Diagenode

DNA Methylation (RRBS Service)

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Catalog Number
Format
G02020000

DNA methylation studies with RRBS

  • The latest bisulfite conversion and Illumina sequencing technologies
  • Single nucleotide resolution through CpG methylation analysis
  • Ready-to-publish data

Reduced representation bisulfite sequencing (RRBS) enables genome-scale DNA methylation analysis in any vertebrate species. The assay benefits from the practical advantages of bisulfite sequencing while avoiding the cost of whole-genome sequencing. The Diagenode RRBS service makes this technology widely available and provides high coverage (up to 4 million CpGs in human samples). Multiplexing prior to bisulfite conversion allows processing of 96 samples per experiment, enabling studies of large cohorts.

TESTIMONIAL

Our laboratory has used RRBS sevice of Diagenode on murine and human samples. The service was impeccable in each phase, from the sample preparation to bionformatic analysis because it was always customer-oriented. I highly recommend my colleagues to use the RRBS service from Diagenode.

Prof. Lucia Altucci, MD, PhD, Seconda Università degli Studi di Napoli, Dipartimento di Biochimica, Biofisica e Patologia generale .

  • Description

    Superior coverage for DNA methylation studies: fast and affordable

    • Superior coverage– 4 million CpGs
    • High throughput - 96 samples processed in one experiment
    • Cost-efficient – Multiplex up to 6-8 samples/sequencing lane
    • High efficiency and minimal bias - Low DNA degradation and reduced amplification
    • Complete solution - Enzymatic digestion, library preparation, bisulfite conversion reagents, sequencing and bioinformatics
    • Results provided within 8-10 weeks (standard project)

    Examples of RRBS data generated with the Diagenode RRBS service using samples from four different species

    Species No. of reads aligned (alignment rate) No. of unique CpGs covered (alignment rate) Bisulfite conversion rate (%) Mean sequencing depth per covered CpG (×)
    Human

    21,787,346 (74%)

    3,913,287

    99.4

    14

    Human FFPE

    27,290,117 (79%)

    2,525,053

    99.7

    28

    Rat

    27,499,313 (86%)

    1,663,104

    99.0

    38

    Dog

    14,053,009 (84%)

    3,572,384

    99.4

    9

    Zebrafish

    29,035,877 (83%)

    1,685,466

    99.3

    48



    Standard analysis

    Standard analysis

    File

    Description

    FastQ file QC

    Trimming report (.txt)

    contains statistics on read trimming

    Read filtering & trimming

    Alignment report (.txt)

    contains mapping statistics and methylation statistics of the reads

    BS-specific alignment

    Alignment file in BAM format (BAM)

    contains all the aligned reads, their positions on the genome and their methylation information, as per the standard SAM/BAM format

    C methylation extraction

    methylation extraction file (.cov)

    contains methylation information on all covered Cs, one line represents one C, each line has 6 fields: chromosome - start - end - methylation percentage - covering methylated reads - covering unmethylated reads.

    QC summary report

    bedGraph file formatted for UCSC browser (bedGraph)

    contains the same methylation information as the previous .cov file. It can be loaded into the UCSC Genome Browser or any similar viewer where the methylation information can be visualized.

    Comparative analysis

    Genome-wide CpG methylation analysis

    Description

    Hierarchical clustering

    Clustering of samples according to DNA methylation level (without heatmap)

    Scatter plot + pearson correlation

    Correlations between the samples

    Histograms

    Distribution of DNA methylation levels  and CpG coverage of each sample

    Principal Components Analysis

    Visualization of principle components that best explain the data

    Pie charts

    Distribution of covered CpGs across  genomic and CpG regions

    Differentially methylated CpG analysis

    Description

    Hierarchical clustering and heatmap

    Clustering of DMRs with heatmap

    Csv file for DMRs

    DMRs identification

    Csv file for DMPs

    DMPs identification

    Bar chart

    DMPs repartition between hyper- and hypo-methylation for each chromosome

    Pie charts

    Distribution of DMPs across  genomic and CpG regions

  • Features

    Chr Shearing


    Superior coverage

    Comparison of CpG coverage between competing technologies.

