Suryawanshi Gajendra W, Xu Song, Xie Yiming, Chou Tom, Kim Namshin, Chen Irvin S Y, Kim Sanggu
UCLA AIDS Institute, University of California at Los Angeles (UCLA); Department of Microbiology, Immunology, & Molecular Genetics, University of California at Los Angeles (UCLA).
Departments of Biomathematics and Mathematics, University of California at Los Angeles (UCLA).
J Vis Exp. 2017 Jun 14(124):55812. doi: 10.3791/55812.
Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral gene therapy studies, IS assays, in combination with next-generation sequencing, have been used as a cell-tracking tool to characterize clonal stem cell populations sharing the same IS. For the accurate comparison of repopulating stem cell clones within and across different samples, the detection sensitivity, data reproducibility, and high-throughput capacity of the assay are among the most important assay qualities. This work provides a detailed protocol and data analysis workflow for bidirectional IS analysis. The bidirectional assay can simultaneously sequence both upstream and downstream vector-host junctions. Compared to conventional unidirectional IS sequencing approaches, the bidirectional approach significantly improves IS detection rates and the characterization of integration events at both ends of the target DNA. The data analysis pipeline described here accurately identifies and enumerates identical IS sequences through multiple steps of comparison that map IS sequences onto the reference genome and determine sequencing errors. Using an optimized assay procedure, we have recently published the detailed repopulation patterns of thousands of Hematopoietic Stem Cell (HSC) clones following transplant in rhesus macaques, demonstrating for the first time the precise time point of HSC repopulation and the functional heterogeneity of HSCs in the primate system. The following protocol describes the step-by-step experimental procedure and data analysis workflow that accurately identifies and quantifies identical IS sequences.
整合位点（IS）分析是逆转录病毒整合位点及其生物学意义研究的关键组成部分。在最近的逆转录病毒基因治疗研究中，IS分析与新一代测序相结合，已被用作一种细胞追踪工具，以表征共享相同IS的克隆干细胞群体。为了在不同样本内和不同样本间准确比较再增殖干细胞克隆，该分析的检测灵敏度、数据可重复性和高通量能力是最重要的分析质量指标。这项工作提供了双向IS分析的详细方案和数据分析工作流程。双向分析可以同时对载体-宿主连接的上游和下游进行测序。与传统的单向IS测序方法相比，双向方法显著提高了IS检测率以及目标DNA两端整合事件的表征。这里描述的数据分析流程通过将IS序列映射到参考基因组并确定测序错误的多个比较步骤，准确识别和枚举相同的IS序列。使用优化的分析程序，我们最近发表了恒河猴移植后数千个造血干细胞（HSC）克隆的详细再增殖模式，首次证明了HSC再增殖的精确时间点以及灵长类系统中HSC的功能异质性。以下方案描述了准确识别和量化相同IS序列的逐步实验过程和数据分析工作流程。
