Assessing Distribution and Variation of Genome-Wide DNA Methylation Using Short-Read Sequencing

  • Jörg Hagmann
  • Claude Becker
Part of the Methods in Molecular Biology book series (MIMB, volume 1610)


DNA methylation is one of the most prominent epigenetic marks and is particularly complex in plant genomes. Whole-genome bisulfite sequencing (WGBS) using short reads has become the standard tool to study genome-wide patterns of DNA methylation. The goal of the present protocol is to enable readers to perform WGBS on both the wet lab and the computational side. We briefly outline important steps in bisulfite library preparation, then focus on the different aspects of DNA methylation analysis, from read mapping to identifying biologically relevant differential methylation between different samples.

Key words

DNA methylation Short-read sequencing Bisulfite Differential methylation Differentially methylated region 



This work was supported by the Max Planck Society.


  1. 1.
    Pikaard CS, Mittelsten Scheid O (2014) Epigenetic regulation in plants. Cold Spring Harb Perspect Biol 6(12):a019315. doi: 10.1101/cshperspect.a019315 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Zhang X, Yazaki J, Sundaresan A, Cokus S, Chan SW, Chen H, Henderson IR, Shinn P, Pellegrini M, Jacobsen SE, Ecker JR (2006) Genome-wide high-resolution mapping and functional analysis of DNA methylation in arabidopsis. Cell 126(6):1189–1201. doi: 10.1016/j.cell.2006.08.003 CrossRefPubMedGoogle Scholar
  3. 3.
    Zilberman D, Gehring M, Tran RK, Ballinger T, Henikoff S (2007) Genome-wide analysis of Arabidopsis thaliana DNA methylation uncovers an interdependence between methylation and transcription. Nat Genet 39(1):61–69. doi: 10.1038/ng1929 CrossRefPubMedGoogle Scholar
  4. 4.
    Frommer M, McDonald LE, Millar DS, Collis CM, Watt F, Grigg GW, Molloy PL, Paul CL (1992) A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci U S A 89(5):1827–1831CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Cokus SJ, Feng SH, Zhang XY, Chen ZG, Merriman B, Haudenschild CD, Pradhan S, Nelson SF, Pellegrini M, Jacobsen SE (2008) Shotgun bisulfite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452(7184):215–219. doi: 10.1038/nature06745 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Lister R, O'Malley RC, Tonti-Filippini J, Gregory BD, Berry CC, Millar AH, Ecker JR (2008) Highly integrated single-base resolution maps of the epigenome in Arabidopsis. Cell 133(3):523–536. doi: 10.1016/j.cell.2008.03.029 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Robinson MD, Kahraman A, Law CW, Lindsay H, Nowicka M, Weber LM, Zhou X (2014) Statistical methods for detecting differentially methylated loci and regions. Front Genet 5:324. doi: 10.3389/fgene.2014.00324 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Lister R, Pelizzola M, Dowen RH, Hawkins RD, Hon G, Tonti-Filippini J, Nery JR, Lee L, Ye Z, Ngo QM, Edsall L, Antosiewicz-Bourget J, Stewart R, Ruotti V, Millar AH, Thomson JA, Ren B, Ecker JR (2009) Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 462(7271):315–322. doi: 10.1038/nature08514 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Becker C, Hagmann J, Muller J, Koenig D, Stegle O, Borgwardt K, Weigel D (2011) Spontaneous epigenetic variation in the Arabidopsis thaliana methylome. Nature 480(7376):245–249. doi: 10.1038/nature10555 CrossRefPubMedGoogle Scholar
  10. 10.
    Hagmann J, Becker C, Muller J, Stegle O, Meyer RC, Wang G, Schneeberger K, Fitz J, Altmann T, Bergelson J, Borgwardt K, Weigel D (2015) Century-scale methylome stability in a recently diverged Arabidopsis thaliana lineage. PLoS Genet 11(1):e1004920. doi: 10.1371/journal.pgen.1004920 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    van Gurp TP, Wagemaker NC, Wouters B, Vergeer P, Ouborg JN, Verhoeven KJ (2016) epiGBS: reference-free reduced representation bisulfite sequencing. Nat Methods 13(4):322–324. doi: 10.1038/nmeth.3763 CrossRefPubMedGoogle Scholar
  12. 12.
    Molaro A, Hodges E, Fang F, Song Q, McCombie WR, Hannon GJ, Smith AD (2011) Sperm methylation profiles reveal features of epigenetic inheritance and evolution in primates. Cell 146(6):1029–1041. doi: 10.1016/j.cell.2011.08.016 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Storey JD, Tibshirani R (2003) Statistical significance for genomewide studies. Proc Natl Acad Sci U S A 100(16):9440–9445. doi: 10.1073/pnas.1530509100 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Computomics GmbHTübingenGermany
  2. 2.Department of Molecular BiologyMax Planck Institute for Developmental BiologyTübingenGermany
  3. 3.Gregor Mendel InstituteAustrian Academy of Sciences, Vienna Biocenter (VBC)ViennaAustria

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