Genome-wide transposon screening and quantitative insertion site sequencing for cancer gene discovery in mice
Genome-wide transposon screening and quantitative insertion site sequencing for cancer gene discovery in mice
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DOI：
10.1038/nprot.2016.164
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发表时间：
2017-02-01
期刊：
影响因子：
14.8
通讯作者：
Rad, Roland
中科院分区：
文献类型：
--
作者：
Friedrich, Mathias J.;Rad, Lena;Rad, Roland
转座子介导的小鼠正向遗传学筛选已成为发现癌症基因的有力工具。它精确定位了其他方法难以找到的癌症驱动因素，从而补充了基于测序的人类癌症基因普查。我们在这里描述了一个大系列的插入诱变的小鼠品系，是兼容的两个转座子系统，PiggyBac和睡美人，并提供指导使用不同的工程转座子变体组成或组织特异性癌症基因发现筛选。我们还描述了半定量转座子插入位点测序（QiSeq）的方法。QiSeq文库制备方案利用声学DNA片段化来减少广泛使用的用于连接介导的插入位点扩增的基于限制性消化的方法所固有的偏差。广泛的多路复用与下一代测序相结合，允许在1周内以高通量形式进行负担得起的超深转座子插入位点恢复。最后，我们描述了数据分析和解释的原则，以获得对癌症基因功能和遗传肿瘤演变的见解。
Transposon-mediated forward genetics screening in mice has emerged as a powerful tool for cancer gene discovery. It pinpoints cancer drivers that are difficult to find with other approaches, thus complementing the sequencing-based census of human cancer genes. We describe here a large series of mouse lines for insertional mutagenesis that are compatible with two transposon systems, PiggyBac and Sleeping Beauty, and give guidance on the use of different engineered transposon variants for constitutive or tissue-specific cancer gene discovery screening. We also describe a method for semiquantitative transposon insertion site sequencing (QiSeq). The QiSeq library preparation protocol exploits acoustic DNA fragmentation to reduce bias inherent to widely used restriction-digestion-based approaches for ligation-mediated insertion site amplification. Extensive multiplexing in combination with next-generation sequencing allows affordable ultra-deep transposon insertion site recovery in high-throughput formats within 1 week. Finally, we describe principles of data analysis and interpretation for obtaining insights into cancer gene function and genetic tumor evolution.