RRHP: a tag-based approach for 5-hydroxymethylcytosine mapping at single-site resolution

Abstract

Current methods for genomic mapping of 5-hydroxymethylcytosine (5hmC) have been limited by either costly sequencing depth, high DNA input, or lack of single-base resolution. We present an approach called Reduced Representation 5-Hydroxymethylcytosine Profiling (RRHP) to map 5hmC sites at single-base resolution by exploiting the use of beta-glucosyltransferase to inhibit enzymatic digestion at the junction where adapters are ligated to a genomic library. Therefore, only library fragments presenting glucosylated 5hmC residues at the junction are sequenced. RRHP can detect sites with low 5hmC abundance, and when combined with RRBS data, 5-methylcytosine and 5-hydroxymethylcytosine can be compared at a specific site.

Single-Base Resolution Mapping of 5-Hydroxymethylcytosine Modifications in Hippocampus of Alzheimer’s Disease Subjects

“…To analyze sites of 5-hmC modification at single base resolution, 1 μg of nDNA was submitted to Zymo Research Corporation (ZRC) (CA, US) for analysis by reduced representation hydroxymethylation profiling (RRHP), which utilizes specific restriction endonuclease enzymes to analyze CCGG sequences throughout the genome (Petterson et al 2014). As described previously by Petterson et al .…”

“…As described previously by Petterson et al . (Petterson et al 2014), the specific restriction endonuclease MspI cuts between the CC bases in the CCGG sequence. If the internal cytosine (C C GG) is modified as 5-hmC, glucosylation of this site protects it from MspI cleavage and the epigenetic marker can be evaluated at each CCGG site.…”

“…Recently, three sequencing techniques, oxBS-seq (oxidative bisulfite sequencing), TAB-seq (TET-assisted bisulfite sequencing), and reduced representation hydroxymethylation profiling (RRHP), have advanced the field by specifically quantifying 5- hmC throughout the genome (Booth et al . 2012) (Petterson et al . 2014) (Schuler & Miller 2012).…”

“…2014) (Schuler & Miller 2012). RRHP is suggested to be the most sensitive technique, utilizing specific restriction endonuclease enzymes to detect 5-hmC modifications throughout the genome without the use of harsh chemical labeling techniques (Petterson et al 2014). …”

New guidelines for DNA methylome studies regarding 5-hydroxymethylcytosine for understanding transcriptional regulation

“…Recently, realizing the importance of 5hmC as a representative of the demethylation forms, a number 3 of methods have been proposed to detect it at single-base resolution (Li et al, 2016;Petterson et al, 2014). Some of these methods are based on oxidative bisulfite sequencing (oxBS), which specifically detects 5mC.…”