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RRBS (Reduced Representation Bisulfite Sequencing)
single base pair resolution DNA methylation analysis
Reduced Representation Bisulfite Sequencing（RRBS）は、400万個を超えるCpG領域において1塩基対の分解能でDNAメチル化データを取得でき、サンプル間でのメチル化の差も容易に同定することが可能です。 Whole Genome Bisulfite Sequencingよりも費用効果が高く、ビーズアレイベースのDNAメチル化プラットフォームよりも広い範囲のデータを取得できるため、RRBSはサンプル間でDNAメチル化をプロファイリングする理想的なプラットフォームです。
What our customers are saying about us ...
"We worked with Active Motif to process some FFPE and frozen liver samples sourced from 25+year old samples for an RRBS assay. The overall process of submitting samples, communicating with Active Motif, receiving the data, and obtaining the summary was quick and painless. The whole operation was smooth and professionally done."
Brian Chorley, PhD
Environmental Protection Agency
View complete list of testimonials >
RRBS の特長:
- サンプルあたり、300-500万のCpGのメチル化データを取得
- プロモーターおよびCpGアイランドを含む生物学的に関連性のある標的領域の情報
- シークエンスの深さは、30,000,000 リード以上
- DNAの必要量はわずか 1 μg
- ランダムなバーコードの組み込みによるライブラリの偏りを軽減
納品するデータ:
- Raw data (FASTQ) output files.
- Aligned data (BAM) files.
- Visualization files
– BAM files can be used for visualization in
IGV (Integrated Genome Viewer) - Methylation table includes percent methylation value for each CpG.
- Differential methylation analysis.
- Figures & graphs
– Includes coverage depth analysis, heatmaps & pie charts
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RRBS Background & Data
RRBSは、DNAエンドヌクレアーゼ消化と、300bpより小さいDNA断片の増幅により、CpGを含むDNAを単離しメチル化の程度を解析する方法である。 簡単に述べると、まずメチル化非感受性エンドヌクレアーゼMsp1およびTaq1を用いて、それぞれC ^ CGGおよびT ^ CGGを切断し、CpGを有するDNA断片を生成します。 CpGアイランドおよびプロモーターは高密度のCpGを有するので高い頻度で切断され、これらの領域由来の断片は短くなる傾向になります。 短いフラグメントのサブセットは、PCRを使ったライブラリー作成の際に優先的に増幅され、シーケンシングクラスター生成中に追加の選択が行われます。DNAフラクションはシーケンスされ、トータルゲノムの中の小さい断片ではあるが、CpGに富んでいる配列が取得できます。これらの断片は、ゲノムにマッピングされ、数百万あるCpGのメチル化状態を明らかにすることが可能となります。
Figure 1: RRBS data using biopsied human kidney tumor and adjacent normal kidney.
The displayed region is a representative region from the genome-wide data set and shows differential DNA methylation at the promoter of the LAT1 gene. Each block is a separate data point with red representing a methylated cytosine and blue representing the unmethylated base.
Figure 2: RRBS data showing methylation values (as a percentage) for promoter regions.
Values 0 (red) to 1 (green) correspond to 0-100% average methylation frequency of covered CpGs within each promoter region, which are indicated by the light blue line within the heatmap.
Figure 3: Graph illustrating sequencing depth coverage for any given CpG.
The minimum sequence depth requirement for a CpG to be included in the analysis is 3. There are over 1 million CpGs that are only covered at a sequencing depth of 1, approximately 100 that have a sequencing depth coverage of 50 and approximately 5000 with depth coverage over 50 sequence tags per CpG location.
Figure 4: RRBS reproducibility.
Three separate RRBS experiments were performed in duplicate. The number of overlapping and unique CpGs covered in the experiments are represented by the circles in the Venn diagrams. DNA methylation data is provided at over 4.5 million CpGs in human and over 2.8 million CpGs in mouse. The overlap in CpG coverage within duplicates is over 89% for all experiments.
Figure 5: RRBS reproducibility.
Two separate RRBS experiments were performed in duplicate. DNA methylation data from individual CpGs were assigned to promoter regions (2Kb upstream of the TSS) to establish an average methylation percentage over promoters. High reproducibility is indicated by Pearson correlation coefficients that are above 0.93 in each experiment.
