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'meta_title' => 'Premium Reduced Representation Bisulfite Sequencing (RRBS) Kit V2 x96 | Diagenode',
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<p><strong>Reduced Representation Bisulfite Sequencing (RRBS)</strong> offers a <strong>cost-effective, focused solution</strong> to perform genome-scale DNA methylation analysis at the <strong>single nucleotide level</strong> in any vertebrate species. The fundamental idea of RRBS is to get a “reduced representation” of the genome. <span>By cutting the genome using the <strong>restriction MspI enzyme</strong> (CCGG target sites) followed by size selection, the DNA sample is enriched with</span><span><span> </span>biologically relevant </span><span><strong>CpG-rich regions</strong> (including<span> </span></span><span>promoters and<span> </span></span><span>CpG islands) in which DNA methylation marks are typically found.</span><span><span> </span></span></p>
<p>With Diagenode’s Premium RRBS Kit V2 perform cost-effective and reliable genome-scale DNA methylation analysis. Detect around <strong>4 million CpGs</strong> (with coverage > 10) in human samples and identify methylation patterns in CpG rich regions including promoters and CpG islands.</p>
<p>Secure high-quality NGS data for DNA methylation analysis at single base resolution and enjoy our specific and upgraded features:</p>
<ul>
<li>Work with <strong>the lowest DNA amounts</strong> from <strong>25 -100ng gDNA</strong></li>
<li>Process <strong>up to 96 sample</strong> in parallel while saving handling-time and cost per sample with early sample pooling strategy</li>
<li>Utilize our <strong>Software for Intelligent Pooling</strong> (SIP) for better pooling strategies</li>
<li>Get <strong>optimal results</strong> from your sequencer with <strong>Unique Dual Indexing</strong> (UDI)</li>
<li><strong>Identify</strong> and <strong>remove PCR duplicates</strong> from your data with <strong>Unique Molecular Identifiers</strong> (UMIs)</li>
</ul>
<p>The Premium RRBS kit V2 includes all reagents necessary for the library preparation. Specific adapters were designed and validated to fit the technology and are available separately as described below.</p>
<p><strong>For RRBS V2 with UDI and UMI library construction:</strong></p>
<ul>
<li><a href="https://www.diagenode.com/en/p/premium-methyl-UDI-UMI-adapters-set-A-x24">C02030040 - Premium Methyl UMI-UDI Adapters - Set A</a></li>
<li><a href="https://www.diagenode.com/en/p/premium-methyl-UDI-UMI-adapters-set-B-x24">C02030041 - Premium Methyl UMI-UDI Adapters - Set B</a></li>
</ul>
<ul class="accordion" data-accordion="">
<li class="accordion-navigation"><a href="#v5" style="color: #13b29c;"><i class="fa fa-caret-right"></i> Software for Intelligent Pooling (SIP)</a>
<div id="v5" class="content">
<p>Diagenode's new online<strong> intelligent pooling aid</strong> (RRBS SIP) provides the <strong>optimal pool design</strong> for RRBS to meet your specific sample and analysis needs:</p>
<ul style="list-style-type: disc;">
<li><strong>Time-saving: </strong>avoid<strong> </strong>complex caculations</li>
<li><strong>Highest pooling efficiency</strong> based on qPCR quantification - elevate your pooling effectiveness</li>
<li><strong>Powerful:</strong> incorporates advanced aspects such as number of samples per pool required and the separation between projects</li>
<li><strong>Accurate:</strong> identifies outliers</li>
</ul>
<p></p>
<p>Get access to Diagenode's <a href="https://diagenode.shinyapps.io/RRBS_SIP/">RRBS Software for Intelligent Pooling (SIP</a>)</p>
<p>Get your RRBS template file - <a href="https://www.diagenode.com/files/products/kits/Template_SIP_RRBS.xlsx">Here</a></p>
</div>
</li>
</ul>
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'label1' => 'Characteristics',
'info1' => '<h4>Sequencing quality</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/fig_RRBS_50ng_1_R1_trimmed_per_base_quality.png" width="850px" alt="DNA methylation" caption="false" /></p>
<p><b>Figure 1. Excellent sequencing quality. </b>RRBS libraries were prepared from different starting amounts of human gDNA using Diagenode’s Premium RRBS V2 kit and sequenced in paired-end 50 bp on Illumina NovaSeq instrument generating 30-40 million read pairs per sample. Sequencing statistics reveal that all samples performed well with mean Phred scores above 30 along the entire reads 1 (A) and 2 (B) (data shown for 50 ng gDNA input after trimming).</p>
<p><br /><br /></p>
<h4>Accurate Coverage of CpGs</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/fig2_table_rrbs.png" width="100%" alt="Reduced Representation Bisulfite Sequencing" caption="false" /></p>
<p><b>Table 1. Examples of Premium RRBS V2 sequencing data.</b> UMI data processing enables accurate estimation of CpG counts.</p>
<p><br /><br /></p>
<h4>Focus on CpG-rich regions</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/fig3_50ng_10X_coverage_annotation_cpg.png" width="400px" alt="single nucleotide resolution" caption="false" /></p>
<p><b>Figure </b><b>2. </b><b>Coverage</b> <b>of </b><b>CpGs</b><b> and genomic regions by Premium RRBS V2</b><b>. </b>Diagenode’s Premium RRBS V2 allows a wide interrogation of CpGs (with a sequencing depth >10) of the human genome with a focus on CpG rich regions, especially CpG islands (data shown for 50 ng gDNA input).</p>
<p><br /><br /></p>
<h4>Compatible with all vertebrates</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/table2-rrbs.png" width="100%" alt="CpG-dense regions" caption="false" /></p>
<p><b>Table 2.</b> The kit RRBS v2 is compatible with all vertebrates. The Table 2 shows some examples of results obtained for different species.</p>
<p><br /><br /></p>
<h4>Superior performance</h4>
<ul>
<li>Lower duplicate rate</li>
<li>More CpGs detected</li>
<li>Higher % of uniquely aligned reads containing CpGs</li>
<li>Higher genome coverage (3,3%)</li>
</ul>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/table3-rrbs.png" width="100%" alt="RRBS Kit V2 x96" caption="false" /></p>
<p><b>Table 3.</b> RRBS was performed using Diagenode RRBS v2 kit on HeLa cells and human blood samples (total 10 samples) as well as using competitor kit on HeLa cells, human blood and brain samples (total 10 samples). The Table 3 shows sequencing parameters for 10 samples processed with each kit.</p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/fig3-rrbs.png" width="500px" alt="Reduced Representation Bisulfite Sequencing" caption="false" /></p>
