Reliable detection of somatic mutations in solid tissues by laser-capture microdissection and low-input DNA sequencing

显微解剖 基因组 计算生物学 激光捕获显微切割 DNA测序 生物 人类基因组 工作流程 DNA 体细胞 遗传学 基因 计算机科学 数据库 基因表达
作者
Peter Ellis,Luiza Moore,Mathijs A. Sanders,Timothy Butler,Simon Brunner,Henry Lee-Six,Robert J. Osborne,Ben W. Farr,Tim H. H. Coorens,Andrew Lawson,Alex Cagan,Mike Stratton,Iñigo Martincorena,Peter J. Campbell
出处
期刊:Nature Protocols [Nature Portfolio]
卷期号:16 (2): 841-871 被引量:123
标识
DOI:10.1038/s41596-020-00437-6
摘要

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.
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