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Reduced Representation Bisulfite Sequencing

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CD Genomics is offering Reduced Representation Bisulfite Sequencing (RRBS) service at single-nucleotide resolution by reducing the amount of sequencing required while capturing the majority of promoters and other relevant genomic regions. RRBS is favorable for biomarker discovery by the identification and analysis of differentially methylated regions (DMRs) between samples.

Reduced Representation Bisulfite Sequencing

DNA methylation occurs predominantly in CpG contexts, and these CpG di-nucleotides are more abundant in selected regions of the genome. RRBS combines restriction enzyme digestion with bisulfite sequencing to enrich for a CpG-dense fraction of the genome. By using the MspI restriction enzyme to cut at CCGG sites in the genome, enriching for CpG-dense regions and sequencing only the fragments pertaining to those regions, the RRBS allows for a maximum amount of methylation data using a minimal amount of sequencing at a significantly reduced cost when compared with whole genome bisulfite sequencing (WGBS). This combination makes RRBS improved the efficiency of the utilization of samples and the perfect platform for pilot studies and clinical applications. The workflow of RRBS is demonstrated in below Figure 1. Briefly, first the DNA is digested, typically with MspI which is methylation insensitive and cuts at CCGG sites, for enriching CpG rich regions of the genome. The fragment ends are then end repaired and A-tailing and adapters are ligated. Next the fragments are size selected, bisulfite converted, amplified and sequenced.

Schematic workflow of RRBS Figure 1. Schematic workflow of RRBS.

Our RRBS covers ≥85% all CpG islands, >60% all gene promoters, and detects 1.5-2 million unique CpG sites at 5-10x average minimum coverage from less sequencing reads.

This technology is not recommended for samples from species with low CpG density and unpredictable restriction enzyme cut sites, i.e. plant species.

Sequencing Strategy and Recommended Sequencing Depth

  • Illumina HiSeq2500 platform, paired-end 125 bp
  • Sequencing depth > 50 M clean reads

Data Analysis

  1. Alignment against reference genome
  2. Promoter and CpG islands coverage analysis
  3. Promoter and CpG islands methylation level analysis
  4. DMRs analysis
  5. Functional annotation of differentially expressed genes
  6. Relation of methylation level and functional elements’ length
  7. CpG region distribution
  8. Regional distribution of genes

Sample Requirements

  1. Sample Type: Genomic DNA without RNA contamination and severe degradation
  2. Amount: DNA ≥ 5 µg; concentration ≥ 100 ng/µl
  3. Purity: OD260/280 = 1.8~2.0

Key Features and Advantages

  • Highly efficient enrichment of CpG-dense regions. Over 85% of CpG islands and 60% of promoters are captured and analyzed.
  • High accuracy and reproducibility. Provides single-nucleotide resolution. Comparison of sample analyses shows highly reproducible results.
  • Economical strategy for genome-wide epigenetic study. Sequencing only GpC-enriched genomic regions dramatically decreases the required volume of sequencing data as well as the cost.
  • Cutting edge tools in bioinformatics analysis. Use widely accepted and updated mainstream software and mature pipeline.

By combining enzyme digestion, bisulfite conversion and NGS technology, CD Genomics is able to provide RRBS as an alternative way to generate and profile comprehensive methylomes at a reduced cost.

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