RRBS (Reduced Representation Bisulfite Sequencing)
single base pair resolution DNA methylation analysis
Reduced Representation Bisulfite Sequencing (RRBS) is a method that facilitates the identification of differentially methylated regions by providing DNA methylation data at single base pair resolution on greater than 4 million CpGs. More cost effective than Whole Genome Bisulfite Sequencing and with greater coverage than bead array based DNA methylation platforms, RRBS is an ideal platform to profile DNA methylation across samples.
- Data on 3 to 5 million CpGs per sample.
- Biologically relevant target regions including promoters and CpG islands.
- Sequencing depth of > 30,000,000 reads.
- Low input DNA requirement of 1 µg.
- Reduced library bias resulting from random bar code incorporation.
- Raw data (FASTQ) output files.
- Aligned data (BAM) files.
- Visualization files
– BAM files can be used for visualization in
IGV (Integrated Genome Viewer)
- Methylation table includes percent methylation value for each CpG.
- Differential methylation analysis.
- Figures & graphs
– Includes coverage depth analysis, heatmaps & pie charts
RRBS Background & Data
RRBS is a method designed to isolate DNA containing CpGs by using DNA restriction endonuclease digestion and amplification of fragments smaller than 300 bp. In Brief, the methylation insensitive endonucleases, Msp1 and Taq1, are used to cut C^CGG and T^CGG respectively, thus generating DNA fragments with CpG at the end. Since CpG islands and promoters have a high density of CpGs they are cut at a high frequency and the fragments derived from these regions tend to be short. This subset of short fragments is preferentially amplified when the library is generated through PCR and additional selection occurs during sequencing cluster generation. The final fraction of DNA that is sequenced represents a small fraction of the total genome and is enriched in CpGs. These fragments are mapped to the genome to reveal the methylation status of millions of individual CpGs.
Figure 1: RRBS data using biopsied human kidney tumor and adjacent normal kidney.
Figure 2: RRBS data showing methylation values (as a percentage) for promoter regions.
Figure 3: Graph illustrating sequencing depth coverage for any given CpG.
Figure 4: RRBS reproducibility.
Figure 5: RRBS reproducibility.