Precise genomic mapping of 5-hydroxymethylcytosine via covalent tether-directed sequencing

PLoS Biol. 2020 Apr 10;18(4):e3000684. doi: 10.1371/journal.pbio.3000684. eCollection 2020 Apr.


5-hydroxymethylcytosine (5hmC) is the most prevalent intermediate on the oxidative DNA demethylation pathway and is implicated in regulation of embryogenesis, neurological processes, and cancerogenesis. Profiling of this relatively scarce genomic modification in clinical samples requires cost-effective high-resolution techniques that avoid harsh chemical treatment. Here, we present a bisulfite-free approach for 5hmC profiling at single-nucleotide resolution, named hmTOP-seq (5hmC-specific tethered oligonucleotide-primed sequencing), which is based on direct sequence readout primed at covalently labeled 5hmC sites from an in situ tethered DNA oligonucleotide. Examination of distinct conjugation chemistries suggested a structural model for the tether-directed nonhomologous polymerase priming enabling theoretical evaluation of suitable tethers at the design stage. The hmTOP-seq procedure was optimized and validated on a small model genome and mouse embryonic stem cells, which allowed construction of single-nucleotide 5hmC maps reflecting subtle differences in strand-specific CG hydroxymethylation. Collectively, hmTOP-seq provides a new valuable tool for cost-effective and precise identification of 5hmC in characterizing its biological role and epigenetic changes associated with human disease.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 5-Methylcytosine / analogs & derivatives*
  • 5-Methylcytosine / chemistry
  • Acetylation
  • Animals
  • Bacteriophage lambda / genetics
  • Cell Line
  • DNA Methylation
  • Embryonic Stem Cells / physiology
  • Genome
  • Histones / metabolism
  • Lysine / metabolism
  • Mice
  • Oligonucleotides
  • Reproducibility of Results
  • Sequence Analysis, DNA / methods*
  • Sulfites


  • Histones
  • Oligonucleotides
  • Sulfites
  • 5-hydroxymethylcytosine
  • 5-Methylcytosine
  • Lysine
  • hydrogen sulfite

Grant support

The work was supported by the Research Council of Lithuania ( (Researcher groups project No. MIP-58-17 to EK) and the European Research Council ( (ERC-AdG-2016/742654 to SK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.