Mirror Bisulfite Sequencing: A Method for Single-Base Resolution of Hydroxymethylcytosine.

Although the role of 5-methylcytosine has been well studied, the biological role of 5-hydroxymethylcytosine still remains unclear because of the limited methods available for single-base detection of 5-hydroxymethylcytosine (5hmC). Here, we present mirror bisulfite sequencing for 5hmC detection at a single CpG site by synthesizing a DNA strand to mirror the parental strand. This semiconservative duplex is sequentially treated with β-glucosyltransferase and M.

Practical guidelines and consideration of using RRHP for 5hmC detection.

5-hydroxymethylcytosine (5hmC) is an epigenetic modification, which has been associated with gene expression in many biological contexts. Reduced representation hydroxymethylation profiling was developed as an enzymatic-based method for genome-wide 5hmC detection. It exploits β-glucosyltransferase to inhibit enzymatic cleavage of adapters ligated to a genomic library, allowing only fragments with glucosylated 5hmC residues at adapter junctions to be amplified and sequenced.

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

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.

JBP1-seq: a fast and efficient method for genome-wide profiling of 5hmC.

We developed a novel approach, J-binding protein 1 sequencing (JBP1-seq), that combines the benefits of an improved recombinant JBP1 protein, Nextera-based library construction, and next-generation sequencing (NGS) for genome-wide profiling of 5-hydroxymethylcytosine (5hmC). Compared with the original JBP1, this new recombinant JBP1 was biotinylated in vivo and conjugated to magnetic beads via biotin-streptavidin interactions. These modifications allowed a more efficient and consistent pull-down of β-glucosyl-5-hydroxymethylcytosine (β-glu-5hmC), and sequence-ready libraries can be generated within 4.