RRBS (Bisulfite Sequencing)

Superior coverage for DNA methylation studies: fast and affordable

  • Superior coverage– 4 million CpGs
  • High throughput - 96 samples processed in one experiment
  • Cost-efficient – Multiplex up to 6 samples/sequencing lane
  • High efficiency and minimal bias - Low DNA degradation and reduced amplification
  • Complete solution - Enzymatic digestion, library preparation, bisulfite conversion reagents, sequencing and bioinformatics
  • Results provided within 8-10 weeks (standard project)

Chr Shearing

Superior coverage

Comparison of CpG coverage between competing technologies.

They love it!
The new Diagenode Premium RRBS Kit makes it easy to use RRBS cost-effectively and with high throughput, using early sample pooling and multiplex sequencing. Most importantly, the method provides an improved coverage of up to 4 million CpGs for the human genome. We successfully used this protocol on more than 1,000 samples comprising of six different species, various cancers, FFPE and lowinput samples.
Paul Datlinger and Christoph Bock, CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria


dna methylation

Accurate determination of DNA methylation level

Excellent results were obtained using Diagenode’s Premium RRBS kit: almost 90% alignment rate, 4.1 million CpGs covered and bisulfite conversion rates around 99.5% for all samples. DNA methylation percentages in the region of IGF2 obtained with the Diagenode’s Premium RRBS Kit. Two human cell lines were analyzed: Gm12878 and MCF7. The MCF7 cell line was studied in duplicates. Each peak represents the DNA methylation percentage at one CpG. The methylated CpGs are shown in red and the unmethylated CpGs in grey.

rrbs how it works

How it works

By cutting the genome using the restriction enzyme MspI (CCGG target sites) followed by size selection, DNA is enriched to represent CpG-rich genomic regions (including CpG islands, CpG island shores, enhancers, and other gene-regulatory elements), which are particularly relevant for epigenetic regulation. Similar to exome-sequencing for mutation discovery, the RRBS protocol enriches for some of the most interesting target regions and thereby achieves a reduction in sequencing cost of a factor of 10-20 compared to whole genome bisulfite sequencing.

Full suite of bioinformatic analysis

Package Analysis Description

• FastQ file QC
• Read filtering and trimming
• BS-specific alignment
• C methylation extraction
• QC summary report

• Trimming report (.txt)
• Alignment report (.txt)
• Alignment file in BAM format (BAM)
• methylation extraction file (.cov)
• bedGraph file formatted for UCSC browser (bedGraph)


• Hierarchical clustering

• Scatter plot + Pearson Correlation
• Histogram
• UCSC genome browser track

• Pie Chart (genomic region)
• Pie Chart (CpG region)

• Clustering of samples according to DNA methylation level (without heatmap)
• Kruskal-Wallis test (probability 2 groups /conditions are the same)
• Distribution of DNA methylation levels of each group/condition
• Genomic and CpG regions identification/annotation on the reference genome
• Genomic regions covered by CpGs
• CpG regions covered by CpGs

• Hierarchical clustering and heatmap
• UCSC genome browser track
• Pie Chart (DMRs repartition)
• Pie Chart (genomic region)
• Pie Chart (CpG region)

• Clustering of DMRs with heatmap
• DMRs identification/annotation on the reference genome
• DMRs repartition between hyper- and hypo-methylation (trend)
• Genomic regions covered by DMRs
• CpG regions covered by DMRs


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