Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine

Nature Protocols volume 8, pages 1841–1851 (2013)Cite this article

Subjects

Abstract

To uncover the function of and interplay between the mammalian cytosine modifications 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), new techniques and advances in current technology are needed. To this end, we have developed oxidative bisulfite sequencing (oxBS-seq), which can quantitatively locate 5mC and 5hmC marks at single-base resolution in genomic DNA. In bisulfite sequencing (BS-seq), both 5mC and 5hmC are read as cytosines and thus cannot be discriminated; however, in oxBS-seq, specific oxidation of 5hmC to 5-formylcytosine (5fC) and conversion of the newly formed 5fC to uracil (under bisulfite conditions) means that 5hmC can be discriminated from 5mC. A positive readout of actual 5mC is gained from a single oxBS-seq run, and 5hmC levels are inferred by comparison with a BS-seq run. Here we describe an optimized second-generation protocol that can be completed in 2 d.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

Buy

All prices are NET prices.

Figure 1: Structures of 5mC, 5hmC, 5fC and 5caC.
Figure 2: After bisulfite treatment of DNA, C reads as T, whereas 5mC and 5hmC read as C.
Figure 3: Workflow of oxBS-seq.
Figure 4: Efficacy of PCR amplification.

References

  1. Deaton, A.M. & Bird, A. CpG islands and the regulation of transcription. Genes Dev. 25, 1010–1022 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Jones, P.A. Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat. Rev. Genet. 13, 484–492 (2012).

    CAS  PubMed  Google Scholar 

  3. Feinberg, A.P. & Tycko, B. The history of cancer epigenetics. Nat. Rev. Cancer 4, 143–153 (2004).

    Article  CAS  PubMed  Google Scholar 

  4. Kriaucionis, S. & Heintz, N. The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science 324, 929–930 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Tahiliani, M. et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324, 930–935 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Ito, S. et al. Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science 333, 1300–1303 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Globisch, D. et al. Tissue distribution of 5-hydroxymethylcytosine and search for active demethylation intermediates. PLoS ONE 5, e15367 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Munzel, M. et al. Quantification of the sixth DNA base hydroxymethylcytosine in the brain. Angew. Chem. Int. Ed. 49, 5375–5377 (2010).

    Article  Google Scholar 

  9. Branco, M.R., Ficz, G. & Reik, W. Uncovering the role of 5-hydroxymethylcytosine in the epigenome. Nat. Rev. Genet. 13, 7–13 (2012).

    Article  CAS  Google Scholar 

  10. Pfaffeneder, T. et al. The discovery of 5-formylcytosine in embryonic stem cell DNA. Angew. Chem. Int. Ed. Engl. 50, 7008–7012 (2011).

    Article  CAS  PubMed  Google Scholar 

  11. He, Y.F. et al. Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science 333, 1303–1307 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Maiti, A. & Drohat, A.C. Thymine DNA glycosylase can rapidly excise 5-formylcytosine and 5-carboxylcytosine: potential implications for active demethylation of CpG sites. J. Biol. Chem. 286, 35334–35338 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Zhang, L. et al. Thymine DNA glycosylase specifically recognizes 5-carboxylcytosine-modified DNA. Nat. Chem. Biol. 8, 328–330 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Booth, M.J. et al. Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution. Science 336, 934–937 (2012).

    Article  CAS  PubMed  Google Scholar 

  15. Frommer, M. et al. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc. Natl. Acad. Sci. USA 89, 1827–1831 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Hayatsu, H. & Shiragami, M. Reaction of bisulfite with the 5-hydroxymethyl group in pyrimidines and in phage DNAs. Biochemistry 18, 632–637 (1979).

    Article  CAS  PubMed  Google Scholar 

  17. Huang, Y. et al. The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing. PLoS ONE 5, e8888 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Meissner, A. et al. Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis. Nucleic Acids Res. 33, 5868–5877 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Yu, M. et al. Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome. Cell 149, 1368–1380 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Ficz, G. et al. Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation. Nature 473, 398–402 (2011).

