REDUCED REPRESENTATION BISULFITE SEQUENCING

REDUCED REPRESENTATION BISULFITE SEQUENCING

Overview – What is reduced representation bisulfite sequencing (RRBS)?

DNA methylation in vertebrates most frequently occurs at cytosines in CpG islands. Reduced representation bisulfite sequencing (RRBS) is a method for enriching these GC-rich parts of mammalian genomes. The technique combines restriction digestion that is specific to the CpG motifs and bisulfite treatment followed by sequencing, in order to economically achieve methylation profiling with single-nucleotide resolution.  

RRBS is beneficial for studies looking at important genomic features like promoters. Clinically, the method can be applied to biomarker discovery by enabling comparative analysis of differentially methylated genomic DNA between sample groups (ex.: diseased versus normal tissue).

Applications – What are the advantages of RRBS?

RRBS is a favoured technique by many epigeneticists because it: 

  • Reduces sequencing costs compared to whole genome bisulfite sequencing
  • Increases sequence coverage of CpG islands
  • Provides base-pair resolution for analysis of cytosine methylation
  • Allows for quantitative measurements of DNA methylation frequency
  • Is suitable for analysis of complex DNA areas like repeat sequences that might be difficult to profile using conventional bisulfite sequencing approaches

Workflow – RRBS methods & technologies

RRBS begins with a restriction digest by an enzyme such as the MspI enzyme. MspI recognises “CCGG” motifs, which are more prevalent in gene-regulatory elements than in the rest of the genome. When MspI digests the whole genomic DNA, the restriction digestion enzyme cuts more frequently at CpG-rich areas, resulting in short DNA fragments from these regions. Since other parts of the genome contain fewer CCGG recognition sites, MspI cuts these sections less frequently, and longer DNA fragments are produced from the rest of the genome. 

The resulting DNA fragments undergo size selection to exclude longer fragments from downstream processing. Only the CpG-rich islands, which are most frequently methylated, are analysed by next-generation sequencing. 

Scientific expertise: RRBS

GATC Biotech quickly recognised the potential of reduced representation bisulfite sequencing and was one of the first to adopt the technique in its laboratories.

Publications

Find here, a list of selected research articles supported by GATC Biotech’s sequencing products, as well as the latest publications on epigenetics.

Related products to RRBS

Is it possible to affordably analyse CpG methylation with single-nucleotide resolution? With our standardised service package for RRBS it is! Try our complete product, including DNA isolation, expert library preparation, sequencing on the Illumina platform, professional BioIT analysis and a final comprehensive GATC Data Analysis report.

See how our INVIEW EPIGENOME product can help advance your mammalian epigenetics project or contact us for more information.

For cases where sequencing data for the entire methylome is needed, whole genome bisulfite sequencing might be the right service for you. 

Real-time detection of base modifications like 5-methylcytosine, 4-methylcytosine, 5-methyladenine from unamplified source material is also possible using PacBio’s sequencing technology with our INVIEW MODIFICATION ANALYSIS product. 

Further reading on reduced representation bisulfite sequencing

Nagarajan, A., Roden, C., Wajapeyee, N. Reduced representation bisulfite sequencing to identify global alteration of DNA methylation. Methods Mol Biol. 1176, 23 – 31 (2014).

Meissner, A. et al. Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis. Nucleic Acids Res. 33(18), 5868-77 (2005).