Oxidative Bisulfite Sequencing: An Experimental and Computational Protocol

Methods Mol Biol. 2021:2198:333-348. doi: 10.1007/978-1-0716-0876-0_26.


Bisulfite sequencing (BS-seq) remains the gold standard technique to quantitively map DNA methylation at a single-base resolution. However, BS-seq cannot discriminate between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Oxidative bisulfite sequencing (oxBS-seq) was one of the first techniques that enabled absolute quantification of 5mC and 5hmC at single-base resolution. OxBS-seq uses chemical oxidation of 5hmC prior to bisulfite treatment to provide a direct readout of 5mC; comparison with BS-seq data can then be used to infer 5hmC levels. Here we describe in detail an updated version of our laboratory's oxBS-seq protocol, which uses potassium perruthenate (KRuO4) as an oxidant. We also describe a bioinformatics pipeline designed to handle Illumina short read sequencing data from whole-genome oxBS-seq.

Keywords: 5-Hydroxymethylcytosine; 5-Methylcytosine; Bisulfite; High-throughput sequencing; Potassium perruthenate.

MeSH terms

  • 5-Methylcytosine / analogs & derivatives*
  • 5-Methylcytosine / analysis
  • Animals
  • Computational Biology / methods*
  • Cytosine / analysis
  • Cytosine / metabolism
  • DNA / genetics
  • DNA Methylation / genetics
  • Dioxygenases / genetics
  • Dioxygenases / metabolism
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Oxidation-Reduction
  • Oxidative Stress
  • Sequence Analysis, DNA / methods*
  • Sulfites / chemistry


  • Sulfites
  • 5-hydroxymethylcytosine
  • 5-Methylcytosine
  • Cytosine
  • DNA
  • Dioxygenases
  • hydrogen sulfite