Ultrafast bisulfite sequencing detection of 5-methylcytosine in DNA and RNA

Nat Biotechnol. 2024 Oct;42(10):1559-1570. doi: 10.1038/s41587-023-02034-w. Epub 2024 Jan 2.

Abstract

Bisulfite sequencing (BS-seq) to detect 5-methylcytosine (5mC) is limited by lengthy reaction times, severe DNA damage, overestimation of 5mC level and incomplete C-to-U conversion of certain DNA sequences. We present ultrafast BS-seq (UBS-seq), which uses highly concentrated bisulfite reagents and high reaction temperatures to accelerate the bisulfite reaction by ~13-fold, resulting in reduced DNA damage and lower background noise. UBS-seq allows library construction from small amounts of purified genomic DNA, such as from cell-free DNA or directly from 1 to 100 mouse embryonic stem cells, with less overestimation of 5mC level and higher genome coverage than conventional BS-seq. Additionally, UBS-seq quantitatively maps RNA 5-methylcytosine (m5C) from low inputs of mRNA and allows the detection of m5C stoichiometry in highly structured RNA sequences. Our UBS-seq results identify NSUN2 as the major 'writer' protein responsible for the deposition of ~90% of m5C sites in HeLa mRNA and reveal enriched m5C sites in 5'-regions of mammalian mRNA, which may have functional roles in mRNA translation regulation.

MeSH terms

  • 5-Methylcytosine* / chemistry
  • Animals
  • DNA Methylation / genetics
  • DNA* / chemistry
  • DNA* / genetics
  • HeLa Cells
  • High-Throughput Nucleotide Sequencing / methods
  • Humans
  • Mice
  • RNA* / chemistry
  • RNA* / genetics
  • Sequence Analysis, DNA / methods
  • Sequence Analysis, RNA / methods
  • Sulfites* / chemistry

Substances

  • 5-Methylcytosine
  • DNA
  • RNA
  • hydrogen sulfite
  • Sulfites