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Reduced-representation Bisulfite Sequencing (RRBS) Service

Introduction Protocol Features Samples Q&As Resource

Reduced-representation bisulfite sequencing (RRBS) is an alternative to whole genome bisulfite sequencing (WGBS), applied for analyzing the genome-wide methylation profiles at single-nucleotide resolution. This method is high-efficiency and low-cost compared with WGBS. Leveraging the method, Creative Biolabs is able to provide unrivaled RRBS services with high quality and low cost, helping you better study the genome-wide methylation state.

Introduction of RRBS

RRBS is an optimized bisulfite-based sequencing method and used for whole-genome DNA methylation analysis. This method uses restriction enzyme digestion and DNA size selection to enrich the regions of the genome containing CpG islands which are the major sites of DNA methylation. Thus, bisulfite sequencing can be performed on the enriched gene regions instead of the whole genome, thereby reducing sequence redundancy and cost. This technique is an efficient and cost-effective technique for analyzing the genome-wide methylation profiles at single-nucleotide resolution.

Protocol

A standard protocol of RRBS is described below.

Enzyme Digestion
Genomic DNA is digested using a methylation-insensitive restriction enzyme, such as MspI, to generate DNA fragments of various sizes, each with a CpG at each end.

End Repair & A-tailing
End repair is required to fill in the 3'-terminal of the ends of the strands. A-Tailing is also necessary for adapter ligation due to the adapters contain a 3'-T overhang.

Adapter Ligation
Adapters are ligated to the DNA fragments. It is noted the adapters must contain methylated cytosines so that they cannot be converted to uracil in the subsequent bisulfite conversion step.

Size Selection
After adapter ligation, different sizes of DNA fragments are separated using gel electrophoresis. 40-120 bp and 120-220 bp length fragments are puried for bisulfite conversion.

Bisulfite Conversion
After bisulfite treatment, non-methylated cytosines are converted to uracil residues, while methylated cytosines remain unchanged.

PCR Amplification
The bisulfite converted DNA is amplified by PCR.

Sequencing
Illumina NGS platform is most commonly performed to sequence the amplified libraries.

Bioinformatics
Sequence alignment and methylation identification.

Fig. 1 Reduced representation bisulfite sequencing protocol.

Features

Highly accurate: single-base resolution CpG detection
High coverage: covering nearly all CpG sites across the entire genome
Cost-effective: a lower DNA methylation sequencing cost compared to WGBS
End-to-end package: from sample preparation to data analysis
Bioinformatics: raw data quality control, sequence alignment, methylation calling, differential methylation analysis, and differentially methylated regions (DMRs) cluster analysis, or custom bioinformatics analysis according to customers’ requirements.
Sequencing: Hiseq 2 × 150, ≥ 30X sequencing depth to generate more than 5 G data per sample, Q30 sequencing score > 80%
Rapid turnaround time: 4~8 weeks from sample submission

Sample Requirements

DNA amount: ≥ 3 μg for regular sample, ≥ 5 μg for FFPE sample
DNA concentration: ≥ 50 ng/μL
Purity: OD260/280 = 1.8-2.0 without degradation, no protein or RNA contamination

RRBS allows genome-scale DNA methylation analysis in a high-accurate and low-cost manner. Creative Biolabs is very skilled in utilizing RRBS technique to detect single-base methylation status across the entire genome. Our service package includes sample preparation, enzyme digestion, adapter ligation, size selection, bisulfite treatment, PCR amplification, sequencing, and bioinformatics analysis. Our professional expert team is devoted to delivering highly reliable and accurate sequencing data to promote your whole-genome methylation researches. Please contact us for more information and a detailed quote.

Q&As

Q: How does RRBS differ from whole genome bisulfite sequencing (WGBS)?

A: RRBS targets specific CpG-rich regions of the genome, reducing the amount of data that needs to be sequenced compared to WGBS. This approach lowers costs and focuses on the most relevant genomic areas for methylation studies, which is particularly useful in cancer research.

Q: What types of cancer samples can be analyzed using RRBS?

A: RRBS can be applied to various cancer sample types, including fresh, frozen, and FFPE tissues. This flexibility makes it suitable for both retrospective and prospective studies, enabling comprehensive methylation profiling across different cancer stages and types.

Q: What is the typical data output from an RRBS experiment?

A: An RRBS experiment typically provides single-base resolution methylation data for millions of CpG sites across the genome. The data includes methylation levels, differentially methylated regions (DMRs), and other relevant epigenetic features that are crucial for cancer research.

Q: How does bioinformatics analysis support RRBS data interpretation?

A: Bioinformatics tools are essential for processing RRBS data, including quality control, alignment to the reference genome, and methylation calling. Advanced analyses can identify differentially methylated regions and correlate them with gene expression changes in cancer.

Q: What are the advantages of using RRBS for cancer studies?

A: RRBS provides high-resolution methylation data with lower costs and smaller sample requirements compared to WGBS. It also allows the use of various sample types, including formalin-fixed paraffin-embedded (FFPE) tissues, making it versatile for different cancer research applications.