全基因组染色质相互作用Hi-C文库制备的优化及其质量控制

摘要

Highest-throughput chromosome conformation capture (Hi-C) is one of the key assays for genomewide chromatin interaction studies.It is a time-consuming process that involves many steps and many different kinds of reagents,consumables,and equipments.At present,the reproducibility is unsatisfactory.By optimizing the key steps of the Hi-C experiment,such as crosslinking,pretreatment of digestion,inactivation of restriction enzyme,and in situ ligation etc.,we established a robust Hi-C procedure and prepared two biological replicates of Hi-C libraries from the GM12878 cells.After preliminary quality control by Sanger sequencing,the two replicates were high-throughput sequenced.The bioinformatics analysis of the raw sequencing data revealed the mapping-ability and pair-mate rate of the raw data were around 90％ and 72％,respectively.Additionally,after removal of self-circular ligations and dangling-end products,more than 96％ of the valid pairs were reached.Genome-wide interactome profiling shows clear topological associated domains (TADs),which is consistent with previous reports.Further correlation analysis showed that the two biological replicates strongly correlate with each other in terms of both bin coverage and all bin pairs.All these results indicated that the optimized Hi-C procedure is robust and stable,which will be very helpful for the wide applications of the Hi-C assay.%Hi-C(highest-throughput chromosome conformation capture)技术是近年出现的一种研究染色质相互作用的关键技术.该技术步骤多、耗时长,涉及的试剂耗材繁杂,目前常规流程还有较多可以改进优化的步骤.本研究以GM12878细胞为材料,通过优化常规Hi-C实验中的交联、酶切、限制性内切酶的失活、末端生物素标记、原位连接等关键步骤,建立了稳健的Hi-C流程,制备了相应的Hi-C文库.文库经初步的Sanger测序等质量控制以后,两个生物学重复文库进行了高通量测序.测序结果利用生物信息学处理发现:原始测序数据中可比对率和配对率分别达到90％和72％左右.此外,去除自连片段(self-circular ligation)和dangling-ends片段以后,可获得超过96％的有效相互作用对,其中染色体内相互作用数据达到60％.进一步的染色体相互作用热图分析可见清晰拓扑学相关结构域TADs(topologically associated domains),与已发表的文献报道一致.而两次生物学重复之间的相关性分析则表明bin coverage和all bin pairs的相关性都极强.上述结果表明通过对常规Hi-C流程关键步骤的优化,本研究建立了高效、稳健、可靠的Hi-C流程,对Hi-C技术的进一步使用与推广具有重要的参考价值.