mauger, Jean-François Deleuze & Jörg Tost

  2. Laboratory for Bioinformatics, Fondation Jean Dausset – CEPH, 27 rue Juliette Dodu, Paris, 75010, France

    Victor Renault & Jean-François Deleuze

Authors
  1. Christian Daviaud
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  2. Victor Renault
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  3. Florence Mauger
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  4. Jean-François Deleuze
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  5. Jörg Tost
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Corresponding author

Correspondence to Jörg Tost .

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Editors and Affiliations

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

    Jörg Tost

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Daviaud, C., Renault, V., Mauger, F., Deleuze, JF., Tost, J. (2018). Whole-Genome Bisulfite Sequencing Using the Ovation® Ultralow Methyl-Seq Protocol. In: Tost, J. (eds) DNA Methylation Protocols. Methods in Molecular Biology, vol 1708. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7481-8_5

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  • DOI: https://doi.org/10.1007/978-1-4939-7481-8_5

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7479-5

  • Online ISBN: 978-1-4939-7481-8

  • eBook Packages: Springer Protocols

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