    Article  CAS  PubMed  Google Scholar 

  21. Pastor, W.A. et al. Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells. Nature 473, 394–397 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Sun, Z. et al. High-resolution enzymatic mapping of genomic 5-hydroxymethylcytosine in mouse embryonic stem cells. Cell Rep. 3, 567–576 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Yu, M. et al. Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine. Nat. Protoc. 7, 2159–2170 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Flusberg, B.A. et al. Direct detection of DNA methylation during single-molecule, real-time sequencing. Nat. Methods 7, 461–465 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Song, C.X. et al. Sensitive and specific single-molecule sequencing of 5-hydroxymethylcytosine. Nat. Methods 9, 75–77 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Wallace, E.V. et al. Identification of epigenetic DNA modifications with a protein nanopore. Chem. Commun. 46, 8195–8197 (2010).

    Article  CAS  Google Scholar 

  27. Krueger, F. & Andrews, S.R. Bismark: a flexible aligner and methylation caller for bisulfite-seq applications. Bioinformatics 27, 1571–1572 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Quinlivan, E.P. & Gregory, J.F. III DNA digestion to deoxyribonucleoside: a simplified one-step procedure. Anal. Biochem. 373, 383–385 (2008).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank the UK Biotechnology and Biological Sciences Research Council for funding this work. S.B. and W.R. are recipients of Wellcome Trust Senior Investigator Awards. CEGX is a Syncona Partners–funded company.

Author information

Authors and Affiliations

  1. Department of Chemistry, University of Cambridge, Cambridge, UK

    Michael J Booth, Dario Beraldi, Neil M Bell & Shankar Balasubramanian

  2. Cambridge Epigenetix Limited, Cambridge, UK

    Tobias W B Ost

  3. Cancer Research UK, Cambridge Institute, Li Ka Shing Centre, Cambridge, UK

    Dario Beraldi & Shankar Balasubramanian

  4. Epigenetics Programme, Babraham Institute, Cambridge, UK

    Miguel R Branco & Wolf Reik

  5. Centre for Trophoblast Research, University of Cambridge, Cambridge, UK

    Miguel R Branco & Wolf Reik

  6. The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, UK

    Wolf Reik

Authors
  1. Michael J Booth
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tobias W B Ost
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dario Beraldi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Neil M Bell
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Miguel R Branco
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wolf Reik
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Shankar Balasubramanian
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.J.B. and S.B. invented the oxBS-seq method. M.J.B. and T.W.B.O. designed the experiments with contributions from all authors. M.J.B., T.W.B.O. and N.M.B. prepared samples and conducted experimental work. D.B. performed data analysis, interpretation and simulations. M.J.B. and T.W.B.O. drafted the manuscript and all authors contributed to writing, reviewing and editing the manuscript.

Corresponding author

Correspondence to Shankar Balasubramanian.

Ethics declarations

Competing interests

T.W.B.O. is an employee of Cambridge Epigenetix, Ltd. M.J.B., N.M.B., D.B. and W.R. are consultants for Cambridge Epigenetix, Ltd. M.J.B., T.W.B.O., M.R.B., W.R. and S.B. are shareholders in Cambridge Epigenetix, Ltd. S.B. is an advisor to and shareholder of Illumina, Inc.

Supplementary information

Supplementary Figure 1

Identification via sequencing of uracil resulting from reacting 5fC from different sources with bisulfite. (PDF 461 kb)

Supplementary Figure 2

HPLC chromatogram of a digested, 5hmC-containing oligonucleotide before and after oxidation. (PDF 386 kb)

Supplementary Figure 3

Color changes of the oxidant solution. (PDF 270 kb)

Supplementary Figure 4

Agilent TapeStation analysis of 100mer digestion control with and without Taq1 digestion. (PDF 117 kb)

Supplementary Table 1

Fisher's test of varying sequence coverage. (PDF 168 kb)

Supplementary Table 2

Sequences of control DNA oligonucleotides. (PDF 357 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Booth, M., Ost, T., Beraldi, D. et al. Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine. Nat Protoc 8, 1841–1851 (2013). https://doi.org/10.1038/nprot.2013.115

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2013.115

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing