University of Vienna, Max Perutz Labs, Department of Chromosome Biology, Vienna, Austria.
Max Perutz Labs, Vienna Biocenter Campus (VBC), Vienna, Austria.
Methods Mol Biol. 2025;2856:401-418. doi: 10.1007/978-1-0716-4136-1_23.
This chapter describes the computational pipeline for the processing and visualization of Protec-Seq data, a method for purification and genome-wide mapping of double-stranded DNA protected by a specific protein at both ends. In the published case, the protein of choice was Saccharomyces cerevisiae Spo11, a conserved topoisomerase-like enzyme that makes meiotic double-strand breaks (DSBs) to initiate homologous recombination, ensuring proper segregation of homologous chromosomes and fertility. The isolated DNA molecules were thus termed double DSB (dDSB) fragments and were found to represent 34 to several hundred base-pair long segments that are generated by Spo11 and are enriched at DSB hotspots, which are sites of topological stress. In order to allow quantitative comparisons between dDSB profiles across experiments, we implemented calibrated chromatin immunoprecipitation sequencing (ChIP-Seq) using the meiosis-competent yeast species Saccharomyces kudriavzevii as calibration strain. Here, we provide a detailed description of the computational methods for processing, analyzing, and visualizing Protec-Seq data, comprising the download of the raw data, the calibrated genome-wide alignments, and the scripted creation of either arc plots or Hi-C-style heatmaps for the illustration of chromosomal regions of interest. The workflow is based on Linux shell scripts (including wrappers for publicly available, open-source software) as well as R scripts and is highly customizable through its modular structure.
本章描述了 Protec-Seq 数据处理和可视化的计算流程，这是一种从两端都被特定蛋白质保护的双链 DNA 中纯化和进行全基因组作图的方法。在已发表的案例中，选择的蛋白质是酿酒酵母 Spo11，一种保守的拓扑异构酶样酶，它会产生减数分裂双链断裂（DSB）以启动同源重组，确保同源染色体的正确分离和生殖能力。因此，分离的 DNA 分子被称为双 DSB（dDSB）片段，它们代表由 Spo11 产生的 34 到几百个碱基对长的片段，并在 DSB 热点（拓扑压力的位点）处富集。为了允许在实验之间对 dDSB 图谱进行定量比较，我们使用具有减数分裂能力的酵母物种酿酒酵母克鲁维酵母作为校准菌株，实现了经过校准的染色质免疫沉淀测序（ChIP-Seq）。在这里，我们提供了 Protec-Seq 数据处理、分析和可视化的详细计算方法描述，包括原始数据的下载、经过校准的全基因组比对，以及用于说明感兴趣的染色体区域的弧形图或 Hi-C 风格热图的脚本创建。该工作流程基于 Linux shell 脚本（包括对公开可用的开源软件的包装）以及 R 脚本，并通过其模块化结构实现高度可定制性。
