4C-seq from beginning to end: A detailed protocol for sample preparation and data analysis
4C-seq from beginning to end: A detailed protocol for sample preparation and data analysis
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DOI：
10.1016/j.ymeth.2019.07.014
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发表时间：
2020-01-01
期刊：
影响因子：
4.8
通讯作者：
de Laat, Wouter
中科院分区：
文献类型：
--
作者：
Krijger, Peter H. L.;Geeven, Geert;de Laat, Wouter
染色体构象捕获（3C）方法基于核邻近连接测量DNA接触频率，以揭示体内基因组折叠模式。4C-seq是一种衍生的3C方法，旨在搜索基因组中与选定的感兴趣基因组位点接触的序列。4C-seq采用反向PCR和下一代测序来扩增、鉴定和定量其邻近连接的DNA片段。它基于有限数量的测序读段为选定的基因组位点生成高分辨率的接触图谱。4C-seq可用于研究基因组组织的多个方面。它主要用于鉴定单个调控DNA模块之间的特异性长距离DNA接触，例如在增强子和启动子之间形成调控染色质环，或在粘着蛋白和CTCF相关结构域边界之间形成结构染色质环。此外，4C-seq接触谱可以揭示接触域的轮廓，并可以鉴定共同占据相同核隔室的结构域。在这里，我们提出了一个改进的分步方案，用于样品制备和4C-seq测序文库的生成，包括优化的PCR和4C模板纯化策略。此外，提供了数据处理流水线，其直接从FASTQ文件处理多路复用的4C-seq读取，并生成与标准基因组浏览器兼容的文件，用于数据的可视化和进一步的统计分析，例如使用peakC的峰调用。所提供的协议和管道应易于允许任何人生成、可视化和解释他们自己的高分辨率4C接触数据集。
Chromosome conformation capture (3C) methods measure DNA contact frequencies based on nuclear proximity ligation, to uncover in vivo genomic folding patterns. 4C-seq is a derivative 3C method, designed to search the genome for sequences contacting a selected genomic site of interest. 4C-seq employs inverse PCR and next generation sequencing to amplify, identify and quantify its proximity ligated DNA fragments. It generates high-resolution contact profiles for selected genomic sites based on limited amounts of sequencing reads. 4C-seq can be used to study multiple aspects of genome organization. It primarily serves to identify specific long-range DNA contacts between individual regulatory DNA modules, forming for example regulatory chromatin loops between enhancers and promoters, or architectural chromatin loops between cohesin- and CTCF-associated domain boundaries. Additionally, 4C-seq contact profiles can reveal the contours of contact domains and can identify the structural domains that co-occupy the same nuclear compartment. Here, we present an improved step-by-step protocol for sample preparation and the generation of 4C-seq sequencing libraries, including an optimized PCR and 4C template purification strategy. In addition, a data processing pipeline is provided which processes multiplexed 4C-seq reads directly from FASTQ files and generates files compatible with standard genome browsers for visualization and further statistical analysis of the data such as peak calling using peakC. The protocols and the pipeline presented should readily allow anyone to generate, visualize and interpret their own high resolution 4C contact datasets.