Comparison of Hi-C results using in-solution versus in-nucleus ligation.
Comparison of Hi-C results using in-solution versus in-nucleus ligation.
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DOI：
10.1186/s13059-015-0753-7
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发表时间：
2015-08-26
期刊：
影响因子：
12.3
通讯作者：
Fraser P
中科院分区：
文献类型：
--
作者：
Nagano T;Várnai C;Schoenfelder S;Javierre BM;Wingett SW;Fraser P
染色体构象捕获和各种衍生方法，如4C，5C和Hi-C已经成为分析细胞核中基因组三维结构的标准工具。这些方法采用稀释的交联染色质复合物的连接，旨在促进邻近依赖性的复合物内连接。在单细胞Hi-C的开发过程中，我们设计了一种替代Hi-C方案，其在保存的细胞核中而不是在溶液中进行连接。在这里，我们直接比较了采用核内连接与标准溶液内连接的Hi-C方法。我们表明，核内连接导致染色体间接触的水平始终较低。通过染色质混合实验，我们表明，一个显着的大部分染色体间的接触是假连接事件在溶液中连接过程中形成的结果。核内连接显著减少了这种实验噪音来源，并导致重复之间的重现性提高。我们还发现，核内连接消除了限制性片段长度的偏见，发现在溶液中连接。这些改进导致长距离染色体内和染色体间接触的更大再现性，以及增强的结构特征如拓扑相关域边界的检测。我们的结论是，核内连接在更宽的距离范围内更一致地捕获染色质相互作用，并显着降低实验噪音和偏差。核内连接产生更高质量的Hi-C文库，同时简化实验程序。我们认为，整个范围的3C应用程序可能会显示类似的好处，从核内结扎。本文的在线版本（doi：10.1186/s13059-015-0753-7）包含补充材料，可供授权用户使用。
Chromosome conformation capture and various derivative methods such as 4C, 5C and Hi-C have emerged as standard tools to analyze the three-dimensional organization of the genome in the nucleus. These methods employ ligation of diluted cross-linked chromatin complexes, intended to favor proximity-dependent, intra-complex ligation. During development of single-cell Hi-C, we devised an alternative Hi-C protocol with ligation in preserved nuclei rather than in solution. Here we directly compare Hi-C methods employing in-nucleus ligation with the standard in-solution ligation. We show in-nucleus ligation results in consistently lower levels of inter-chromosomal contacts. Through chromatin mixing experiments we show that a significantly large fraction of inter-chromosomal contacts are the result of spurious ligation events formed during in-solution ligation. In-nucleus ligation significantly reduces this source of experimental noise, and results in improved reproducibility between replicates. We also find that in-nucleus ligation eliminates restriction fragment length bias found with in-solution ligation. These improvements result in greater reproducibility of long-range intra-chromosomal and inter-chromosomal contacts, as well as enhanced detection of structural features such as topologically associated domain boundaries. We conclude that in-nucleus ligation captures chromatin interactions more consistently over a wider range of distances, and significantly reduces both experimental noise and bias. In-nucleus ligation creates higher quality Hi-C libraries while simplifying the experimental procedure. We suggest that the entire range of 3C applications are likely to show similar benefits from in-nucleus ligation. The online version of this article (doi:10.1186/s13059-015-0753-7) contains supplementary material, which is available to authorized users.