Determining DNA Methylation Profiles Using Sequencing
Determining DNA Methylation Profiles Using Sequencing
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DOI：
10.1007/978-1-61779-089-8_16
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发表时间：
2011-01-01
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影响因子：
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通讯作者：
Pellegrini, Matteo
中科院分区：
文献类型：
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作者：
Feng, Suhua;Rubbi, Liudmilla;Pellegrini, Matteo
胞嘧啶甲基化是一种表观遗传标记，对DNA转录和复制的调控具有重要影响。DNA甲基化模式在细胞分裂中高度保守，因此具有高度遗传性。此外，在多细胞生物体中，DNA甲基化模式是细胞分化和组织特异性表达模式的关键决定因素。最后，DNA去甲基化酶可以在特定的发育阶段影响DNA甲基化的整体水平。亚硫酸氢盐测序被认为是测量胞嘧啶甲基化状态的金标准。亚硫酸氢钠将未甲基化的胞嘧啶转化为尿嘧啶（PCR后转化为胸腺嘧啶），而甲基化的胞嘧啶则不被转化。通过将亚硫酸氢盐处理的DNA映射回原始参考基因组，然后可以确定单个胞嘧啶的甲基化状态。随着下一代测序仪在过去几年中的出现，现在可以确定整个基因组的甲基化状态。在这里，我们详细描述了用于制备亚硫酸氢盐处理的文库的两种方案，其可以使用Illumina GAII测序仪进行测序。其中第一个使用预甲基化的衔接子，这是不受亚硫酸氢盐处理，而第二个使用两阶段的衔接子策略，不需要预甲基化的衔接子。我们还描述了专门的协议映射亚硫酸氢盐转换读取。这些方法允许确定基因组中每个胞嘧啶的甲基化状态。
Cytosine methylation is an epigenetic mark that has a significant impact on the regulation of transcription and replication of DNA. DNA methylation patterns are highly conserved across cell divisions and arc therefore highly heritable. Furthermore, in multicellular organisms, DNA methylation patterning is a key determinant of cellular differentiation and tissue-specific expression patterns. Lastly, DNA demethylases can affect global levels of DNA methylation during specific stages of development. Bisulfite sequencing is considered the gold standard for measuring the methylation state of cytosines. Sodium bisulfite converts unmethylated cytosines to uracils (which after PCR are converted to thymines), while leaving methylated cytosines unconverted. By mapping bisulfite treated DNA back to the original reference genome, it is then possible to determine the methylation state of individual cytosines. With the advent of next-generation sequencers during the past few years, it is now possible to determine tit:: methylation state of an entire genome. Here, we describe in detail two protocols for preparing bisulfite treated libraries, which may be sequenced using Illumina GAII sequencers. The first of these uses premethylated adapters, which are not affected by bisulfite treatments, while the second uses a two-stage adapter strategy and does not require premethylation of the adapters. We also describe the specialized protocol for mapping bisulfite converted reads. These approaches allow one to determine the methylation state of each cytosine in the genome.