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

  • Christian Daviaud
  • Victor Renault
  • Florence Mauger
  • Jean-François Deleuze
  • Jörg Tost
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

Whole-genome bisulfite sequencing DNA methylation Data analysis Bisulfite conversion Low-input Spike-in 

Notes

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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Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Christian Daviaud
    • 1
  • Victor Renault
    • 2
  • Florence Mauger
    • 1
  • Jean-François Deleuze
    • 1
    • 2
  • Jörg Tost
    • 1
  1. 1.Laboratory for Epigenetics and Environment, Centre National de Recherche en Génomique HumaineCEA-Institut de Biologie Francois JacobEvryFrance
  2. 2.Laboratory for BioinformaticsFondation Jean Dausset – CEPHParisFrance

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