Reliable detection of somatic mutations in solid tissues by laser-capture microdissection and low-input DNA sequencing
Reliable detection of somatic mutations in solid tissues by laser-capture microdissection and low-input DNA sequencing
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DOI：
10.1038/s41596-020-00437-6
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发表时间：
2020-12-14
期刊：
影响因子：
14.8
通讯作者：
Campbell, Peter J.
中科院分区：
文献类型：
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作者：
Ellis, Peter;Moore, Luiza;Campbell, Peter J.
随着我们年龄的增长，体细胞突变会在健康的组织中积累，从而引发癌症，并可能导致衰老。为了研究非肿瘤组织中的体细胞突变，我们开发了一系列方案来对从组织切片中分离的小群体细胞的基因组进行排序。在这里，我们描述了一个完整的工作流程，它结合了激光捕获显微切割(LCM)和低输入基因组测序，同时绕过了全基因组扩增(WGA)的使用。该协议大致分为四个步骤：组织处理、LCM、低输入文库生成和突变调用和筛选。组织处理和LCM步骤是作为一般指南提供的，可能需要根据手头研究的具体要求进行调整。我们的低输入文库生成协议使用酶而不是声波碎裂来生成不含WGA的全基因组文库。最后，突变调用和过滤策略已经从以前发布的协议中进行了调整，以解决通过库创建引入的人工产物。到目前为止，我们已经使用这个工作流程对来自广泛范围的人体组织的数千个显微活检中的小细胞群体(通常是100-1000个细胞)进行了有针对性的全基因组测序。低输入DNA协议被设计为与液体处理平台兼容，并利用任何核心测序设施的设备和专业技术标准。然而，通过LCM获得低投入的DNA材料需要专门的设备和专业知识。从组织接收到全基因组文库生成的整个过程可以在短短1周内完成，尽管2-3周可能是更典型的周转时间。该协议描述了一种将激光捕获显微切割与低输入基因组测序相结合的工作流程，同时绕过了全基因组扩增的使用，以准确检测非肿瘤组织中的体细胞突变。
Somatic mutations accumulate in healthy tissues as we age, giving rise to cancer and potentially contributing to ageing. To study somatic mutations in non-neoplastic tissues, we developed a series of protocols to sequence the genomes of small populations of cells isolated from histological sections. Here, we describe a complete workflow that combines laser-capture microdissection (LCM) with low-input genome sequencing, while circumventing the use of whole-genome amplification (WGA). The protocol is subdivided broadly into four steps: tissue processing, LCM, low-input library generation and mutation calling and filtering. The tissue processing and LCM steps are provided as general guidelines that might require tailoring based on the specific requirements of the study at hand. Our protocol for low-input library generation uses enzymatic rather than acoustic fragmentation to generate WGA-free whole-genome libraries. Finally, the mutation calling and filtering strategy has been adapted from previously published protocols to account for artifacts introduced via library creation. To date, we have used this workflow to perform targeted and whole-genome sequencing of small populations of cells (typically 100-1,000 cells) in thousands of microbiopsies from a wide range of human tissues. The low-input DNA protocol is designed to be compatible with liquid handling platforms and make use of equipment and expertise standard to any core sequencing facility. However, obtaining low-input DNA material via LCM requires specialized equipment and expertise. The entire protocol from tissue reception through whole-genome library generation can be accomplished in as little as 1 week, although 2-3 weeks would be a more typical turnaround time.This protocol describes a workflow that combines laser-capture microdissection with low-input genome sequencing, while circumventing the use of whole-genome amplification, for accurate detection of somatic mutations in non-neoplastic tissues.