Liu, Y. et al. Nat. Biotechnol. https://doi.org/10.1038/s41587-019-0041-2 (2019).
Bisulfite sequencing uncovers DNA modifications. However, it requires the conversion of unmodified cytosine to uracil, which can reduce DNA sequence richness, and when paired with harsh reaction conditions, it can substantially degrade DNA. To address these limitations, Liu et al. attempted to sequence 5mC and 5hmC directly and to retain unmodified cytosine intact. To this end, they used TET enzymes to oxidize 5mC and 5hmC to 5-carboxylcytosine (5caC), and then induced conversion to dihydrouracil (DHU) via borane reduction. A subsequent PCR reaction enabled the conversion of DHU to thymine. The researchers termed this 5mC/5hmC-to-T method TET-assisted pyridine borane sequencing (TAPS). An advantage is that the mild reaction conditions can preserve DNA fragments more than 10 kb long. To sequence 5mC alone, they used β-glucosyltransferase to protect 5hmC from TET oxidation and borane reduction so that only 5mC was converted to T. Alternatively, one could use potassium perruthenate to specifically oxidize 5hmC, thus enabling 5hmC-to-T conversion.
About this article
Cite this article
Tang, L. Bisulfite-free epigenetic sequencing. Nat Methods 16, 286 (2019). https://doi.org/10.1038/s41592-019-0387-x
Acta Clinica Belgica (2020)