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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.

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
View complete list of testimonials >

 

RRBS Highlights:

  • 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.

Deliverables:

  • 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 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.

 
Name Cat No. Price  
RRBS 25069 Request Quote

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.

FFPE ChIP-Seq data generated from rat brain by Active Motif Epigenetic Services compared to ENCODE ChIP-Seq data from mouse tissue
Figure 1: RRBS data using biopsied human kidney tumor and adjacent normal kidney.

The displayed region is a representative region from the genome-wide data set and shows differential DNA methylation at the promoter of the LAT1 gene. Each block is a separate data point with red representing a methylated cytosine and blue representing the unmethylated base.

FFPE ChIP-Seq data generated from rat brain by Active Motif Epigenetic Services compared to ENCODE ChIP-Seq data from mouse tissue
Figure 2: RRBS data showing methylation values (as a percentage) for promoter regions.

Values 0 (red) to 1 (green) correspond to 0-100% average methylation frequency of covered CpGs within each promoter region, which are indicated by the light blue line within the heatmap.

FFPE ChIP-Seq data generated from rat brain by Active Motif Epigenetic Services compared to ENCODE ChIP-Seq data from mouse tissue
Figure 3: Graph illustrating sequencing depth coverage for any given CpG.

The minimum sequence depth requirement for a CpG to be included in the analysis is 3. There are over 1 million CpGs that are only covered at a sequencing depth of 1, approximately 100 that have a sequencing depth coverage of 50 and approximately 5000 with depth coverage over 50 sequence tags per CpG location.

RRBS reproducibility data
Figure 4: RRBS reproducibility.

Three separate RRBS experiments were performed in duplicate. The number of overlapping and unique CpGs covered in the experiments are represented by the circles in the Venn diagrams. DNA methylation data is provided at over 4.5 million CpGs in human and over 2.8 million CpGs in mouse. The overlap in CpG coverage within duplicates is over 89% for all experiments.

RRBS reproducibility data
Figure 5: RRBS reproducibility.

Two separate RRBS experiments were performed in duplicate. DNA methylation data from individual CpGs were assigned to promoter regions (2Kb upstream of the TSS) to establish an average methylation percentage over promoters. High reproducibility is indicated by Pearson correlation coefficients that are above 0.93 in each experiment.