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. The simple workflow and high sensitivity make it an efficient assay for 5hmC mapping. In this review, we discuss some technical consideration in applying reduced representation hydroxymethylation profiling, such as the use of alternative restriction enzymes for increased genomic coverage in different species, application of control libraries and specifications for multiplexing, data processing and normalization.