Reduced-representation Bisulfite Sequencing (RRBS) Service

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. 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.

1. Whole Genome Bisulfite Sequencing (WGBS) Service
2. Targeted Methylation Sequencing Service
3. Chromatin Immunoprecipitation (ChIP) Sequencing Service
4. Chromatin Accessibility Sequencing Service
5. Epigenomic Sequencing Service for Formalin-Fixed Paraffin-Embedded (FFPE) Tissue