Life Technologies, Foster City, California, United States of America.
PLoS One. 2010 Feb 22;5(2):e9320. doi: 10.1371/journal.pone.0009320.
Methylation, the addition of methyl groups to cytosine (C), plays an important role in the regulation of gene expression in both normal and dysfunctional cells. During bisulfite conversion and subsequent PCR amplification, unmethylated Cs are converted into thymine (T), while methylated Cs will not be converted. Sequencing of this bisulfite-treated DNA permits the detection of methylation at specific sites. Through the introduction of next-generation sequencing technologies (NGS) simultaneous analysis of methylation motifs in multiple regions provides the opportunity for hypothesis-free study of the entire methylome. Here we present a whole methylome sequencing study that compares two different bisulfite conversion methods (in solution versus in gel), utilizing the high throughput of the SOLiD System. Advantages and disadvantages of the two different bisulfite conversion methods for constructing sequencing libraries are discussed. Furthermore, the application of the SOLiD bisulfite sequencing to larger and more complex genomes is shown with preliminary in silico created bisulfite converted reads.
甲基化，即在胞嘧啶（C）上添加甲基基团，在正常和功能失调细胞中的基因表达调控中起着重要作用。在亚硫酸氢盐转化和随后的 PCR 扩增过程中，未甲基化的 C 被转化为胸腺嘧啶（T），而甲基化的 C 则不会被转化。对这种经亚硫酸氢盐处理的 DNA 进行测序可以检测到特定位点的甲基化。通过引入下一代测序技术（NGS），可以同时分析多个区域的甲基化模式，从而提供了对整个甲基组进行无假设研究的机会。在这里，我们展示了一项全甲基组测序研究，该研究比较了两种不同的亚硫酸氢盐转化方法（溶液法与胶内法），并利用 SOLiD 系统的高通量特性。讨论了两种不同的亚硫酸氢盐转化方法在构建测序文库方面的优缺点。此外，还展示了 SOLiD 亚硫酸氢盐测序在更大、更复杂基因组上的应用，包括初步通过计算机生成的亚硫酸氢盐转化读取序列。
