Genome Wide Mapping of Methylated and Hydroxyl-Methylated Cytosines Using a Modified HpaII Tiny Fragment Enrichment by Ligation Mediated PCR Tagged Sequencing Protocol

Methods Mol Biol. 2018:1792:167-177. doi: 10.1007/978-1-4939-7865-6_12.

Abstract

Here we describe a method for genome wide investigation of methylation and hydroxymethylation status of cytosines. This protocol is an improvement of the HELP-tagging protocol previously described by Suzuki et al. It involves the glucosylation of 5-hydroxymethylcytosines (5-hmC) with β-glucosyl transferase (β-GT), thus rendering them resistant to digestion by MspI. Parallel digestion of β-GT treated samples with MspI, untreated sample with MspI and another untreated sample with HpaII, followed by adapter ligation, parallel sequencing and bioinformatics processing results in a differential display of MspI digestion sites that allows the determination of the distribution of 5-methylcytosines (5-mC) and 5-hmC at these sites.

Keywords: 5-hydroxymethylcytosine; 5-methylcytosine; DNA methylation; HpaII; MspI; Sequencing; β-glucosyl transferase.

MeSH terms

  • Chromosome Mapping*
  • Cytosine / metabolism*
  • DNA Methylation*
  • DNA-Cytosine Methylases / metabolism*
  • Genome-Wide Association Study* / methods
  • Humans
  • Polymerase Chain Reaction* / methods
  • Sequence Analysis, DNA* / methods

Substances

  • Cytosine
  • DNA modification methylase HpaII
  • DNA-Cytosine Methylases