Sequence analysis and editing for bisulphite genomic sequencing projects
Sequence analysis and editing for bisulphite genomic sequencing projects
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DOI：
10.1093/nar/gkm330
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发表时间：
2007-05-01
影响因子：
14.9
通讯作者：
Bonthron, David T.
中科院分区：
文献类型：
--
作者：
Carr, Ian M.;Valleley, Elizabeth M. A.;Bonthron, David T.
亚硫酸氢盐基因组测序是用于详细分析DNA区域甲基化状态的广泛使用的技术。它依赖于用亚硫酸氢钠处理后未甲基化的胞嘧啶选择性脱氨基为尿嘧啶，通常随后对所选靶区域进行PCR扩增。由于这两步程序用胸腺嘧啶取代了所有未甲基化的胞嘧啶碱基，因此来自未甲基化模板的PCR产物仅含有三种不同比例的核苷酸。这可能会造成一些技术问题（如：G.对于某些碱基识别方法），并且妨碍了测序结果的手动分析（因为T或A残基的长运行难以与亲本序列视觉比对）。为了便于亚硫酸氢盐PCR产物的详细分析（特别是使用多个克隆模板），我们开发了一个直观的程序，通过分析MegaBace或ABI测序仪产生的原始序列数据文件以及Staden SCF跟踪文件和纯文本文件来识别CpG二核苷酸的甲基化状态。然后，该程序还整理和呈现来自独立模板的数据（例如，G.单独的克隆）。这导致完成详细的基因组甲基化项目所需的时间大大减少。
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.