Garrett-Bakelman Francine E, Sheridan Caroline K, Kacmarczyk Thadeous J, Ishii Jennifer, Betel Doron, Alonso Alicia, Mason Christopher E, Figueroa Maria E, Melnick Ari M
Department of Medicine, Weill Cornell Medical College;
Department of Medicine, Weill Cornell Medical College.
J Vis Exp. 2015 Feb 24(96):e52246. doi: 10.3791/52246.
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.
DNA甲基化模式图谱在正常组织和病变组织中得到了广泛研究。已经建立了多种方法来探究细胞中的胞嘧啶甲基化模式。全基因组亚硫酸氢盐测序的简化表征技术被开发出来，用于检测富含GC的基因组位点上的定量碱基对分辨率的胞嘧啶甲基化模式。这是通过结合使用限制性内切酶和随后的亚硫酸氢盐转化来实现的。增强型简化表征亚硫酸氢盐测序（ERRBS）增加了所覆盖的生物学相关基因组位点，并已用于分析来自人类、小鼠和其他生物体的DNA中的胞嘧啶甲基化。ERRBS首先通过对DNA进行限制性内切酶消化来产生低分子量片段，用于文库制备。这些片段经过标准的文库构建用于下一代测序。在最终扩增步骤之前对未甲基化的胞嘧啶进行亚硫酸氢盐转化，能够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分辨率分析。该方案可在四天内完成。尽管测序的前三个碱基复杂度较低，但使用指定的测序对照泳道时，ERRBS文库可产生高质量数据。然后进行图谱绘制和生物信息学分析，并产生可轻松与各种全基因组平台整合的数据。ERRBS可以利用少量的输入材料，使得处理人类临床样本成为可能，并适用于一系列研究应用。所制作的视频展示了ERRBS方案的关键步骤。
