- Scale: RRBS is a high-throughput method well suited to genome-wide DNA methylation studies.
- Cost: The RRBS method uses restriction enzymes that lead to amplification of CpG-rich regions, such as CpG islands and promoter regions. The result is focused sequencing efforts that provide good sequencing depth over regions of interest. Resources are spared by under-representing CpG-suppressed regions.
- Reproducibility: Comparison of sample analyses shows highly reproducible results: 85-95% overlap between multiple samples, which enables comparative studies.
- Single nucleotide resolution: RRBS localizes methylation status to individual CpG sites within each DNA fragment, providing superior data to alternative profiling methods such as MeDIP-Seq, which do not provide equivalent resolution.
- Coverage: RRBS has very good coverage for more than 5 million CpG sites at the whole-genome scale, 65–75% of which are covered by ≥10x coverage depth.
- Sample size and quality: Analysis can be accomplished with very limited genomic DNA, even if it is degraded, making it possible for our collaborators to focus on their precious human tissue samples and is idea for clinical sample detection (including FFPE samples).
Systematic Assessment of Reduced Representation Bisulfite Sequencing to Human Blood Samples: A Promising Method for Large-Sample-Scale Epigenomic Studies. J Biotechnol. 6 Jul 2011.
Complementary to the time- and cost-intensive direct bisulfite sequencing, we applied RRBS to the human peripheral blood mononuclear cells from YH, the Asian individual whose genome and epigenome has been deciphered in the YH project and systematically assessed the genomic coverage, coverage depth and reproducibility of this technology as well as the concordance of DNA methylation levels measured by RRBS and direct bisulfite sequencing for the detected CpG sites. Our result suggests that RRBS can cover more than half of CpG islands and promoter regions with a good coverage depth and the proportion of the CpG sites covered by the biological replicates reaches 80-90%, indicating good reproducibility.
- Data Filtering (removing adaptor sequences, contamination and low-quality reads from raw reads) and Production Statistics
- Reads Alignment
- Coverage analysis for Promoter and CpG Islands
- Covered Promoter and CpG Islands
- Theoretical and Experimental Coverage of Cytosine
- Cumulative Distribution of Effective Sequencing Depth in Cytosine
- Coverage of CpG sites
- Comparison of theoretical coverage and coverage in each different depth
- Methylation Analysis for Promoter and CGI
- Purity:OD260/280=1.8-2.0, without RNA contamination
- Concentration: ≥50ng/μl
- DNA amount: single library preparation starts from at least 3μg, the more the better. For FFPE or other degraded DNA samples, 5 μg is recommended.
RRBS is available for both human and mouse samples