Abstract
Active DNA demethylation is mediated by ten-eleven translocation (TET) proteins that progressively oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). We have developed a methylation-assisted bisulfite sequencing (MAB-seq) method that enables direct genome-scale mapping and quantification of 5fC and 5caC marks together at single-base resolution. In bisulfite sequencing (BS), unmethylated cytosine residues (Cs), 5fCs and 5caCs, are converted to uracil and cannot be discriminated from each other. The pretreatment of the DNA with the CpG methylation enzyme M.SssI, which converts only the Cs to 5mCs, protects Cs but not 5fCs and 5caCs, which enables direct detection of 5fCs and 5caCs as uracils. Here we also describe an adapted version of the protocol to perform reduced-representation MAB-seq (RRMAB-seq) that provides increased coverage on CpG-rich regions, thus reducing the execution costs and increasing the feasibility of the technique. The main advantage of MAB-seq is to reduce the number of chemical/enzymatic DNA treatments required before bisulfite treatment and to avoid the need for prohibitive sequencing coverage, thus making it more reliable and affordable than subtractive approaches. The method presented here is the ideal tool for studying DNA demethylation dynamics in any biological system. Overall timing is ∼3 d for library preparation.
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Acknowledgements
This work was supported by the Associazione Italiana Ricerca sul Cancro (AIRC) IG 2011 11982 to S.O.
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F.N. conceived the MAB-seq. D.I. and F.N. developed the protocol and the computational methods, and analyzed the data. A.K. and C.P. contributed to the MAB optimization. S.O. supervised the experiments. F.N., D.I. and S.O. wrote the manuscript. All authors read and approved the final manuscript.
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Supplementary information
Supplementary Data 1 — Bsmap_postprocess (step 99).
This custom perl script elaborates the output SAM file derived from Bsmap software and it generates two independent files: a SAM file of the mapped reads and a FastQ file of the unmapped reads. (TXT 3 kb)
Supplementary Data 2 — 5fcaC_bintest (step 104).
This custom perl script elaborates the methratio file generated in step 103 and estimates the significance (p-value) of each cytosine to be 5fC/5caC taking into account the M.SssI methylation fail rate calculated in step 102. (TXT 3 kb)
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Neri, F., Incarnato, D., Krepelova, A. et al. Methylation-assisted bisulfite sequencing to simultaneously map 5fC and 5caC on a genome-wide scale for DNA demethylation analysis. Nat Protoc 11, 1191–1205 (2016). https://doi.org/10.1038/nprot.2016.063
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DOI: https://doi.org/10.1038/nprot.2016.063
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