Abstract

Here we present APOBEC-coupled epigenetic sequencing (ACE-seq), a bisulfite-free method for localizing 5-hydroxymethylcytosine (5hmC) at single-base resolution with low DNA input. The method builds on the observation that AID/APOBEC family DNA deaminase enzymes can potently discriminate between cytosine modification states and exploits the non-destructive nature of enzymatic, rather than chemical, deamination. ACE-seq yielded high-confidence 5hmC profiles with at least 1,000-fold less DNA input than conventional methods. Applying ACE-seq to generate a base-resolution map of 5hmC in tissue-derived cortical excitatory neurons, we found that 5hmC was almost entirely confined to CG dinucleotides. The whole-genome map permitted cytosine, 5-methylcytosine (5mC) and 5hmC to be parsed and revealed genomic features that diverged from global patterns, including enhancers and imprinting control regions with high and low 5hmC/5mC ratios, respectively. Enzymatic deamination overcomes many challenges posed by bisulfite-based methods, thus expanding the scope of epigenome profiling to include scarce samples and opening new lines of inquiry regarding the role of cytosine modifications in genome biology.

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Acknowledgements

We are grateful to Z. Zhou, M. Fasolino, A. Bryson and J.M. SanMiguel for discussion and reagents. This work was supported by the US National Institutes of Health through R21-HG009545 (to R.M.K.) and by the Penn Epigenetics Institute. Additional support included R00-HG007982 (to H.W.), DP2-HL142044 (to H.W.) and R01-GM118501 (to R.M.K.). E.K.S. and J.E.D. are NSF Graduate Research Fellows. J.E.D. and E.B.F. were supported by NIH training grant T32-GM07229, and M.Y.L. by F30-CA196097.

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Affiliations

  1. Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Emily K Schutsky
    • , Jamie E DeNizio
    • , Monica Yun Liu
    • , Christopher S Nabel
    •  & Rahul M Kohli
  2. Department of Genetics, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Peng Hu
    • , Emily B Fabyanic
    •  & Hao Wu
  3. Department of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Young Hwang
    •  & Frederic D Bushman
  4. Penn Epigenetics Institute, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Hao Wu
    •  & Rahul M Kohli

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Contributions

E.K.S., C.S.N., R.M.K. and H.W. conceived of and developed the ACE-seq approach. E.K.S. conducted all of the experiments, with assistance from J.E.D., M.Y.L., E.B.F., P.H. and Y.H. F.D.B. contributed to phage experiment design. H.W. performed computational analysis. E.K.S., H.W. and R.M.K. analyzed the results and wrote the manuscript, with contributions from all of the authors.

Competing interests

Aspects of the ACE-seq protocol have been non-exclusively licensed.

Corresponding authors

Correspondence to Hao Wu or Rahul M Kohli.

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https://doi.org/10.1038/nbt.4204