Carr Ian M, Valleley Elizabeth M A, Cordery Sarah F, Markham Alexander F, Bonthron David T
Leeds Institute for Molecular Medicine, University of Leeds, Leeds, UK.
Nucleic Acids Res. 2007;35(10):e79. doi: 10.1093/nar/gkm330. Epub 2007 May 21.
Bisulphite genomic sequencing is a widely used technique for detailed analysis of the methylation status of a region of DNA. It relies upon the selective deamination of unmethylated cytosine to uracil after treatment with sodium bisulphite, usually followed by PCR amplification of the chosen target region. Since this two-step procedure replaces all unmethylated cytosine bases with thymine, PCR products derived from unmethylated templates contain only three types of nucleotide, in unequal proportions. This can create a number of technical difficulties (e.g. for some base-calling methods) and impedes manual analysis of sequencing results (since the long runs of T or A residues are difficult to align visually with the parent sequence). To facilitate the detailed analysis of bisulphite PCR products (particularly using multiple cloned templates), we have developed a visually intuitive program that identifies the methylation status of CpG dinucleotides by analysis of raw sequence data files produced by MegaBace or ABI sequencers as well as Staden SCF trace files and plain text files. The program then also collates and presents data derived from independent templates (e.g. separate clones). This results in a considerable reduction in the time required for completion of a detailed genomic methylation project.
亚硫酸氢盐基因组测序是一种广泛应用的技术，用于详细分析DNA区域的甲基化状态。它依赖于用亚硫酸氢钠处理后，将未甲基化的胞嘧啶选择性脱氨为尿嘧啶，通常随后对选定的目标区域进行PCR扩增。由于这个两步过程用胸腺嘧啶取代了所有未甲基化的胞嘧啶碱基，从未甲基化模板衍生的PCR产物只包含三种类型的核苷酸，比例不等。这可能会产生一些技术难题（例如对于某些碱基识别方法），并阻碍对测序结果的人工分析（因为长串的T或A残基很难与亲本序列进行视觉比对）。为了便于对亚硫酸氢盐PCR产物进行详细分析（特别是使用多个克隆模板），我们开发了一个视觉直观的程序，通过分析由MegaBace或ABI测序仪产生的原始序列数据文件以及Staden SCF跟踪文件和平文本文档，来识别CpG二核苷酸的甲基化状态。该程序还会整理并呈现来自独立模板（例如单独的克隆）的数据。这使得完成一个详细的基因组甲基化项目所需的时间大幅减少。
