Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany.
Nat Protoc. 2013 Oct;8(10):2022-32. doi: 10.1038/nprot.2013.118. Epub 2013 Sep 26.
Epigenetic modifications such as carbon 5 methylation of the cytosine base in a CpG dinucleotide context are involved in the onset and progression of human diseases. A comprehensive understanding of the role of genome-wide DNA methylation patterns, the methylome, requires quantitative determination of the methylation states of all CpG sites in a genome. So far, analyses of the complete methylome by whole-genome bisulfite sequencing (WGBS) are rare because of the required large DNA quantities, substantial bioinformatic resources and high sequencing costs. Here we describe a detailed protocol for tagmentation-based WGBS (T-WGBS) and demonstrate its reliability in comparison with conventional WGBS. In T-WGBS, a hyperactive Tn5 transposase fragments the DNA and appends sequencing adapters in a single step. T-WGBS requires not more than 20 ng of input DNA; hence, the protocol allows the comprehensive methylome analysis of limited amounts of DNA isolated from precious biological specimens. The T-WGBS library preparation takes 2 d.
表观遗传修饰，如 CpG 二核苷酸序列中胞嘧啶碱基的 5 位碳甲基化，参与了人类疾病的发生和发展。全面了解全基因组 DNA 甲基化模式（甲基组）需要定量测定基因组中所有 CpG 位点的甲基化状态。到目前为止，由于需要大量的 DNA 数量、大量的生物信息学资源和高昂的测序成本，全基因组亚硫酸氢盐测序（WGBS）对完整甲基组的分析很少。在这里，我们描述了一种基于标签酶切的 WGBS（T-WGBS）的详细方案，并证明了与传统 WGBS 相比其可靠性。在 T-WGBS 中，一种超活性 Tn5 转座酶在一步中即可将 DNA 片段化并添加测序接头。T-WGBS 所需的输入 DNA 不超过 20ng；因此，该方案允许从珍贵的生物样本中有限量的 DNA 中进行全面的甲基组分析。T-WGBS 文库制备需要 2 天。
