Thorson John A, Murray Sarah S
Department of Pathology, University of California San Diego, La Jolla, CA, USA.
Center for Advanced Laboratory Medicine, University of California San Diego Health, La Jolla, CA, USA.
Methods Mol Biol. 2019;1908:19-36. doi: 10.1007/978-1-4939-9004-7_2.
The use of next generation sequencing (NGS) to profile tumor genomes for the presence of diagnostic, prognostic, or therapeutically targetable variants is revolutionizing the practice of oncology and is increasingly utilized in clinical laboratory settings. Beginning with the isolation of DNA of sufficient quality and quantity from a tumor specimen, the creation of a library of genomic fragments representing the portion of the genome of interest, ranging from a few genes to the entire exome, is the first step required in the sequencing process. Fixed tumor tissue in the form of a tissue block is the most commonly encountered specimen for analysis in a clinical setting. Special precautions must be employed to ensure that material isolated from these specimens is suitable for use. Once DNA is obtained, one of the most commonly used methods for library preparation involves fluid phase hybridization-based capture of the genomic regions to be interrogated. This multistep process involves fragmentation of the DNA to a uniform size distribution, ligating adapter molecules which are labeled with specific barcodes to enable downstream sequencing and sample identification, and the use of a multiplexed pool of biotinylated single stranded RNA or DNA hybridization probes to recognize and capture the targeted genomic regions. Fragments which are not specifically captured during the hybridization process are removed via a series of wash steps, and a final low cycle amplification is used to prepare the library of captured fragments for sequencing. In this chapter, we provide a step-by-step guide to the preparation of fixed tissue-derived DNA libraries for sequencing via the Illumina process and highlight some of the precautions necessary when working with these types of specimens.
利用新一代测序（NGS）对肿瘤基因组进行分析，以检测诊断、预后或可治疗的靶向变异，正在彻底改变肿瘤学的实践，并在临床实验室环境中得到越来越广泛的应用。从从肿瘤标本中分离出质量和数量足够的DNA开始，创建一个代表感兴趣基因组部分的基因组片段文库，范围从几个基因到整个外显子组，是测序过程中所需的第一步。组织块形式的固定肿瘤组织是临床环境中最常遇到的分析标本。必须采取特殊预防措施，以确保从这些标本中分离的材料适合使用。一旦获得DNA，文库制备最常用的方法之一涉及基于液相杂交的待检测基因组区域捕获。这个多步骤过程包括将DNA片段化至均匀的大小分布，连接用特定条形码标记的接头分子，以便进行下游测序和样品识别，以及使用生物素化单链RNA或DNA杂交探针的多重池来识别和捕获靶向基因组区域。在杂交过程中未被特异性捕获的片段通过一系列洗涤步骤去除，最后进行低循环扩增，以制备用于测序的捕获片段文库。在本章中，我们提供了一份通过Illumina流程制备用于测序的固定组织来源DNA文库的分步指南，并强调了处理这类标本时一些必要的预防措施。
