Application of microdroplet PCR for large-scale targeted bisulfite sequencing
Application of microdroplet PCR for large-scale targeted bisulfite sequencing
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DOI：
10.1101/gr.116863.110
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发表时间：
2011-10-01
期刊：
影响因子：
7
通讯作者：
Salomon, Daniel R.
中科院分区：
文献类型：
--
作者：
Komori, H. Kiyomi;LaMere, Sarah A.;Salomon, Daniel R.
基因启动子中DNA CpG二核苷酸的胞嘧啶甲基化是一种调节基因转录的表观遗传修饰。虽然存在许多方法来询问甲基化状态，但目前很少有方法提供大规模的、靶向的、单个CpG解析。我们报告了一种结合亚硫酸氢盐处理，然后微滴PCR与下一代测序的方法，以测定其转录起始位点上游和下游1 kb区域中50个基因的甲基化状态。该方法获得了96%的靶向CpG覆盖率，并证明了CpG岛（CGI）DNA甲基化与转录调控之间的高度相关性。该方法被缩放以询问原代CD4 T细胞中2100个基因的启动子区域中的77，674个CpG的甲基化状态。2100基因文库产生了97%的所有靶向CpG的覆盖率和99%的靶扩增子。
Cytosine methylation of DNA CpG dinucleotides in gene promoters is an epigenetic modification that regulates gene transcription. While many methods exist to interrogate methylation states, few current methods offer large-scale, targeted, single CpG resolution. We report an approach combining bisulfite treatment followed by microdroplet PCR with next-generation sequencing to assay the methylation state of 50 genes in the regions 1 kb upstream of and downstream from their transcription start sites. This method yielded 96% coverage of the targeted CpGs and demonstrated high correlation between CpG island (CGI) DNA methylation and transcriptional regulation. The method was scaled to interrogate the methylation status of 77,674 CpGs in the promoter regions of 2100 genes in primary CD4 T cells. The 2100 gene library yielded 97% coverage of all targeted CpGs and 99% of the target amplicons.