Houghton Jeffrey, Hadd Andrew G, Zeigler Robert, Haynes Brian C, Latham Gary J
Asuragen, Inc.
Asuragen, Inc.;
J Vis Exp. 2016 Apr 11(110):e53836. doi: 10.3791/53836.
All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench ("wet bench") and data analyses conducted using bioinformatics pipelines ("dry bench"). Both elements are essential to produce accurate and reliable results, which are particularly critical for clinical laboratories. Targeted NGS technologies have increasingly found favor in oncology applications to help advance precision medicine objectives, yet the methods often involve disconnected and variable wet and dry bench workflows and uncoordinated reagent sets. In this report, we describe a method for sequencing challenging cancer specimens with a 21-gene panel as an example of a comprehensive targeted NGS system. The system integrates functional DNA quantification and qualification, single-tube multiplexed PCR enrichment, and library purification and normalization using analytically-verified, single-source reagents with a standalone bioinformatics suite. As a result, accurate variant calls from low-quality and low-quantity formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration (FNA) tumor biopsies can be achieved. The method can routinely assess cancer-associated variants from an input of 400 amplifiable DNA copies, and is modular in design to accommodate new gene content. Two different types of analytically-defined controls provide quality assurance and help safeguard call accuracy with clinically-relevant samples. A flexible "tag" PCR step embeds platform-specific adaptors and index codes to allow sample barcoding and compatibility with common benchtop NGS instruments. Importantly, the protocol is streamlined and can produce 24 sequence-ready libraries in a single day. Finally, the approach links wet and dry bench processes by incorporating pre-analytical sample quality control results directly into the variant calling algorithms to improve mutation detection accuracy and differentiate false-negative and indeterminate calls. This targeted NGS method uses advances in both wetware and software to achieve high-depth, multiplexed sequencing and sensitive analysis of heterogeneous cancer samples for diagnostic applications.
所有下一代测序（NGS）程序都包括在实验室工作台上进行的检测（“湿实验台”）以及使用生物信息学流程进行的数据分析（“干实验台”）。这两个要素对于产生准确可靠的结果至关重要，而这对于临床实验室尤为关键。靶向NGS技术在肿瘤学应用中越来越受到青睐，以助力推进精准医学目标，然而这些方法通常涉及不连贯且可变的湿实验台和干实验台工作流程以及不协调的试剂套装。在本报告中，我们以一个21基因检测板对具有挑战性的癌症标本进行测序的方法为例，介绍一种全面的靶向NGS系统。该系统集成了功能性DNA定量和鉴定、单管多重PCR富集以及文库纯化和标准化，使用经过分析验证的单源试剂以及独立的生物信息学套件。结果，可以从低质量和低数量的福尔马林固定石蜡包埋（FFPE）及细针穿刺（FNA）肿瘤活检样本中获得准确的变异检测结果。该方法可以常规地从400个可扩增DNA拷贝的输入量中评估癌症相关变异，并且在设计上具有模块化特点，以适应新的基因内容。两种不同类型的经过分析定义的对照提供质量保证，并有助于保障临床相关样本的检测准确性。一个灵活的“标签”PCR步骤嵌入平台特异性接头和索引代码，以实现样本条形码标记并与常见的台式NGS仪器兼容。重要的是，该方案经过简化，一天内可产生24个可用于测序的文库。最后，该方法通过将分析前样本质量控制结果直接纳入变异检测算法，将湿实验台和干实验台流程联系起来，以提高突变检测准确性并区分假阴性和不确定结果。这种靶向NGS方法利用硬件和软件方面的进展，实现对异质性癌症样本的高深度、多重测序和灵敏分析，以用于诊断应用。
