High-throughput bisulfite sequencing in mammalian genomes.
High-throughput bisulfite sequencing in mammalian genomes.
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DOI：
10.1016/j.ymeth.2009.05.003
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发表时间：
2009-07
期刊：
影响因子：
4.8
通讯作者：
Meissner, Alexander
中科院分区：
文献类型：
--
作者：
Smith, Zachary D.;Gu, Hongcang;Bock, Christoph;Gnirke, Andreas;Meissner, Alexander
DNA甲基化是哺乳动物发育所必需的一个重要的表观遗传学标志，在包括癌症在内的许多疾病中都存在异常。在过去的十年里，已经开发和应用了多种方法来表征它在全基因组的分布。其中，简化代表性亚硫酸盐测序(RRBS)产生基于Illumina的核苷酸分辨文库，该文库通过甲基化不敏感的限制性内切酶消化来丰富CpG密集区域。在这里，我们提供了一个广泛的、优化的协议来生成RRBS文库，并讨论了这种策略在甲基组分析中的威力。我们包括关于序列分析的信息和对具有代表性的小鼠MspI产生的RRBS文库感兴趣的基因组区域的相对覆盖率。当代测序和基于阵列的技术与样本吞吐量和覆盖率进行了比较，突出了可用于在基因组规模上研究甲基化的各种选择。
DNA methylation is a critical epigenetic mark that is essential for mammalian development and aberrant in many diseases including cancer. Over the past decade multiple methods have been developed and applied to characterize its genome-wide distribution. Of these, Reduced Representation Bisulfite Sequencing (RRBS) generates nucleotide resolution Illumina-based libraries that enrich for CpG-dense regions by methylation-insensitive restriction digestion. Here we provide an extensive, optimized protocol for generating RRBS libraries and discuss the power of this strategy for methylome profiling. We include information on sequence analysis and the relative coverage over genomic regions of interest for a representative mouse MspI generated RRBS library. Contemporary sequencing and array-based technologies are compared against sample throughput and coverage, highlighting the variety of options available to investigate methylation on the genome-scale.
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