Hi-CO: 3D genome structure analysis with nucleosome resolution
Hi-CO: 3D genome structure analysis with nucleosome resolution
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DOI：
10.1038/s41596-021-00543-z
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发表时间：
2021-05-28
期刊：
影响因子：
14.8
通讯作者：
Taniguchi, Yuichi
中科院分区：
文献类型：
--
作者：
Ohno, Masae;Ando, Tadashi;Taniguchi, Yuichi
核小体是基因组的基本组织单位。核小体的折叠结构与基因组功能密切相关，并且已被报道与各种核蛋白与基因组位点的结合动态相互作用。在这里，我们描述了我们的高通量染色体构象捕获与核小体取向（Hi-CO）技术，以获得3D核小体的位置与它们在细胞核中的每个基因组位点的方向。该技术包括核小体邻近分析的实验过程和3D建模的计算过程。实验过程是基于高通量染色体构象捕获（Hi-C）分析的改进方法。传统的Hi-C允许在1-10 kbp分辨率的基因组位点之间进行空间邻近分析，而我们的Hi-CO允许在每个核小体位点的DNA进入或退出点之间进行邻近分析。该分析通过在微球菌核酸酶片段化基因组中的每个核小体中的进入/退出点之间进行连接，并通过用下一代测序定量连接产物的频率来实现。我们的方案通过干净地排除由于微球菌核酸酶的频繁基因组片段化而丰富的不需要的非连接产物来实现这种分析。计算过程是基于模拟退火分子动力学，它允许确定优化的3D位置和方向的每个核小体，满足邻近连接数据足够好。通常，用1.3亿个测序读数检查酿酒酵母基因组有助于以6.8 nm分辨率分析总共66，360个核小体基因座。该技术需要2-3周的测序文库制备和2周的simulation.The作者描述了一个逐步的工作流程，用于生成全基因组染色质相互作用图，核小体分辨率使用邻近连接和深度测序，然后建模最佳核小体位置和方向。
The nucleosome is the basic organizational unit of the genome. The folding structure of nucleosomes is closely related to genome functions, and has been reported to be in dynamic interplay with binding of various nuclear proteins to genomic loci. Here, we describe our high-throughput chromosome conformation capture with nucleosome orientation (Hi-CO) technology to derive 3D nucleosome positions with their orientations at every genomic locus in the nucleus. This technology consists of an experimental procedure for nucleosome proximity analysis and a computational procedure for 3D modeling. The experimental procedure is based on an improved method of high-throughput chromosome conformation capture (Hi-C) analysis. Whereas conventional Hi-C allows spatial proximity analysis among genomic loci with 1-10 kbp resolution, our Hi-CO allows proximity analysis among DNA entry or exit points at every nucleosome locus. This analysis is realized by carrying out ligations among the entry/exit points in every nucleosome in a micrococcal-nuclease-fragmented genome, and by quantifying frequencies of ligation products with next-generation sequencing. Our protocol has enabled this analysis by cleanly excluding unwanted non-ligation products that are abundant owing to the frequent genome fragmentation by micrococcal nuclease. The computational procedure is based on simulated annealing-molecular dynamics, which allows determination of optimized 3D positions and orientations of every nucleosome that satisfies the proximity ligation data sufficiently well. Typically, examination of the Saccharomyces cerevisiae genome with 130 million sequencing reads facilitates analysis of a total of 66,360 nucleosome loci with 6.8 nm resolution. The technique requires 2-3 weeks for sequencing library preparation and 2 weeks for simulation.The authors describe a step-by-step workflow for generating genome-wide chromatin interaction maps with nucleosome resolution using proximity ligation and deep sequencing followed by modeling optimal nucleosome position and orientation.