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Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution


An Addendum to this article was published on 21 May 2014

This article has been updated


Recently, the cytosine modifications 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) were found to exist in the genomic deoxyribonucleic acid (DNA) of a wide range of mammalian cell types. It is now important to understand their role in normal biological function and disease. Here we introduce reduced bisulfite sequencing (redBS-Seq), a quantitative method to decode 5fC in DNA at single-base resolution, based on a selective chemical reduction of 5fC to 5hmC followed by bisulfite treatment. After extensive validation on synthetic and genomic DNA, we combined redBS-Seq and oxidative bisulfite sequencing (oxBS-Seq) to generate the first combined genomic map of 5-methylcytosine, 5hmC and 5fC in mouse embryonic stem cells. Our experiments revealed that in certain genomic locations 5fC is present at comparable levels to 5hmC and 5mC. The combination of these chemical methods can quantify and precisely map these three cytosine derivatives in the genome and will help provide insights into their function.

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Figure 1: Outline of reduced bisulfite sequencing to map 5fC in DNA.
Figure 2: Validation of redBS-Seq methodology on synthetic DNA.
Figure 3: Outline of BS-Seq, oxBS-Seq and redBS-Seq and internal control DNA conversion rates.
Figure 4: Quantification, asymmetry and interplay of 5mC, 5hmC and 5fC in mES cells.
Figure 5: Visual representation of 5mC, 5hmC and 5fC contexts at three genomic locations.

Change history

  • 06 May 2014

    The raw data including sequencing files (fastq format), processed data including counts of modified bases for each library preparation (reduced, oxidative and simple bisulfite) and the estimation of significant sites as reported in the paper, together with some explanation and a summary of the experimental and analytical protocols, are available here:


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We thank the Biotechnology and Biological Sciences Research Council for a studentship to M.J.B. and Cancer Research UK for a studentship to M.B. S.B. is a Senior Investigator of The Wellcome Trust and the Balasubramanian group is core-funded by Cancer Research UK.

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M.J.B. and S.B. co-invented the redBS-Seq method. M.J.B. and S.B. conceived the experiments with contributions from all authors. M.J.B. and M.B. performed experimental work. G.M. and D.B. performed bioinformatics analysis. M.J.B. and S.B. wrote the manuscript with contributions from all the authors.

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Correspondence to Shankar Balasubramanian.

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The authors declare no competing financial interests.

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Booth, M., Marsico, G., Bachman, M. et al. Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution. Nature Chem 6, 435–440 (2014).

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