Low Input Whole-Genome Bisulfite Sequencing Using a Post-Bisulfite Adapter Tagging Approach

  • Julian R. Peat
  • Sébastien A. SmallwoodEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1708)


The epigenetic mark 5-methylcytosine confers heritable regulation of gene expression that can be dynamically modulated during transitions in cell fate. With the development of high-throughput sequencing technologies, it is now possible to obtain comprehensive genome-wide maps of the mammalian DNA methylation landscape, but the application of these techniques to limited material remains challenging. Here, we present an optimized protocol to perform whole-genome bisulfite sequencing on low inputs (100–5000 somatic cells) using a post-bisulfite adapter tagging approach. In this strategy, bisulfite treatment is performed prior to library generation in order to both convert unmethylated cytosines and fragment DNA to an appropriate size. Then sequencing adapters are added by complementary strand synthesis using random tetramer priming, and libraries are subsequently amplified by PCR.

Key words

DNA methylation High-throughput sequencing Bisulfite sequencing Low input Epigenetics 


  1. 1.
    Smith ZD, Meissner A (2013) DNA methylation: roles inmammalian development. Nat Rev Genet 14:204–220CrossRefPubMedGoogle Scholar
  2. 2.
    Ferguson-Smith AC (2011) Genomic imprinting: the emergence of an epigenetic paradigm. Nat Rev Genet 12:565–575CrossRefPubMedGoogle Scholar
  3. 3.
    Smallwood SA, Kelsey G (2012) De novo DNA methylation: a germ cell perspective. Trends Genet 28:33–42CrossRefPubMedGoogle Scholar
  4. 4.
    Seisenberger S, Peat JR, Hore TA et al (2012) Reprogramming DNA methylation in the mammalian life cycle: building and breaking epigenetic barriers. Philos Trans R Soc Lond Ser B Biol Sci 368:20110330CrossRefGoogle Scholar
  5. 5.
    Cedar H, Bergman Y (2012) Programming of DNA methylation patterns. Annu Rev Biochem 81:97–117CrossRefPubMedGoogle Scholar
  6. 6.
    Weber M, Davies JJ, Wittig D et al (2005) Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat Genet 37:853–862CrossRefPubMedGoogle Scholar
  7. 7.
    Harris RA, Wang T, Coarfa C et al (2010) Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications. Nat Biotechnol 28:1097–1105CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Smallwood SA, Tomizawa S-I, Krueger F et al (2011) Dynamic CpG island methylation landscape in oocytes and preimplantation embryos. Nat Genet 43:811–814CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Smallwood SA, Kelsey G (2012) Genome-wide analysis of DNA methylation in low cell numbers by reduced representation bisulfite sequencing. In: Genomic imprinting. Humana, Totowa, NJ, pp 187–197CrossRefGoogle Scholar
  10. 10.
    Miura F, Enomoto Y, Dairiki R et al (2012) Amplification-free whole-genome bisulfite sequencing by post-bisulfite adaptor tagging. Nucleic Acids Res 40:e136CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Shirane K, Toh H, Kobayashi H et al (2013) Mouse oocyte methylomes at base resolution reveal genome-wide accumulation of non-CpG methylation and role of DNA methyltransferases. PLoS Genet 9:e1003439CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Kobayashi H, Sakurai T, Imai M et al (2012) Contribution of intragenic DNA methylation in mouse gametic DNA methylomes to establish oocyte-specific heritable marks. PLoS Genet 8:e1002440CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Peat JR, Dean W, Clark SJ et al (2014) Genome-wide bisulfite sequencing in zygotes identifies demethylation targets and maps the contribution of TET3 oxidation. Cell Rep 9(6):1990–2000CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Stewart KR, Veselovska L, Kim J et al (2015) Dynamic changes in histone modifications precede de novo DNA methylation in oocytes. Genes Dev 29(23):2449–2462CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Quail MA, Otto TD, Gu Y et al (2012) Optimal enzymes for amplifying sequencing libraries. Nat Methods 9:10–11CrossRefGoogle Scholar
  16. 16.
    Krueger F, Andrews SR (2011) Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics 27:1571–1572CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.Epigenetics ProgrammeBabraham InstituteCambridgeUK
  2. 2.Friedrich Miescher Institute for Biomedical ResearchBaselSwitzerland

Personalised recommendations