Diagenode Premium RRBS technology: Cost-effective DNA methylation mapping with superior CpG resolution and coverage

DNA methylation is an important epigenetic mark with broad relevance in development and disease. Reduced representation bisulfite sequencing (RRBS) enables genome-scale DNA methylation analysis with all the advantages of sequencing technology (accuracy, robustness, flexibility) at a fraction of the cost of whole genome bisulfite sequencing. Diagenode Premium RRBS kit makes this powerful technology readily available to any interested laboratory. Its optimized protocol provides genomic coverage for 3.5 to 4 million CpG dinucleotides in the human genome, which is 5-10 fold higher than the coverage of commercially available DNA methylation microarrays. The method covers not only CpG islands and promoter regions, but also a broad sampling of other functional elements including enhancers, CpG island shores and non-coding RNAs. Finally, its optimized workflow with multiplexing prior to bisulfite conversion allows for convenient processing of up to 96 samples in a single experiment, thus enabling epigenome-wide association studies in large cohorts and in any vertebrate species using an optimized and easy-to-use kit. Introduction DNA methylation provides an essential mechanism of epigenetic gene regulation in embryonic development, with an important role for lineage commitment, genomic imprinting and chromosome stability. DNA methylation patterns are deregulated in many diseases and most strongly in cancer. Several methods exist for mapping DNA methylation patterns throughout the genome. DNA methylation microarrays such as the Infinium assay make it easy to study large cohorts, but they provide low genome-wide coverage, are susceptible to batch effects and available only for the human genome. Whole genome bisulfite sequencing (WGBS) provides accurate and robust coverage for most CpGs in the genome, but the associated sequencing costs are limiting its application to small sample sizes, making it hard to draw biologically relevant conclusions. The RRBS technology combines the strengths of both assays, providing an accurate and cost-effective assay that has all the advantages of bisulfite sequencing, including its robustness and flexible use in any species. RRBS is based on the observation that DNA methylation in vertebrates occurs mainly at CpG dinucleotides1, and it uses the restriction enzyme MspI (target site: C^CGG) to generate genomic fragments starting and ending with a CpG dinucleotide independently of their DNA methylation status. Subsequent size selection and bisulfite sequencing provides high coverage for CpG islands and promoter regions, and also subset of other genomic elements such as enhancers, CpG island shores and non-coding RNAs. RRBS has seen a recent rise in popularity as researchers switch from microarray-based assays to next generation sequencing. Diagenode Premium RRBS kit overcomes many limitations of the original RRBS protocol, which was time-consuming and Diagenode Premium RRBS technology: Cost-effective DNA methylation mapping with superior CpG resolution and coverage Anne-Clémence Veillard (1), Paul Datlinger (2), Miklos Laczik (1), Sharon Squazzo (3) and Christoph Bock (2) (1) Diagenode, Liège, Belgium. (2) CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria. (3) Diagenode Inc., Denville, USA. Correspondence should be addressed to: anne-clemence.veillard@diagenode.com