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Whole Genome Bisulfite Sequencing (WGBS)

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Whole Genome Bisulfite Sequencing (WGBS)

CD Genomics is providing whole genome sequencing of bisulfite-converted DNA, as an effective and reliable strategy to identify individually methylated cytosines on a genome-wide scale.

DNA methylation at the C5 position of cytosine is one of the major pathways of gene expression regulation in chromosomal DNA, which is essential for normal development1 and is uniquely distributed in all cell types. Detection and quantification of methylation is critical to understanding gene expression and other processes subject to epigenetic regulation. Whole genome bisulfite sequencing is the gold-standard approach for acquiring comprehensive base-pair resolution and quantitative information at most genomic methylated cytosines, it allows for unbiased genome-wide DNA methylation profiling.

In the WGBS process, methylated DNA is treated with bisulfite which converts un-methylated cytosines to uracil. Methylated cytosines remain unchanged. The methylation state on the target regions, (e.g. CpG islands) can be quantitatively characterized. Comparison of the sequence obtained from the bisulfite-treated amplicon to the published sequence enables the identification of differential methylations. DNA methylation pattern is provided in CpG context as well as in the less common CHG and CHH contexts. The outline of bisulfite conversion is shown in Figure 1.

Figure 1. Outline of bisulfite conversion. Nucleotides in blue are unmethylated  cytosines that can be converted to uracils by bisulfite, while red nucleotides  are 5-methylcytosines and are resistant to conversion.Figure 1. Outline of bisulfite conversion. Nucleotides in blue are unmethylated cytosines that can be converted to uracils by bisulfite, while red nucleotides are 5-methylcytosines and are resistant to conversion.

Workflow

The workflow of WGBS is illustrated in Figure 2. Briefly, the DNA sample is processed with cytosine bisulfite conversion followed by random priming, tagging at the 5’ and 3’ end and introduction of Illumina adapters by PCR amplification.

Figure 2. Schematic workflow of whole genome bisulfite sequencing. Figure 2. Schematic workflow of whole genome bisulfite sequencing.

Sequencing Strategy and Recommended Sequencing Depth

  • 250~300-bp insert bisulfite treated DNA library
  • HiSeq platform, paired-end 150 bp
  • Sequencing depth > 20X

Data Analysis

  1. Alignment against reference genome
  2. Sequence depth and coverage analysis
  3. mC calling
  4. Methylation level analysis
  5. Global trends of methylome
  6. Methylation density analysis
  7. Differentially Methylated Regions (DMRs) analysis
  8. DMR annotation and enrichment analysis (GO/KEGG)
  9. Clustering analysis

Sample Requirements

  1. Sample Type: Genomic DNA without RNA contamination and severe degradation
  2. Amount: DNA ≥ 5 µg; concentration ≥ 50 ng/µl
  3. Purity: OD260/280 = 1.8~2.0

Key Features and Advantages

  • Exceptionally high bisulfite conversion rate at true base level resolution
  • Applicable to a broad range of sample sources
  • Comprehensive bioinformatics analysis and high quality data delivery
  • Extensive experience in diverse species including human, mouse, Arabidopsis, rice, maize and et al.

By integrating the whole genome bisulfite treatment, next-generation sequencing technology and bioinformatics analysis techniques, CD Genomics is providing WGBS to enable a sophisticated genome-wide DNA methylation profiling at single-base resolution. Our full service package includes sample standardization, library construction, Next-Generation Sequencing on Illumina Hiseq, raw data alignment, down-stream bioinformatics processing and statistical analysis. We are very flexible, and our services can be customized to fit your project needs, including customizable analysis specifically tailored to your needs.

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