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

• Published in: BMC genomics 2013-01, Vol.14 (1), p.11-11, Article 11
• Main Authors: Wang, Junwen, Xia, Yudong, Li, Lili, Gong, Desheng, Yao, Yu, Luo, Huijuan, Lu, Hanlin, Yi, Na, Wu, Honglong, Zhang, Xiuqing, Tao, Qian, Gao, Fei
• Format: Article
• Language: English
• Subjects: Analysis; Animals; Bisulfite; Colorectal cancer; CpG coverage; CpG islands; CpG Islands - genetics; Deoxyribonucleic acid; Digestion; Digestive enzymes; DNA; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases - deficiency; DNA (Cytosine-5-)-Methyltransferases - genetics; DNA methylation; DNA Methylation - drug effects; DNA Methyltransferase 3B; DNA sequencing; Double-enzyme RRBS; Enzymes; Gene Knockout Techniques; Genetic research; Genetics; Genomes; Genomics; Genomics - methods; HCT116 Cells; Humans; Instrument industry; Methodology; Methods; Methylation; Mice; Nucleotide sequencing; Restriction Mapping - methods; Sequence Analysis, DNA - methods; Single-enzyme RRBS; Sulfites; Sulfites - pharmacology; Tumor cell lines
• ISSN: 1471-2164; 1471-2164
• DOI: 10.1186/1471-2164-14-11

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.