RRBS

Reduced Representation Bisulfite Sequencing (RRBS) is a genome-wide DNA methylation profiling method that provides single-base resolution methylation data at a fraction of the cost of whole-genome bisulfite sequencing. RRBS enriches for CpG-dense regions by digesting genomic DNA with a restriction enzyme such as MspI, selecting a size range of fragments (for example 40–220 bp), and performing bisulfite conversion followed by PCR amplification and sequencing. The approach yields high coverage of CpG islands, promoters, and other CpG-rich regions while reducing representation of most of the genome.

Workflow and analysis generally involve digesting genomic DNA with MspI, end-repair and ligation of methylated adapters, size selection of fragments, and bisulfite treatment to convert unmethylated cytosines. PCR amplification then creates the sequencing library. Sequencing reads are aligned with bisulfite-aware aligners (for example Bismark or BS-Seeker), and methylation at CpG sites is called. RRBS provides methylation data at single-CpG resolution for the covered sites, enabling comparisons of methylation across tissues, developmental stages, or disease states.

Applications and limitations: RRBS is cost-effective for profiling DNA methylation in mammals and other organisms, delivering dense CpG coverage in CpG-rich regions. It is not genome-wide and underrepresents distal or CpG-poor regions, which can bias interpretation. It requires relatively good DNA quality and careful library preparation to minimize bias, and bisulfite conversion can reduce library complexity.

Variants and improvements: Enhanced RRBS (ERRBS) broadens CpG coverage by incorporating additional restriction enzymes and adjusted fragment selection. Such refinements aim to balance coverage and cost while extending utility to a wider range of samples.