Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution
Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution
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DOI：
10.3791/52246
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发表时间：
2015-02-01
影响因子：
1.2
通讯作者：
Melnick, Ari M.
中科院分区：
文献类型：
--
作者：
Garrett-Bakelman, Francine E.;Sheridan, Caroline K.;Melnick, Ari M.
DNA 甲基化模式图谱在正常和患病组织中得到了深入研究。已经建立了多种方法来检测细胞中的胞嘧啶甲基化模式。开发了全基因组亚硫酸氢盐测序的简化表示，以检测富含 GC 的基因组位点的定量碱基对分辨率胞嘧啶甲基化模式。这是通过结合使用限制性酶和亚硫酸氢盐转化来实现的。增强型简化代表性亚硫酸氢盐测序 (ERRBS) 增加了所覆盖的生物学相关基因组位点，并已用于分析人类、小鼠和其他生物体 DNA 中的胞嘧啶甲基化。 ERRBS 首先对 DNA 进行限制性内切酶消化，生成用于文库制备的低分子量片段。这些片段经过标准文库构建以进行下一代测序。在最终扩增步骤之前对未甲基化胞嘧啶进行亚硫酸氢盐转化，可以对所覆盖的基因组位点中的胞嘧啶甲基化水平进行定量碱基解析。该协议可以在四天内完成。尽管前三个碱基测序的复杂性较低，但 ERRBS 文库在使用指定的测序控制通道时会产生高质量的数据。然后进行绘图和生物信息学分析，并产生可以轻松与各种全基因组平台集成的数据。 ERRBS 可以利用少量的输入材料，从而可以处理人类临床样本并适用于一系列研究应用。制作的视频演示了 ERRBS 协议的关键步骤。
DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.