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Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine

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.

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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.

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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

Authors

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)

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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

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

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