RRBS (Reduced Representation Bisulfite Sequencing)

single base pair resolution DNA methylation analysis

Reduced Representation Bisulfite Sequencing (RRBS) provides DNA methylation data at single base pair resolution on up to 5 million CpGs. More cost effective than Whole Genome Bisulfite Sequencing and with greater coverage and less bias than bead array-based DNA methylation platforms, RRBS is an ideal platform to profile DNA methylation, with a focus on functional loci, genome-wide.

RRBS Highlights:

  • DNA methylation data on up to 5 million CpGs per sample.
  • Biologically relevant target regions including promoters and CpG islands.
  • Single-end sequencing depth of >30,000,000 reads.
  • Low input DNA requirement of 100 ng.
  • Reduced library bias through from removal of PCR duplicates by random bar code incorporation.

Learn More About RRBS

Deliverables:

  • Raw data (FASTQ) output files
  • Aligned data (BAM) files
  • Methylation table includes percent methylation value for each CpG
  • Differential methylation analysis
  • Figures & graphs
    – Includes coverage depth analysis, correlation plots, and pie charts
RRBS Overview flow chart

To learn more, please give us a call or send us an Epigenetic Services Information Request. You can also download Active Motif’s Epigenetic Services Profile.

What our customers are saying about us...

"We worked with Active Motif to process some FFPE and frozen liver samples sourced from 25+year old samples for an RRBS assay. The overall process of submitting samples, communicating with Active Motif, receiving the data, and obtaining the summary was quick and painless. The whole operation was smooth and professionally done."

Brian Chorley, PhD
Environmental Protection Agency
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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.

RRBS data from human samples
Figure 1: RRBS data from human samples.

The displayed regions are representative regions from the genome-wide data set and shows differential DNA methylation at 1A) an exon of CD19 and 1B) an intron of PAX5. Each block is a separate data point with red representing a methylated cytosine and blue representing an unmethylated base.


RRBS data illustrating sequencing depth coverage for any given CpG
Figure 2: Graph illustrating sequencing depth coverage for any given CpG.

The minimum sequence depth for a CpG to be included in the analysis is 10. Over 1 million CpG sites have a coverage of at least 10 reads.


RRBS data illustrating the correlation between each sample analyzed
Figure 3: Graph illustrating the correlation between each sample analyzed.

The upper right corner shows the Pearson correlation coefficient for the two samples. The bar plots show the distribution of the percent methylation in each sample, with the majority of analyzed CpG sites having 0 or 100% methylation. The scatter plot in the lower left corner shows the level of methylation at shared CpG sites for each sample.


RRBS data showing the assignment of differentially methylated cytosines to genic features
Figure 4: Pie chart showing the assignment of differentially methylated cytosines to genic features.

A differential methylation analysis was performed on a per CpG basis, and identified sites were then aligned to annotated gene features such as promoters and exons.