Abstract：
Short tandem repeat (STR) expansions are associated with more than 60 genetic disorders. The size and stability of these expansions correlate with the severity and age of onset of the disease. Therefore, being able to accurately detect the absolute length of STRs is important. Current diagnostic assays include laborious lab experiments, including repeat-primed PCR and Southern blotting, that still cannot precisely determine the exact length of very long repeat expansions. Optical genome mapping (OGM) is a cost-effective and easy-to-use alternative to traditional cytogenetic techniques and allows the comprehensive detection of chromosomal aberrations and structural variants >500 bp in length, including insertions, deletions, duplications, inversions, translocations, and copy number variants. Here, we provide methodological guidance for preparing samples and performing OGM as well as running the analysis pipelines and using the specific repeat expansion workflows to determine the exact repeat length of repeat expansions expanded beyond 500 bp. Together these protocols provide all details needed to analyze the length and stability of any repeat expansion with an expected repeat size difference from the expected wild-type allele of >500 bp. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Genomic ultra-high-molecular-weight DNA isolation, labeling, and staining Basic Protocol 2: Data generation and genome mapping using the Bionano Saphyr® System Basic Protocol 3: Manual De Novo Assembly workflow Basic Protocol 4: Local guided assembly workflow Basic Protocol 5: EnFocus Fragile X workflow Basic Protocol 6: Molecule distance script workflow.
摘要：
短串联重复序列（STR）扩增与60多种遗传性疾病有关。这些扩张的大小和稳定性与疾病发作的严重程度和年龄相关。因此,能够准确地检测STR的绝对长度是重要的。目前的诊断化验包括费力的实验室实验，包括重复引物PCR和Southern印迹，这仍然不能精确确定非常长的重复扩展的确切长度。光学基因组作图（OGM）是传统细胞遗传学技术的一种经济有效且易于使用的替代方法，可以全面检测长度>500bp的染色体畸变和结构变异，包括插入，删除,重复,倒置,易位,和拷贝数变体。这里,我们为制备样品和执行OGM以及运行分析管道和使用特定重复扩增工作流程来确定扩增超过500bp的重复扩增的确切重复长度提供方法学指导.这些方案一起提供了分析任何重复扩增的长度和稳定性所需的所有细节，与预期的野生型等位基因的预期重复大小差异>500bp。©2024作者WileyPeriodicalsLLC出版的当前协议。基本方案1：基因组超高分子量DNA分离，标签,和染色基本方案2：使用BionanoSaphyr®System基本方案3：手动DeNovo组装工作流程基本方案4：本地引导组装工作流程基本方案5：聚焦脆性X工作流程基本方案6：分子距离脚本工作流程。
