Identification of differentially methylated regions using streptavidin bisulfite ligand methylation enrichment (SuBLiME), a new method to enrich for methylated DNA prior to deep bisulfite genomic sequencing

We sought to develop a method that can partition bisulfite treated DNA into methylated and unmethylated fractions. The method, Streptavidin bisulfite ligand methylation enrichment (SuBLiME) involves specific labelling with a biotin-labelled nucleotide ligand at, or opposite, the site of a methylated cytosine in a bisulfite-converted nucleic acid and then subsequent affinity capture using streptavidin-coupled magnetic beads. This method is highly adaptable and can be combined with deep sequencing library generation and/or genomic complexity reduction. It can also be applied to the study of plant and insect genomes, where methylation often exists outside of a CpG site context. It should also be possible to apply SuBLiME to the enrichment of RNA cytosine methylation. In this pilot study we enriched methylated DNA from the Csp6I cut complexity-reduced genomes of the colorectal cancer cell lines HCT-116, HT-29 and SW-480 and normal blood leukocytes with the aim of discovering colorectal cancer biomarkers. Enriched libraries were sequenced with SOLiD-3 50-bp technology. In pairwise comparisons we scored a total of 1,769 gene loci and 33 miRNA loci as differentially methylated. Of these, 516 loci were differently methylated in at least two promoter-proximal (PP) CpG sites over two discrete Csp6I fragments. Methylated gene loci were found to be associated with anatomical development, differentiation and cell signalling. The SuBLIME data correlated with good agreement to a number of published colorectal cancer DNA methylation biomarkers and genomic datasets. Interestingly, we discovered the highest density of differentially methylated CpG sites (DMCs) was directly downstream of gene loci transcription start sites.