Open Access Highly Accessed Methodology article

Double restriction-enzyme digestion improves the coverage and accuracy of genome-wide CpG methylation profiling by reduced representation bisulfite sequencing

Junwen Wang1, Yudong Xia1, Lili Li2, Desheng Gong1, Yu Yao1, Huijuan Luo1, Hanlin Lu1, Na Yi1, Honglong Wu1, Xiuqing Zhang1, Qian Tao2* and Fei Gao1*

Author affiliations

1 Science & Technology Department, BGI-Shenzhen, No.11, Bei Shan Industrial Zone, Yantian District, Shenzhen, China

2 Cancer Epigenetics Laboratory, Department of Clinical Oncology, State Key Laboratory of Oncology in South China, Sir YK Pao Center for Cancer and Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, SAR, China

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Citation and License

BMC Genomics 2013, 14:11  doi:10.1186/1471-2164-14-11

Published: 16 January 2013



Reduced representation bisulfite sequencing (RRBS) was developed to measure DNA methylation of high-CG regions at single base-pair resolution, and has been widely used because of its minimal DNA requirements and cost efficacy; however, the CpG coverage of genomic regions is restricted and important regions with low-CG will be ignored in DNA methylation profiling. This method could be improved to generate a more comprehensive representation.


Based on in silico simulation of enzyme digestion of human and mouse genomes, we have optimized the current single-enzyme RRBS by applying double enzyme digestion in the library construction to interrogate more representative regions. CpG coverage of genomic regions was considerably increased in both high-CG and low-CG regions using the double-enzyme RRBS method, leading to more accurate detection of their average methylation levels and identification of differential methylation regions between samples. We also applied this double-enzyme RRBS method to comprehensively analyze the CpG methylation profiles of two colorectal cancer cell lines.


The double-enzyme RRBS increases the CpG coverage of genomic regions considerably over the previous single-enzyme RRBS method, leading to more accurate detection of their average methylation levels. It will facilitate genome-wide DNA methylation studies in multiple and complex clinical samples.

Single-enzyme RRBS; Double-enzyme RRBS; DNA methylation; CpG coverage