Mirror bisulfite sequencing – a method for single-base resolution of hydroxymethylcytosine. Homo sapiens

While the role of 5-methylcytosine has been well studied, the biological role of 5-hydroxymethylcytosine still remains unclear due to the limited methods available for single-base detection of 5-hydroxymethylcytosine (5hmC). Here we present Mirror bisulfite sequencing for the detection of 5hmC at a single CpG site by synthesizing a DNA strand to mirror the parental strand. This semi-conservative duplex is sequentially treated with β-glucosyltransferase and M.SssI methylase. A glucosyl-5hmCpG in the parental strand inhibits methylation of the mirroring CpG site, and after bisulfite conversion, a thymine in the mirroring strand indicates a 5hmCpG site in the parental strand whereas a cytosine indicates a non-5hmC site. Using this method, the 5hmC levels of various human tissues and paired tumor liver tissues were mapped genome-wide. Overall design: developing a new assay for genomic profiling of 5hmC

尽管5-甲基胞嘧啶的生物学功能已得到充分解析，但由于可用于单碱基水平检测5-羟甲基胞嘧啶（5-hydroxymethylcytosine，5hmC）的方法较为匮乏，5hmC的生理功能仍未完全阐明。本研究报道了一种镜像亚硫酸氢盐测序（Mirror bisulfite sequencing）技术，通过合成与亲本链镜像匹配的DNA链，实现单CpG位点的5hmC精准检测。该半保守双链依次经β-葡萄糖基转移酶与M.SssI甲基转移酶处理。亲本链上的葡糖基修饰5hmCpG会抑制镜像链对应CpG位点的甲基化；经亚硫酸氢盐转化后，镜像链中出现的胸腺嘧啶即对应亲本链中的5hmCpG位点，而胞嘧啶则代表该位点为非5hmC位点。利用该方法，我们对多种人体组织及配对的肿瘤肝脏组织开展了全基因组范围的5hmC水平图谱绘制。实验整体设计：开发一种用于5hmC全基因组图谱分析的新型检测方法。