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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-014-0456-5) contains supplementary material, which is available to authorized users.

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.SssI methylase. The glucosyl-5hmCpG in the parental strand inhibits methylation of the mirroring CpG site, and after bisulfite conversion, a thymine in the mirroring strand indicates a 5hmCpG site in the parental strand, whereas a cytosine indicates a non-5hmC site. Using this method, the 5hmC levels of various human tissues and paired liver tissues were mapped genomewide.

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 4.5h from DNA inputs as low as 50ng. 5hmC enrichment of human brain DNA using the new JBP1 resulted in over 25,000 peaks called, which is significantly higher than the 4003 peaks enriched using the old JBP1. Comparison of the technical duplicates and validations with other platforms indicated the results are reproducible and reliable. Thus, JBP1-seq provides a fast, efficient, and cost-effective method for accurate 5hmC genome-wide profiling.

Background/AimsThe role of DHRS3 in human cancer remains unclear. Our study explored the role of DHRS3 in gastric cancer (GC) and its clinicopathological significance and associated mechanisms.MaterialsBisulfite-assisted genomic sequencing PCR and a Mass-Array system were used to evaluate and quantify the methylation levels of the promoter. The expression levels and biological function of DHRS3 was examined by both in vitro and in vivo assays. A two-way hierarchical cluster analysis was used to classify the methylation profiles, and the correlation between the methylation status of the DHRS3 promoter and the clinicopathological characteristics of GC were then assessed.ResultsThe DHRS3 promoter was hypermethylated in GC samples, while the mRNA and protein levels of DHRS3 were significantly downregulated. Ectopic expression of DHRS3 in GC cells inhibited cell proliferation and migration in vitro, decreased tumor growth in vivo. DHRS3 methylation was correlated with histological type and poor differentiation of tumors. GC patients with high degrees of CpG 9.10 methylation had shorter survival times than those with lower methylation.ConclusionDHRS3 was hypermethylated and downregulated in GC patients. Reduced expression of DHRS3 is implicated in gastric carcinogenesis, which suggests DHRS3 is a tumor suppressor.