<p><b>Figure 3.</b> Comparison of CpG coverage between competing technologies.</p>
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<p>By cutting the genome using the <strong>restriction enzyme MspI</strong> (CCGG target sites) followed by size selection, DNA is enriched to represent <strong>CpG-rich genomic regions</strong> (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.</p>
<p><span class="label-green" style="margin-bottom: 16px; margin-left: -22px;">NATURE METHODS</span></p>
<p></p>
<h4 class="advertising-feature"><strong>Premium RRBS technology: cost-effective DNA methylation mapping with superior coverage</strong></h4>
<p><a href="#auth-5" class="name"><span class="fn">Christoph Bock</span></a>,<sup href="#affil-auth"> </sup><a href="#auth-4" class="name"><span class="fn">Sharon Squazzo</span></a><span style="font-size: 13.3333px;">, </span><a href="#auth-3" class="name"><span class="fn">Miklos Laczik</span></a><span style="font-size: 13.3333px;">, </span><a href="#auth-2" class="name"><span class="fn">Paul Datlinger</span></a><span style="font-size: 13.3333px;">, </span><a href="#auth-1" class="name"><span class="fn">Anne-Clémence Veillard</span></a></p>
<p>Nature Methods 13 (2016)</p>
<p>Published online <time datetime="2016-01-28">28 January 2016 </time></p>
<p><a href="https://www.nature.com/articles/nmeth.f.391" class="alert button"><span style="font-weight: 400;">CHECK OUT THE NATURE METHODS PAPER</span></a></p>
<p><iframe width="320" height="180" src="https://www.youtube.com/embed/CJn3XEAznu0?rel=0" frameborder="0" allowfullscreen="allowfullscreen"></iframe> <iframe width="320" height="180" src="https://www.youtube.com/embed/4xgRG9qVT5E"></iframe></p>
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'info3' => '<h4>Comparative analysis using DNA methylation data from RRBS V2:</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/cluster-dendrogram-rrbs.png" width="500px" alt="DNA methylation data from RRBS V2" caption="false" /></p>
<p><b>Figure 1. Example of cluster dendrogram</b><br />Samples: WT – Wild type; Single KO – Single knock-out; Double KO – Double knock-out</p>
<p>After performing alignment to the reference genome, the methylation calling step provides the methylation percentages for every detected CpG. This methylation rate profile, available now for each sample, can be used to visually explore sample distance by hierarchical clustering. The main branches in this type of dendograms show how the clusters are distinct from each other, and how the objects within each cluster are broadly similar to each other.</p>
<p></p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/clustering-heatmap-rrbs.png" width="850px" alt="DNA methylation data from Premium RRBS kit V2" caption="false" /></p>
<p><b>Figure 2. Example of results: Clustering heatmap of significant DMRs (left) and Volcano plot of DMR (right)</b><br />Samples: WT – Wild type; Single KO – Single knock-out; Double KO – Double knock-out</p>
<p>If the groups of interest contain two or more replicates, a differential methylation analysis can be performed. This type of analysis will identify individual CpGs or regions in the genome that are hyper or hypomethylated in the study group with respect to the control group. A heatmap (left) can be used to visualize the overall distribution of differential methylation between the groups. A volcano plot (right) will show more precisely, the mehtylation difference (x axis) and level of significance of that difference (y axis) allowing the researcher to identify CpGs or regions with large and statistically signifcant differences.</p>
<p></p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/percentage-hypo-rrbs.png" width="500px" alt="CpG-dense regions" caption="false" /></p>
<p><b>Figure 3. Example of results: </b><strong>Percentage of hypo and hypermethylated regions in the samples per chromosome</strong></p>
<p>Horizontal bar plots show the number of hyper and hypomethylated events per chromosome, as a percentage of the sites with 10X coverage and a 25% difference of methylation status.</p>
<p></p>
<h4>QC of the samples</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/per_base_sequence_content-rrbs.png" width="500px" alt="single nucleotide resolution" caption="false" /></p>
<p><b>Figure 4. Example of samples QC:</b> <strong>Per base sequence content</strong></p>
<p>A sequencing quality control software such as FastQC will show the per base sequence content of the sequenced reads. With Diagenode’s RRBS Premium kit, read 2 should show a specific profile for the first three bases that correspond to the MspI restrictive site CGG. The first base being a cytosine followed by a guanine can be either methylated or not; 5mC will remain as C after bisulfite conversion whereas unmethylated C will be converted to T after PCR amplification. The first base in the FastQC per base content plot will therefore show a percentage of reads that vary at that position. In this example, 70% of the reads are methylated at position 1 (C) and 30% are unmethylated. Positions 2 and 3 should show 100% G content.</p>
<p></p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/histogram-cpg-rrbs.png" width="500px" alt="DNA methylation" caption="false" /></p>
<p><b>Figure 5. Example of samples QC: </b><strong>Histogram of % CpG methylation</strong></p>
<p>Another quality control step consists in looking at the frequency of methylation percentages for all CpGs detected. This step can only be performed after alignment and methylation call. The histogram shows a bimodal distribution, where most of the cytosines are either fully methylated or unmethylated. Some cytosines have intermediate methylation levels. A bump in the middle of the plot or a non-bimodal distribution would suggest an inefficiency of the bisulfite treatment. With Diagenode’s RRBS Premium kit, the exact conversion rate can be calculated thanks to the spike-in control sequences that are included in the kit.</p>
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$meta_description = 'Premium RRBS kit V2 analyzes DNA methylation at the single nucleotide level for 4 million CpGs. Efficient and accurate quantification of DNA methylation through the use of UMI.'
