Subtraction-free and bisulfite-free specific sequencing of 5-methylcytosine and its oxidized derivatives at base resolution

Nat Commun. 2021 Jan 27;12(1):618. doi: 10.1038/s41467-021-20920-2.

Abstract

Although various methods have been developed for sequencing cytosine modifications, it is still challenging for specific and quantitative sequencing of individual modification at base-resolution. For example, to obtain both true 5-methylcytosine (5mC) and true 5-hydroxymethylcytosine (5hmC) information, the two major epigenetic modifications, it usually requires subtraction of two methods, which increases noise and requires high sequencing depth. Recently, we developed TET-assisted pyridine borane sequencing (TAPS) for bisulfite-free direct sequencing of 5mC and 5hmC. Here we demonstrate that two sister methods, TAPSβ and chemical-assisted pyridine borane sequencing (CAPS), can be effectively used for subtraction-free and specific whole-genome sequencing of 5mC and 5hmC, respectively. We also demonstrate pyridine borane sequencing (PS) for whole-genome profiling of 5-formylcytosine and 5-carboxylcytosine, the further oxidized derivatives of 5mC and 5hmC. This work completes the set of versatile borane reduction chemistry-based methods as a comprehensive toolkit for direct and quantitative sequencing of all four cytosine epigenetic modifications.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 5-Methylcytosine / analogs & derivatives
  • 5-Methylcytosine / metabolism*
  • Animals
  • Base Sequence
  • Mice
  • Mouse Embryonic Stem Cells / metabolism
  • Oxidation-Reduction
  • Pyridines / metabolism
  • Sequence Analysis, DNA*
  • Sulfites / metabolism*

Substances

  • Pyridines
  • Sulfites
  • 5-hydroxymethylcytosine
  • 5-Methylcytosine
  • pyridine
  • hydrogen sulfite