title: Double restriction-enzyme digestion improves the coverage and accuracy of genome-wide CpG methylation profiling by reduced representation bisulfite sequencing.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors: Science & Technology Department, BGI-Shenzhen, No.11, Bei Shan Industrial Zone, Yantian District, Shenzhen, China.
authors: Junwen
authors: J
authors: Wang
authors: Yudong
authors: Y
authors: Xia
authors: Lili
authors: L
authors: Li
authors: Desheng
authors: D
authors: Gong
authors: Yu
authors: Y
authors: Yao
authors: Huijuan
authors: H
authors: Luo
authors: Hanlin
authors: H
authors: Lu
authors: Na
authors: N
authors: Yi
authors: Honglong
authors: H
authors: Wu
authors: Xiuqing
authors: X
authors: Zhang
authors: Qian
authors: Q
authors: Tao
authors: Fei
authors: F
authors: Gao
mesh_terms: D000818:Animals; D018899:CpG Islands / Q000235:genetics*; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000172:deficiency / Q000235:genetics; D019175:DNA Methylation / Q000187:drug effects*; D055786:Gene Knockout Techniques; D023281:Genomics / Q000379:methods*; D045325:HCT116 Cells; D006801:Humans; D051379:Mice; D015183:Restriction Mapping / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites / Q000494:pharmacology*; D000097584:DNA Methyltransferase 3B
chemical_list: D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D013447:Sulfites; D000097584:DNA Methyltransferase 3B; C042345:hydrogen sulfite