$meta_title = 'Premium Reduced Representation Bisulfite Sequencing (RRBS) Kit V2 x96 | Diagenode'
$product = array(
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'name' => 'Premium RRBS kit V2 x96<br /> RRBS for low DNA amounts and accurate analysis',
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<p><strong>Reduced Representation Bisulfite Sequencing (RRBS)</strong> offers a <strong>cost-effective, focused solution</strong> to perform genome-scale DNA methylation analysis at the <strong>single nucleotide level</strong> in any vertebrate species. The fundamental idea of RRBS is to get a “reduced representation” of the genome. <span>By cutting the genome using the <strong>restriction MspI enzyme</strong> (CCGG target sites) followed by size selection, the DNA sample is enriched with</span><span><span> </span>biologically relevant </span><span><strong>CpG-rich regions</strong> (including<span> </span></span><span>promoters and<span> </span></span><span>CpG islands) in which DNA methylation marks are typically found.</span><span><span> </span></span></p>
<p>With Diagenode’s Premium RRBS Kit V2 perform cost-effective and reliable genome-scale DNA methylation analysis. Detect around <strong>4 million CpGs</strong> (with coverage > 10) in human samples and identify methylation patterns in CpG rich regions including promoters and CpG islands.</p>
<p>Secure high-quality NGS data for DNA methylation analysis at single base resolution and enjoy our specific and upgraded features:</p>
<ul>
<li>Work with <strong>the lowest DNA amounts</strong> from <strong>25 -100ng gDNA</strong></li>
<li>Process <strong>up to 96 sample</strong> in parallel while saving handling-time and cost per sample with early sample pooling strategy</li>
<li>Utilize our <strong>Software for Intelligent Pooling</strong> (SIP) for better pooling strategies</li>
<li>Get <strong>optimal results</strong> from your sequencer with <strong>Unique Dual Indexing</strong> (UDI)</li>
<li><strong>Identify</strong> and <strong>remove PCR duplicates</strong> from your data with <strong>Unique Molecular Identifiers</strong> (UMIs)</li>
</ul>
<p>The Premium RRBS kit V2 includes all reagents necessary for the library preparation. Specific adapters were designed and validated to fit the technology and are available separately as described below.</p>
<p><strong>For RRBS V2 with UDI and UMI library construction:</strong></p>
<ul>
<li><a href="https://www.diagenode.com/en/p/premium-methyl-UDI-UMI-adapters-set-A-x24">C02030040 - Premium Methyl UMI-UDI Adapters - Set A</a></li>
<li><a href="https://www.diagenode.com/en/p/premium-methyl-UDI-UMI-adapters-set-B-x24">C02030041 - Premium Methyl UMI-UDI Adapters - Set B</a></li>
</ul>
<ul class="accordion" data-accordion="">
<li class="accordion-navigation"><a href="#v5" style="color: #13b29c;"><i class="fa fa-caret-right"></i> Software for Intelligent Pooling (SIP)</a>
<div id="v5" class="content">
<p>Diagenode's new online<strong> intelligent pooling aid</strong> (RRBS SIP) provides the <strong>optimal pool design</strong> for RRBS to meet your specific sample and analysis needs:</p>
<ul style="list-style-type: disc;">
<li><strong>Time-saving: </strong>avoid<strong> </strong>complex caculations</li>
<li><strong>Highest pooling efficiency</strong> based on qPCR quantification - elevate your pooling effectiveness</li>
<li><strong>Powerful:</strong> incorporates advanced aspects such as number of samples per pool required and the separation between projects</li>
<li><strong>Accurate:</strong> identifies outliers</li>
</ul>
<p></p>
<p>Get access to Diagenode's <a href="https://diagenode.shinyapps.io/RRBS_SIP/">RRBS Software for Intelligent Pooling (SIP</a>)</p>
<p>Get your RRBS template file - <a href="https://www.diagenode.com/files/products/kits/Template_SIP_RRBS.xlsx">Here</a></p>
</div>
</li>
</ul>
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'info1' => '<h4>Sequencing quality</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/fig_RRBS_50ng_1_R1_trimmed_per_base_quality.png" width="850px" alt="DNA methylation" caption="false" /></p>
<p><b>Figure 1. Excellent sequencing quality. </b>RRBS libraries were prepared from different starting amounts of human gDNA using Diagenode’s Premium RRBS V2 kit and sequenced in paired-end 50 bp on Illumina NovaSeq instrument generating 30-40 million read pairs per sample. Sequencing statistics reveal that all samples performed well with mean Phred scores above 30 along the entire reads 1 (A) and 2 (B) (data shown for 50 ng gDNA input after trimming).</p>
<p><br /><br /></p>
<h4>Accurate Coverage of CpGs</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/fig2_table_rrbs.png" width="100%" alt="Reduced Representation Bisulfite Sequencing" caption="false" /></p>
<p><b>Table 1. Examples of Premium RRBS V2 sequencing data.</b> UMI data processing enables accurate estimation of CpG counts.</p>
<p><br /><br /></p>
<h4>Focus on CpG-rich regions</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/fig3_50ng_10X_coverage_annotation_cpg.png" width="400px" alt="single nucleotide resolution" caption="false" /></p>
<p><b>Figure </b><b>2. </b><b>Coverage</b> <b>of </b><b>CpGs</b><b> and genomic regions by Premium RRBS V2</b><b>. </b>Diagenode’s Premium RRBS V2 allows a wide interrogation of CpGs (with a sequencing depth >10) of the human genome with a focus on CpG rich regions, especially CpG islands (data shown for 50 ng gDNA input).</p>
<p><br /><br /></p>
<h4>Compatible with all vertebrates</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/table2-rrbs.png" width="100%" alt="CpG-dense regions" caption="false" /></p>
<p><b>Table 2.</b> The kit RRBS v2 is compatible with all vertebrates. The Table 2 shows some examples of results obtained for different species.</p>
<p><br /><br /></p>
<h4>Superior performance</h4>
<ul>
<li>Lower duplicate rate</li>
<li>More CpGs detected</li>
<li>Higher % of uniquely aligned reads containing CpGs</li>
<li>Higher genome coverage (3,3%)</li>
</ul>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/table3-rrbs.png" width="100%" alt="RRBS Kit V2 x96" caption="false" /></p>
<p><b>Table 3.</b> RRBS was performed using Diagenode RRBS v2 kit on HeLa cells and human blood samples (total 10 samples) as well as using competitor kit on HeLa cells, human blood and brain samples (total 10 samples). The Table 3 shows sequencing parameters for 10 samples processed with each kit.</p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/fig3-rrbs.png" width="500px" alt="Reduced Representation Bisulfite Sequencing" caption="false" /></p>
<p><b>Figure 3.</b> Comparison of CpG coverage between competing technologies.</p>
