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Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine

Nature Protocols volume 8, pages 18411851 (2013) | Download Citation


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.

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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


  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


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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.

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.

Corresponding author

Correspondence to Shankar Balasubramanian.

Supplementary information

PDF files

  1. 1.

    Supplementary Figure 1

    Identification via sequencing of uracil resulting from reacting 5fC from different sources with bisulfite.

  2. 2.

    Supplementary Figure 2

    HPLC chromatogram of a digested, 5hmC-containing oligonucleotide before and after oxidation.

  3. 3.

    Supplementary Figure 3

    Color changes of the oxidant solution.

  4. 4.

    Supplementary Figure 4

    Agilent TapeStation analysis of 100mer digestion control with and without Taq1 digestion.

  5. 5.

    Supplementary Table 1

    Fisher's test of varying sequence coverage.

  6. 6.

    Supplementary Table 2

    Sequences of control DNA oligonucleotides.

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