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

  • Sanchari BattachariyyaEmail author
  • Ruslana Tytarenko
  • Christoph Heuck
  • John Greally
  • Amit Verma
Part of the Methods in Molecular Biology book series (MIMB, volume 1792)


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.

Key words

5-methylcytosine 5-hydroxymethylcytosine Sequencing β-glucosyl transferase HpaII MspI DNA methylation 


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Sanchari Battachariyya
    • 1
    Email author
  • Ruslana Tytarenko
    • 2
  • Christoph Heuck
    • 3
  • John Greally
    • 1
  • Amit Verma
    • 1
  1. 1.Albert Einstein College of MedicineBronxUSA
  2. 2.University of Arkansas for Medical SciencesLittle RockUSA
  3. 3.Janssen R&DSpring HouseUSA

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