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<p>By cutting the genome using the <strong>restriction enzyme MspI</strong> (CCGG target sites) followed by size selection, DNA is enriched to represent <strong>CpG-rich genomic regions</strong> (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.</p>
<p><span class="label-green" style="margin-bottom: 16px; margin-left: -22px;">NATURE METHODS</span></p>
<p></p>
<h4 class="advertising-feature"><strong>Premium RRBS technology: cost-effective DNA methylation mapping with superior coverage</strong></h4>
<p><a href="#auth-5" class="name"><span class="fn">Christoph Bock</span></a>,<sup href="#affil-auth"> </sup><a href="#auth-4" class="name"><span class="fn">Sharon Squazzo</span></a><span style="font-size: 13.3333px;">, </span><a href="#auth-3" class="name"><span class="fn">Miklos Laczik</span></a><span style="font-size: 13.3333px;">, </span><a href="#auth-2" class="name"><span class="fn">Paul Datlinger</span></a><span style="font-size: 13.3333px;">, </span><a href="#auth-1" class="name"><span class="fn">Anne-Clémence Veillard</span></a></p>
<p>Nature Methods 13 (2016)</p>
<p>Published online <time datetime="2016-01-28">28 January 2016 </time></p>
<p><a href="https://www.nature.com/articles/nmeth.f.391" class="alert button"><span style="font-weight: 400;">CHECK OUT THE NATURE METHODS PAPER</span></a></p>
<p><iframe width="320" height="180" src="https://www.youtube.com/embed/CJn3XEAznu0?rel=0" frameborder="0" allowfullscreen="allowfullscreen"></iframe> <iframe width="320" height="180" src="https://www.youtube.com/embed/4xgRG9qVT5E"></iframe></p>
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'info3' => '<h4>Comparative analysis using DNA methylation data from RRBS V2:</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/cluster-dendrogram-rrbs.png" width="500px" alt="DNA methylation data from RRBS V2" caption="false" /></p>
<p><b>Figure 1. Example of cluster dendrogram</b><br />Samples: WT – Wild type; Single KO – Single knock-out; Double KO – Double knock-out</p>
<p>After performing alignment to the reference genome, the methylation calling step provides the methylation percentages for every detected CpG. This methylation rate profile, available now for each sample, can be used to visually explore sample distance by hierarchical clustering. The main branches in this type of dendograms show how the clusters are distinct from each other, and how the objects within each cluster are broadly similar to each other.</p>
<p></p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/clustering-heatmap-rrbs.png" width="850px" alt="DNA methylation data from Premium RRBS kit V2" caption="false" /></p>
<p><b>Figure 2. Example of results: Clustering heatmap of significant DMRs (left) and Volcano plot of DMR (right)</b><br />Samples: WT – Wild type; Single KO – Single knock-out; Double KO – Double knock-out</p>
<p>If the groups of interest contain two or more replicates, a differential methylation analysis can be performed. This type of analysis will identify individual CpGs or regions in the genome that are hyper or hypomethylated in the study group with respect to the control group. A heatmap (left) can be used to visualize the overall distribution of differential methylation between the groups. A volcano plot (right) will show more precisely, the mehtylation difference (x axis) and level of significance of that difference (y axis) allowing the researcher to identify CpGs or regions with large and statistically signifcant differences.</p>
<p></p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/percentage-hypo-rrbs.png" width="500px" alt="CpG-dense regions" caption="false" /></p>
<p><b>Figure 3. Example of results: </b><strong>Percentage of hypo and hypermethylated regions in the samples per chromosome</strong></p>
<p>Horizontal bar plots show the number of hyper and hypomethylated events per chromosome, as a percentage of the sites with 10X coverage and a 25% difference of methylation status.</p>
<p></p>
<h4>QC of the samples</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/per_base_sequence_content-rrbs.png" width="500px" alt="single nucleotide resolution" caption="false" /></p>
<p><b>Figure 4. Example of samples QC:</b> <strong>Per base sequence content</strong></p>
<p>A sequencing quality control software such as FastQC will show the per base sequence content of the sequenced reads. With Diagenode’s RRBS Premium kit, read 2 should show a specific profile for the first three bases that correspond to the MspI restrictive site CGG. The first base being a cytosine followed by a guanine can be either methylated or not; 5mC will remain as C after bisulfite conversion whereas unmethylated C will be converted to T after PCR amplification. The first base in the FastQC per base content plot will therefore show a percentage of reads that vary at that position. In this example, 70% of the reads are methylated at position 1 (C) and 30% are unmethylated. Positions 2 and 3 should show 100% G content.</p>
<p></p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/histogram-cpg-rrbs.png" width="500px" alt="DNA methylation" caption="false" /></p>
<p><b>Figure 5. Example of samples QC: </b><strong>Histogram of % CpG methylation</strong></p>
<p>Another quality control step consists in looking at the frequency of methylation percentages for all CpGs detected. This step can only be performed after alignment and methylation call. The histogram shows a bimodal distribution, where most of the cytosines are either fully methylated or unmethylated. Some cytosines have intermediate methylation levels. A bump in the middle of the plot or a non-bimodal distribution would suggest an inefficiency of the bisulfite treatment. With Diagenode’s RRBS Premium kit, the exact conversion rate can be calculated thanks to the spike-in control sequences that are included in the kit.</p>
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<div class="small-12 medium-8 large-8 columns"><br />
<p><strong>Reduced Representation Bisulfite Sequencing (RRBS)</strong> offers a <strong>cost-effective, focused solution</strong> to perform genome-scale DNA methylation analysis at the <strong>single nucleotide level</strong> in any vertebrate species. The fundamental idea of RRBS is to get a “reduced representation” of the genome. <span>By cutting the genome using the <strong>restriction MspI enzyme</strong> (CCGG target sites) followed by size selection, the DNA sample is enriched with</span><span><span> </span>biologically relevant </span><span><strong>CpG-rich regions</strong> (including<span> </span></span><span>promoters and<span> </span></span><span>CpG islands) in which DNA methylation marks are typically found.</span><span><span> </span></span></p>