    They love it!
    The new Diagenode Premium RRBS Kit makes it easy to use RRBS cost-effectively and with high throughput, using early sample pooling and multiplex sequencing. Most importantly, the method provides an improved coverage of up to 4 million CpGs for the human genome. We successfully used this protocol on more than 1,000 samples comprising of six different species, various cancers, FFPE and lowinput samples.
    Paul Datlinger and Christoph Bock, CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria

     

    dna methylation

    Accurate determination of DNA methylation level

    Excellent results were obtained using Diagenode’s Premium RRBS kit: almost 90% alignment rate, 4.1 million CpGs covered and bisulfite conversion rates around 99.5% for all samples. DNA methylation percentages in the region of IGF2 obtained with the Diagenode’s Premium RRBS Kit. Two human cell lines were analyzed: Gm12878 and MCF7. The MCF7 cell line was studied in duplicates. Each peak represents the DNA methylation percentage at one CpG. The methylated CpGs are shown in red and the unmethylated CpGs in grey.



    rrbs how it works

    How it works

    By cutting the genome using the restriction enzyme MspI (CCGG target sites) followed by size selection, DNA is enriched to represent CpG-rich genomic regions (including CpG islands, CpG island shores, enhancers, and other gene-regulatory elements), which are particularly relevant for epigenetic regulation. Similar to exome-sequencing for mutation discovery, the RRBS protocol enriches for some of the most interesting target regions and thereby achieves a reduction in sequencing cost of a factor of 10-20 compared to whole genome bisulfite sequencing.

  • Testimonials

    Our laboratory has used RRBS sevice of Diagenode on murine and human samples. The service was impeccable in each phase, from the sample preparation to bionformatic analysis because it was always customer-oriented. I highly recommend my colleagues to use the RRBS service from Diagenode.

    Prof. Lucia Altucci, MD, PhD, Seconda Università degli Studi di Napoli, Dipartimento di Biochimica, Biofisica e Patologia generale .

    The new Diagenode Premium RRBS Kit makes it easy to use RRBS cost-effectively and with high throughput, using early sample pooling and multiplex sequencing. Most importantly, the method provides an improved coverage of up to 4 million CpGs for the human genome. We successfully used this protocol on more than 1,000 samples comprising of six different species, various cancers, FFPE and lowinput samples..

    Paul Datlinger and Christoph Bock, CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria
  • Applications
    Bisulfite conversion
    Bisulfite modification of DNA is the most commonly used, "gold standard" method for DNA methylation studies providing single nucleotide resolution. This technology is based on the chemical conversion of unmethylated cytosine to uracil. Methylate... Read more
  • Documents
    Diagenode Premium RRBS technology: Cost-effective DNA methylation mapping with superior CpG resolution and coverage APPLICATION NOTE
    DNA methylation is an important epigenetic mark with broad relevance in development and disease. ...
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    The Diagenode Epigenetics custom service POSTER
    Complete workflows for genome-scale DNA methylation and histone marks analysis Epigenetics is cr...
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  • Publications

    How to properly cite this product in your work

    Diagenode strongly recommends using this: DNA Methylation (RRBS Service) (Diagenode Cat# G02020000). Click here to copy to clipboard.

    Using our products in your publication? Let us know!

    Diagenode® Premium RRBS technology: cost-effective DNA methylation mapping with superior coverage
    Anne-Clémence Veillard, Paul Datlinger, Miklos Laczik, Sharon Squazzo & Christoph Bock
    Reduced representation bisulfite sequencing (RRBS) enables genome-scale DNA methylation analysis in any vertebrate species. The assay benefits from the practical advantages of bisulfite sequencing while avoiding the cost of whole-genome sequencing. The Diagenode Premium RRBS kit makes this technology widely availabl...

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