A concern in the field of genomics is the proper interpretation of large, high-throughput sequencing datasets. The use of DNA FISH followed by high-content microscopy is a valuable tool for validation and contextualization of frequently occurring gene pairing events at the single-cell level identified by deep sequencing. However, these techniques possess certain limitations. Firstly, they do not permit the study of colocalization of many gene loci simultaneously. Secondly, the direct assessment of the relative position of many clustered gene loci within their respective chromosome territories is impossible. Thus, methods are required to advance the study of higher-order nuclear and cellular organization. Here, we describe a multiplexed DNA FISH technique combined with indirect immunofluorescence to study the relative position of 6 distinct genomic or cellular structures. This can be achieved in a single hybridization step using spectral imaging during image acquisition and linear unmixing. Here, we detail the use of this method to quantify gene pairing between highly expressed spliceosomal genes and compare these data to randomly positioned in silico simulated gene clusters. This is a potentially universally applicable approach for the validation of 3C-based technologies, deep imaging of spatial organization within the nucleus and global cellular organization.
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机译：基因组学领域的一个问题是对大型，高通量测序数据集的正确解释。 DNA FISH的使用以及高含量显微镜检查是一种有价值的工具，可用于在通过深度测序鉴定的单细胞水平上验证和关联频繁发生的基因配对事件。但是，这些技术具有某些局限性。首先，它们不允许同时研究许多基因位点的共定位。其次，不可能直接评估许多簇基因位点在其各自染色体区域内的相对位置。因此，需要方法来推进对高级核和细胞组织的研究。在这里，我们描述了一种结合间接免疫荧光的多重DNA FISH技术来研究6种不同基因组或细胞结构的相对位置。这可以在图像采集和线性解混过程中使用光谱成像的单个杂交步骤中实现。在这里，我们详细介绍了使用这种方法来量化高度表达的剪接体基因之间的基因配对，并将这些数据与计算机模拟的基因簇中随机定位的数据进行比较。这是一种潜在的普遍适用的方法，用于验证基于3C的技术，核内空间组织和全球细胞组织的深度成像。
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