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<p>With Diagenode’s Premium RRBS Kit V2 perform cost-effective and reliable genome-scale DNA methylation analysis. Detect around <strong>4 million CpGs</strong> (with coverage > 10) in human samples and identify methylation patterns in CpG rich regions including promoters and CpG islands.</p>
<p>Secure high-quality NGS data for DNA methylation analysis at single base resolution and enjoy our specific and upgraded features:</p>
<ul>
<li>Work with <strong>the lowest DNA amounts</strong> from <strong>25 -100ng gDNA</strong></li>
<li>Process <strong>up to 96 sample</strong> in parallel while saving handling-time and cost per sample with early sample pooling strategy</li>
<li>Utilize our <strong>Software for Intelligent Pooling</strong> (SIP) for better pooling strategies</li>
<li>Get <strong>optimal results</strong> from your sequencer with <strong>Unique Dual Indexing</strong> (UDI)</li>
<li><strong>Identify</strong> and <strong>remove PCR duplicates</strong> from your data with <strong>Unique Molecular Identifiers</strong> (UMIs)</li>
</ul>
<p>The Premium RRBS kit V2 includes all reagents necessary for the library preparation. Specific adapters were designed and validated to fit the technology and are available separately as described below.</p>
<p><strong>For RRBS V2 with UDI and UMI library construction:</strong></p>
<ul>
<li><a href="https://www.diagenode.com/en/p/premium-methyl-UDI-UMI-adapters-set-A-x24">C02030040 - Premium Methyl UMI-UDI Adapters - Set A</a></li>
<li><a href="https://www.diagenode.com/en/p/premium-methyl-UDI-UMI-adapters-set-B-x24">C02030041 - Premium Methyl UMI-UDI Adapters - Set B</a></li>
</ul>
<ul class="accordion" data-accordion="">
<li class="accordion-navigation"><a href="#v5" style="color: #13b29c;"><i class="fa fa-caret-right"></i> Software for Intelligent Pooling (SIP)</a>
<div id="v5" class="content">
<p>Diagenode's new online<strong> intelligent pooling aid</strong> (RRBS SIP) provides the <strong>optimal pool design</strong> for RRBS to meet your specific sample and analysis needs:</p>
<ul style="list-style-type: disc;">
<li><strong>Time-saving: </strong>avoid<strong> </strong>complex caculations</li>
<li><strong>Highest pooling efficiency</strong> based on qPCR quantification - elevate your pooling effectiveness</li>
<li><strong>Powerful:</strong> incorporates advanced aspects such as number of samples per pool required and the separation between projects</li>
<li><strong>Accurate:</strong> identifies outliers</li>
</ul>
<p></p>
<p>Get access to Diagenode's <a href="https://diagenode.shinyapps.io/RRBS_SIP/">RRBS Software for Intelligent Pooling (SIP</a>)</p>
<p>Get your RRBS template file - <a href="https://www.diagenode.com/files/products/kits/Template_SIP_RRBS.xlsx">Here</a></p>
</div>
</li>
</ul>
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'label1' => 'Characteristics',
'info1' => '<h4>Sequencing quality</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/fig_RRBS_50ng_1_R1_trimmed_per_base_quality.png" width="850px" alt="DNA methylation" caption="false" /></p>
<p><b>Figure 1. Excellent sequencing quality. </b>RRBS libraries were prepared from different starting amounts of human gDNA using Diagenode’s Premium RRBS V2 kit and sequenced in paired-end 50 bp on Illumina NovaSeq instrument generating 30-40 million read pairs per sample. Sequencing statistics reveal that all samples performed well with mean Phred scores above 30 along the entire reads 1 (A) and 2 (B) (data shown for 50 ng gDNA input after trimming).</p>
<p><br /><br /></p>
<h4>Accurate Coverage of CpGs</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/fig2_table_rrbs.png" width="100%" alt="Reduced Representation Bisulfite Sequencing" caption="false" /></p>
<p><b>Table 1. Examples of Premium RRBS V2 sequencing data.</b> UMI data processing enables accurate estimation of CpG counts.</p>
<p><br /><br /></p>
<h4>Focus on CpG-rich regions</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/fig3_50ng_10X_coverage_annotation_cpg.png" width="400px" alt="single nucleotide resolution" caption="false" /></p>
<p><b>Figure </b><b>2. </b><b>Coverage</b> <b>of </b><b>CpGs</b><b> and genomic regions by Premium RRBS V2</b><b>. </b>Diagenode’s Premium RRBS V2 allows a wide interrogation of CpGs (with a sequencing depth >10) of the human genome with a focus on CpG rich regions, especially CpG islands (data shown for 50 ng gDNA input).</p>
<p><br /><br /></p>
<h4>Compatible with all vertebrates</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/table2-rrbs.png" width="100%" alt="CpG-dense regions" caption="false" /></p>
<p><b>Table 2.</b> The kit RRBS v2 is compatible with all vertebrates. The Table 2 shows some examples of results obtained for different species.</p>
<p><br /><br /></p>
<h4>Superior performance</h4>
<ul>
<li>Lower duplicate rate</li>
<li>More CpGs detected</li>
<li>Higher % of uniquely aligned reads containing CpGs</li>
<li>Higher genome coverage (3,3%)</li>
</ul>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/table3-rrbs.png" width="100%" alt="RRBS Kit V2 x96" caption="false" /></p>
<p><b>Table 3.</b> RRBS was performed using Diagenode RRBS v2 kit on HeLa cells and human blood samples (total 10 samples) as well as using competitor kit on HeLa cells, human blood and brain samples (total 10 samples). The Table 3 shows sequencing parameters for 10 samples processed with each kit.</p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/fig3-rrbs.png" width="500px" alt="Reduced Representation Bisulfite Sequencing" caption="false" /></p>
<p><b>Figure 3.</b> Comparison of CpG coverage between competing technologies.</p>',
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'info2' => '<center><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/workflow-RRBS_2021.png" width="700px" alt="Premium RRBS kit" caption="false" /></center>
<p>By cutting the genome using the <strong>restriction enzyme MspI</strong> (CCGG target sites) followed by size selection, DNA is enriched to represent <strong>CpG-rich genomic regions</strong> (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.</p>
<p><span class="label-green" style="margin-bottom: 16px; margin-left: -22px;">NATURE METHODS</span></p>
<p></p>
<h4 class="advertising-feature"><strong>Premium RRBS technology: cost-effective DNA methylation mapping with superior coverage</strong></h4>
<p><a href="#auth-5" class="name"><span class="fn">Christoph Bock</span></a>,<sup href="#affil-auth"> </sup><a href="#auth-4" class="name"><span class="fn">Sharon Squazzo</span></a><span style="font-size: 13.3333px;">, </span><a href="#auth-3" class="name"><span class="fn">Miklos Laczik</span></a><span style="font-size: 13.3333px;">, </span><a href="#auth-2" class="name"><span class="fn">Paul Datlinger</span></a><span style="font-size: 13.3333px;">, </span><a href="#auth-1" class="name"><span class="fn">Anne-Clémence Veillard</span></a></p>
