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CD Genomics is providing a novel, flexible, and scalable genome-wide DNA methylation profiling method MethylRAD to allow for de novo methylation analysis with extremely low DNA input.

DNA methylation is a heritable epigenetic mark involving the covalent transfer of a methyl group to the C-5 position of the cytosine ring of DNA by DNA methyltransferases. In plants, cytosines are methylated in both symmetrical (CG or CHG) or asymmetrical (CHH) context, while in mammals, DNA methylation occurs at cytosines in any context of the genome. DNA methylation plays a vital role in many biological processes such as embryogenesis, cellular differentiation, X-chromosome inactivation, genomic imprinting and transposon silencing, perturbed methylation patterns are sometimes a hallmark of important human diseases. Profiling the DNA methylation landscape and its dynamics enables researchers to look deeply into key epigenetic mechanisms that modulate development and diseases.

MethylRAD uses Mrr-like enzymes FspEI to perform reduced methylome sequencing for cost-efficient DNA methylation profiling. FspEI can recognize 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in the CmC and mCDS sites (in the presence of an activator; D = A or G or T; S = C or G). FspEI generates a double-stranded DNA break on the 3' side of the modified cytosine at a fixed distance, which allows nearly every restriction site in the genome to be screened in parallel, without the limitation of sequenceable fragment size. MethylRAD can also discriminate between CG and non-CG methylation, because the methylation status of each site is interrogated independently. The library preparation procedure for MethylRAD is outlined in the below Figure 1. And the workflow of MethylRAD sequencing and data analysis are demonstrated in Figure 2. Briefly, MethylRAD library preparation began with digestion of gDNA with FspE1 and adaptor ligation. The ligation product is then amplified with RCR and barcoded for multiplex sequencing.

Figure  1. Schematic overview of the procedure for MethylRAD library preparation (Wang <em>et al</em>., 2015). Figure 1. Schematic overview of the procedure for MethylRAD library preparation (Wang et al., 2015).

 Figure  2. Workflow of MethylRAD library preparation, sequencing and data analysis. Figure 2. Workflow of MethylRAD library preparation, sequencing and data analysis.

Sequencing Strategy and Recommended Sequencing Depth

  • Illumina HiSeq X-ten PE 150
  • 30 M reads per Gb genome size

Data Analysis

Our standard bioinformatics analysis for MethylRAD includes removal of adapters and low-quality reads, assembly and mapping the reads to reference genome, genome-wide distribution of methylation sites, distribution of methylation site on the function elements, density of methylation sites, relative quantification of DNA methylation levels, DNA methylation pattern profiling, comparisons of methylation levels between different group, and analysis of methylation differential genes.

Sample Requirements

  1. Sample Type: Genomic DNA without RNA contamination and severe degradation
  2. Amount: DNA ≥ 1 µg; concentration ≥ 25 ng/µl
  3. Purity: OD260/280 = 1.8~2.0

Key Features and Advantages

  • High specificity, sensitivity and reproducibility
  • Adjustable tags density
  • Allows for de novo methylation analysis
  • Ideally suited for large-scale methylation profiling
  • Extremely low amount of input DNA required
  • Comprehensive bioinformatics analysis
  • Suitable for most species especially plant species

CD Genomics uses a simple and flexible method for genome-wide DNA methylation profiling with high specificity, sensitivity and reproducibility, enabling de novo methylation analysis with extremely low DNA input, and flexible adjustment of tag density.


  1. Wang S., et al. MethylRAD: a simple and scalable method for genome-wide DNA methylation profiling using methylat ion-dependent restriction enzymes. Open Biol. 2015, 5(11): 150130.
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