<p>Nature Methods 13 (2016)</p>
<p>Published online <time datetime="2016-01-28">28 January 2016 </time></p>
<p><a href="https://www.nature.com/articles/nmeth.f.391" class="alert button"><span style="font-weight: 400;">CHECK OUT THE NATURE METHODS PAPER</span></a></p>
<p><iframe width="320" height="180" src="https://www.youtube.com/embed/CJn3XEAznu0?rel=0" frameborder="0" allowfullscreen="allowfullscreen"></iframe> <iframe width="320" height="180" src="https://www.youtube.com/embed/4xgRG9qVT5E"></iframe></p>
<script src="chrome-extension://hhojmcideegachlhfgfdhailpfhgknjm/web_accessible_resources/index.js"></script>
<script src="chrome-extension://hhojmcideegachlhfgfdhailpfhgknjm/web_accessible_resources/index.js"></script>',
'label3' => 'Examples of data analysis ',
'info3' => '<h4>Comparative analysis using DNA methylation data from RRBS V2:</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/cluster-dendrogram-rrbs.png" width="500px" alt="DNA methylation data from RRBS V2" caption="false" /></p>
<p><b>Figure 1. Example of cluster dendrogram</b><br />Samples: WT – Wild type; Single KO – Single knock-out; Double KO – Double knock-out</p>
<p>After performing alignment to the reference genome, the methylation calling step provides the methylation percentages for every detected CpG. This methylation rate profile, available now for each sample, can be used to visually explore sample distance by hierarchical clustering. The main branches in this type of dendograms show how the clusters are distinct from each other, and how the objects within each cluster are broadly similar to each other.</p>
<p></p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/clustering-heatmap-rrbs.png" width="850px" alt="DNA methylation data from Premium RRBS kit V2" caption="false" /></p>
<p><b>Figure 2. Example of results: Clustering heatmap of significant DMRs (left) and Volcano plot of DMR (right)</b><br />Samples: WT – Wild type; Single KO – Single knock-out; Double KO – Double knock-out</p>
<p>If the groups of interest contain two or more replicates, a differential methylation analysis can be performed. This type of analysis will identify individual CpGs or regions in the genome that are hyper or hypomethylated in the study group with respect to the control group. A heatmap (left) can be used to visualize the overall distribution of differential methylation between the groups. A volcano plot (right) will show more precisely, the mehtylation difference (x axis) and level of significance of that difference (y axis) allowing the researcher to identify CpGs or regions with large and statistically signifcant differences.</p>
<p></p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/percentage-hypo-rrbs.png" width="500px" alt="CpG-dense regions" caption="false" /></p>
<p><b>Figure 3. Example of results: </b><strong>Percentage of hypo and hypermethylated regions in the samples per chromosome</strong></p>
<p>Horizontal bar plots show the number of hyper and hypomethylated events per chromosome, as a percentage of the sites with 10X coverage and a 25% difference of methylation status.</p>
<p></p>
<h4>QC of the samples</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/per_base_sequence_content-rrbs.png" width="500px" alt="single nucleotide resolution" caption="false" /></p>
<p><b>Figure 4. Example of samples QC:</b> <strong>Per base sequence content</strong></p>
<p>A sequencing quality control software such as FastQC will show the per base sequence content of the sequenced reads. With Diagenode’s RRBS Premium kit, read 2 should show a specific profile for the first three bases that correspond to the MspI restrictive site CGG. The first base being a cytosine followed by a guanine can be either methylated or not; 5mC will remain as C after bisulfite conversion whereas unmethylated C will be converted to T after PCR amplification. The first base in the FastQC per base content plot will therefore show a percentage of reads that vary at that position. In this example, 70% of the reads are methylated at position 1 (C) and 30% are unmethylated. Positions 2 and 3 should show 100% G content.</p>
<p></p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/histogram-cpg-rrbs.png" width="500px" alt="DNA methylation" caption="false" /></p>
<p><b>Figure 5. Example of samples QC: </b><strong>Histogram of % CpG methylation</strong></p>
<p>Another quality control step consists in looking at the frequency of methylation percentages for all CpGs detected. This step can only be performed after alignment and methylation call. The histogram shows a bimodal distribution, where most of the cytosines are either fully methylated or unmethylated. Some cytosines have intermediate methylation levels. A bump in the middle of the plot or a non-bimodal distribution would suggest an inefficiency of the bisulfite treatment. With Diagenode’s RRBS Premium kit, the exact conversion rate can be calculated thanks to the spike-in control sequences that are included in the kit.</p>
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<p>Diagenode’s MicroChIP DiaPure columns have been optimized for the purification and elution of very low amounts of DNA. This rapid method has been validated for epigenetic applications like low input ChIP (e.g. using the True MicroChIP kit) and CUT&Tag (e.g. using Diagenode’s pA-Tn5), but is also compatible with many other applications. The DNA can be eluted at high concentrations in volumes down to 6 μl and it is suitable for any downstream application (e.g. NGS).</p>
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<p>Successful ChIP-seq results generated on 50,000 of K562 cells using True MicroChIP technology. ChIP has been performed accordingly to True MicroChIP protocol (Diagenode, Cat. No. C01010130), including DNA purification using the MicroChIP DiaPure columns. For the library preparation the MicroPlex Library Preparation Kit (Diagenode, Cat. No. C05010001) has been used. The below figure shows the peaks from ChIP-seq experiments using the following Diagenode antibodies: H3K4me1 (C15410194), H3K9/14ac (C15410200), H3K27ac (C15410196) and H3K36me3 (C15410192).</p>
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<h2 style="text-align: center;">MicroChIP DiaPure columns after CUT&Tag</h2>
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<p>メチル化アレイとWGBS技術の長所を有するReduced representation bisulfite sequencing (RRBS) 技術は、あらゆる脊椎動物種で柔軟に使用できるため、大規模コホートの研究においても正確で費用対効果の高いアッセイを行えます。制限MspI酵素(CCGGターゲットサイト)を使用してゲノムを切断し、次にサイズを選択することで、濃縮されたDNAはプロモーターやCpGアイランドを含む関連するターゲットCpGリッチ領域を表します。またRRBS技術はゲノムの一部分を濃縮してメチル化解析を行うことによってシーケンス量を減らす手法でカバレッジが非常に高く、CpGメチル化解析に適しています。(ヒトサンプルで最大700万CpGが検出)。</p>
<h2>Genome-scale DNA methylation analysis</h2>
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<li>High coverage - up to 7 million CpGs detected in human samples</li>
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<h2>Complete end-to-end service</h2>
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<li>Leveraging our <a href="https://www.diagenode.com/en/p/premium-rrbs-kit-V2-x24">Premium RRBS kit V2 </a>widely adopted</li>
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<h2></h2>
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<p><span><i class="fa fa-arrow-circle-right"></i> </span><a href="https://www.diagenode.com/en/categories/dna-methylation-profiling-services">See our other DNA Methylation Profiling Services</a></p>
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<li>Get functional insights</li>
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<p><b>Figure 1</b>. <b>Premium RRBS V2 construct</b>. Integrated UMIs enable removal of PCR duplicates during the data analysis.</p>
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<p><b>Figure 1</b>. <b>Premium RRBS V2 construct</b>. Integrated UMIs enable removal of PCR duplicates during the data analysis.</p>
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'description' => '<p><a href="https://www.diagenode.com/files/products/kits/RRBS-KIT-V2_manual.pdf"><img src="https://www.diagenode.com/img/buttons/bt-manual.png" /></a></p>
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<p><strong>Reduced Representation Bisulfite Sequencing (RRBS)</strong> offers a <strong>cost-effective, focused solution</strong> to perform genome-scale DNA methylation analysis at the <strong>single nucleotide level</strong> in any vertebrate species. The fundamental idea of RRBS is to get a “reduced representation” of the genome. <span>By cutting the genome using the <strong>restriction MspI enzyme</strong> (CCGG target sites) followed by size selection, the DNA sample is enriched with</span><span><span> </span>biologically relevant </span><span><strong>CpG-rich regions</strong> (including<span> </span></span><span>promoters and<span> </span></span><span>CpG islands) in which DNA methylation marks are typically found.</span><span><span> </span></span></p>
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<p>With Diagenode’s Premium RRBS Kit V2 perform cost-effective and reliable genome-scale DNA methylation analysis. Detect around <strong>4 million CpGs</strong> (with coverage > 10) in human samples and identify methylation patterns in CpG rich regions including promoters and CpG islands.</p>
<p>Secure high-quality NGS data for DNA methylation analysis at single base resolution and enjoy our specific and upgraded features:</p>
<ul>
<li>Work with <strong>the lowest DNA amounts</strong> from <strong>25 -100ng gDNA</strong></li>
<li>Process <strong>up to 96 sample</strong> in parallel while saving handling-time and cost per sample with early sample pooling strategy</li>
<li>Utilize our <strong>Software for Intelligent Pooling</strong> (SIP) for better pooling strategies</li>
<li>Get <strong>optimal results</strong> from your sequencer with <strong>Unique Dual Indexing</strong> (UDI)</li>
<li><strong>Identify</strong> and <strong>remove PCR duplicates</strong> from your data with <strong>Unique Molecular Identifiers</strong> (UMIs)</li>
</ul>
<p>The Premium RRBS kit V2 includes all reagents necessary for the library preparation. Specific adapters were designed and validated to fit the technology and are available separately as described below.</p>
<p><strong>For RRBS V2 with UDI and UMI library construction:</strong></p>
<ul>
<li><a href="https://www.diagenode.com/en/p/premium-methyl-UDI-UMI-adapters-set-A-x24">C02030040 - Premium Methyl UMI-UDI Adapters - Set A</a></li>
<li><a href="https://www.diagenode.com/en/p/premium-methyl-UDI-UMI-adapters-set-B-x24">C02030041 - Premium Methyl UMI-UDI Adapters - Set B</a></li>
</ul>
<ul class="accordion" data-accordion="">
<li class="accordion-navigation"><a href="#v5" style="color: #13b29c;"><i class="fa fa-caret-right"></i> Software for Intelligent Pooling (SIP)</a>
<div id="v5" class="content">
<p>Diagenode's new online<strong> intelligent pooling aid</strong> (RRBS SIP) provides the <strong>optimal pool design</strong> for RRBS to meet your specific sample and analysis needs:</p>
<ul style="list-style-type: disc;">
<li><strong>Time-saving: </strong>avoid<strong> </strong>complex caculations</li>
<li><strong>Highest pooling efficiency</strong> based on qPCR quantification - elevate your pooling effectiveness</li>
<li><strong>Powerful:</strong> incorporates advanced aspects such as number of samples per pool required and the separation between projects</li>
<li><strong>Accurate:</strong> identifies outliers</li>
</ul>
<p></p>
<p>Get access to Diagenode's <a href="https://diagenode.shinyapps.io/RRBS_SIP/">RRBS Software for Intelligent Pooling (SIP</a>)</p>
<p>Get your RRBS template file - <a href="https://www.diagenode.com/files/products/kits/Template_SIP_RRBS.xlsx">Here</a></p>
</div>
</li>
</ul>
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'label1' => 'Characteristics',
'info1' => '<h4>Sequencing quality</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/fig_RRBS_50ng_1_R1_trimmed_per_base_quality.png" width="850px" alt="DNA methylation" caption="false" /></p>
<p><b>Figure 1. Excellent sequencing quality. </b>RRBS libraries were prepared from different starting amounts of human gDNA using Diagenode’s Premium RRBS V2 kit and sequenced in paired-end 50 bp on Illumina NovaSeq instrument generating 30-40 million read pairs per sample. Sequencing statistics reveal that all samples performed well with mean Phred scores above 30 along the entire reads 1 (A) and 2 (B) (data shown for 50 ng gDNA input after trimming).</p>
<p><br /><br /></p>
<h4>Accurate Coverage of CpGs</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/fig2_table_rrbs.png" width="100%" alt="Reduced Representation Bisulfite Sequencing" caption="false" /></p>
<p><b>Table 1. Examples of Premium RRBS V2 sequencing data.</b> UMI data processing enables accurate estimation of CpG counts.</p>
<p><br /><br /></p>
<h4>Focus on CpG-rich regions</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/fig3_50ng_10X_coverage_annotation_cpg.png" width="400px" alt="single nucleotide resolution" caption="false" /></p>
<p><b>Figure </b><b>2. </b><b>Coverage</b> <b>of </b><b>CpGs</b><b> and genomic regions by Premium RRBS V2</b><b>. </b>Diagenode’s Premium RRBS V2 allows a wide interrogation of CpGs (with a sequencing depth >10) of the human genome with a focus on CpG rich regions, especially CpG islands (data shown for 50 ng gDNA input).</p>
<p><br /><br /></p>
<h4>Compatible with all vertebrates</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/table2-rrbs.png" width="100%" alt="CpG-dense regions" caption="false" /></p>
<p><b>Table 2.</b> The kit RRBS v2 is compatible with all vertebrates. The Table 2 shows some examples of results obtained for different species.</p>
<p><br /><br /></p>
<h4>Superior performance</h4>
<ul>
<li>Lower duplicate rate</li>
<li>More CpGs detected</li>
<li>Higher % of uniquely aligned reads containing CpGs</li>
<li>Higher genome coverage (3,3%)</li>
</ul>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/table3-rrbs.png" width="100%" alt="RRBS Kit V2 x96" caption="false" /></p>
<p><b>Table 3.</b> RRBS was performed using Diagenode RRBS v2 kit on HeLa cells and human blood samples (total 10 samples) as well as using competitor kit on HeLa cells, human blood and brain samples (total 10 samples). The Table 3 shows sequencing parameters for 10 samples processed with each kit.</p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/fig3-rrbs.png" width="500px" alt="Reduced Representation Bisulfite Sequencing" caption="false" /></p>
<p><b>Figure 3.</b> Comparison of CpG coverage between competing technologies.</p>',
'label2' => 'How it works?',
'info2' => '<center><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/workflow-RRBS_2021.png" width="700px" alt="Premium RRBS kit" caption="false" /></center>
<p>By cutting the genome using the <strong>restriction enzyme MspI</strong> (CCGG target sites) followed by size selection, DNA is enriched to represent <strong>CpG-rich genomic regions</strong> (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.</p>
<p><span class="label-green" style="margin-bottom: 16px; margin-left: -22px;">NATURE METHODS</span></p>
<p></p>
<h4 class="advertising-feature"><strong>Premium RRBS technology: cost-effective DNA methylation mapping with superior coverage</strong></h4>
<p><a href="#auth-5" class="name"><span class="fn">Christoph Bock</span></a>,<sup href="#affil-auth"> </sup><a href="#auth-4" class="name"><span class="fn">Sharon Squazzo</span></a><span style="font-size: 13.3333px;">, </span><a href="#auth-3" class="name"><span class="fn">Miklos Laczik</span></a><span style="font-size: 13.3333px;">, </span><a href="#auth-2" class="name"><span class="fn">Paul Datlinger</span></a><span style="font-size: 13.3333px;">, </span><a href="#auth-1" class="name"><span class="fn">Anne-Clémence Veillard</span></a></p>
<p>Nature Methods 13 (2016)</p>
<p>Published online <time datetime="2016-01-28">28 January 2016 </time></p>
<p><a href="https://www.nature.com/articles/nmeth.f.391" class="alert button"><span style="font-weight: 400;">CHECK OUT THE NATURE METHODS PAPER</span></a></p>
<p><iframe width="320" height="180" src="https://www.youtube.com/embed/CJn3XEAznu0?rel=0" frameborder="0" allowfullscreen="allowfullscreen"></iframe> <iframe width="320" height="180" src="https://www.youtube.com/embed/4xgRG9qVT5E"></iframe></p>
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'label3' => 'Examples of data analysis ',
'info3' => '<h4>Comparative analysis using DNA methylation data from RRBS V2:</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/cluster-dendrogram-rrbs.png" width="500px" alt="DNA methylation data from RRBS V2" caption="false" /></p>
<p><b>Figure 1. Example of cluster dendrogram</b><br />Samples: WT – Wild type; Single KO – Single knock-out; Double KO – Double knock-out</p>
<p>After performing alignment to the reference genome, the methylation calling step provides the methylation percentages for every detected CpG. This methylation rate profile, available now for each sample, can be used to visually explore sample distance by hierarchical clustering. The main branches in this type of dendograms show how the clusters are distinct from each other, and how the objects within each cluster are broadly similar to each other.</p>
<p></p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/clustering-heatmap-rrbs.png" width="850px" alt="DNA methylation data from Premium RRBS kit V2" caption="false" /></p>
<p><b>Figure 2. Example of results: Clustering heatmap of significant DMRs (left) and Volcano plot of DMR (right)</b><br />Samples: WT – Wild type; Single KO – Single knock-out; Double KO – Double knock-out</p>
<p>If the groups of interest contain two or more replicates, a differential methylation analysis can be performed. This type of analysis will identify individual CpGs or regions in the genome that are hyper or hypomethylated in the study group with respect to the control group. A heatmap (left) can be used to visualize the overall distribution of differential methylation between the groups. A volcano plot (right) will show more precisely, the mehtylation difference (x axis) and level of significance of that difference (y axis) allowing the researcher to identify CpGs or regions with large and statistically signifcant differences.</p>
<p></p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/percentage-hypo-rrbs.png" width="500px" alt="CpG-dense regions" caption="false" /></p>
<p><b>Figure 3. Example of results: </b><strong>Percentage of hypo and hypermethylated regions in the samples per chromosome</strong></p>
<p>Horizontal bar plots show the number of hyper and hypomethylated events per chromosome, as a percentage of the sites with 10X coverage and a 25% difference of methylation status.</p>
<p></p>
<h4>QC of the samples</h4>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/per_base_sequence_content-rrbs.png" width="500px" alt="single nucleotide resolution" caption="false" /></p>
<p><b>Figure 4. Example of samples QC:</b> <strong>Per base sequence content</strong></p>
<p>A sequencing quality control software such as FastQC will show the per base sequence content of the sequenced reads. With Diagenode’s RRBS Premium kit, read 2 should show a specific profile for the first three bases that correspond to the MspI restrictive site CGG. The first base being a cytosine followed by a guanine can be either methylated or not; 5mC will remain as C after bisulfite conversion whereas unmethylated C will be converted to T after PCR amplification. The first base in the FastQC per base content plot will therefore show a percentage of reads that vary at that position. In this example, 70% of the reads are methylated at position 1 (C) and 30% are unmethylated. Positions 2 and 3 should show 100% G content.</p>
<p></p>
<p><img src="https://www.diagenode.com/img/product/kits/rrbs_v2/histogram-cpg-rrbs.png" width="500px" alt="DNA methylation" caption=&q