Quantitative insertion-site sequencing (QIseq) for high throughput phenotyping of transposon mutants.
Quantitative insertion-site sequencing (QIseq) for high throughput phenotyping of transposon mutants.
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DOI：
10.1101/gr.200279.115
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发表时间：
2016-07
期刊：
影响因子：
7
通讯作者：
Rayner JC
中科院分区：
文献类型：
--
作者：
Bronner IF;Otto TD;Zhang M;Udenze K;Wang C;Quail MA;Jiang RH;Adams JH;Rayner JC
使用随机转座子插入的遗传筛选已经成为揭示原核生物生物学的有力工具，其中全基因组饱和筛选已在多种生物中进行。在真核生物中，这种筛选被证明更有问题，部分原因是缺乏一种敏感而强大的系统来识别转座子插入位点。我们在这里描述了定量插入位点测序（QIseq），它使用定制文库制备和Illumina测序技术，能够从转座子的5 ‘和3 ’端识别插入位点，提供了一个内置的验证水平。该方法是利用人类疟原虫恶性疟原虫中的piggyBac突变体开发的，但应该适用于许多其他真核生物基因组。QIseq被证明是准确的，确认了bb100个突变体中的已知位点，并且是敏感的，识别和监测了超过bb1000万倍的序列计数动态范围内的位点。在转染后不久将QIseq应用于非克隆寄生虫，发现在混合种群中有多个插入，这表明相对适度的转染规模可以产生bbbb4000个独立突变体，为恶性疟原虫基因组规模的筛选提供了明确的途径。QIseq还用于监测先前克隆的突变体池的生长，并在竞争生长中可重复区分有害突变和中性突变。在具有适应度缺陷的突变体中，有一个突变体的piggyBac插入与青蒿素耐药性相关的kelch蛋白K13基因的上游，这意味着该基因的突变体可能具有竞争性适应度成本。QIseq有潜力在多个真核系统中扩大piggybac介导的遗传学。
Genetic screening using random transposon insertions has been a powerful tool for uncovering biology in prokaryotes, where whole-genome saturating screens have been performed in multiple organisms. In eukaryotes, such screens have proven more problematic, in part because of the lack of a sensitive and robust system for identifying transposon insertion sites. We here describe quantitative insertion-site sequencing, or QIseq, which uses custom library preparation and Illumina sequencing technology and is able to identify insertion sites from both the 5′ and 3′ ends of the transposon, providing an inbuilt level of validation. The approach was developed using piggyBac mutants in the human malaria parasite Plasmodium falciparum but should be applicable to many other eukaryotic genomes. QIseq proved accurate, confirming known sites in >100 mutants, and sensitive, identifying and monitoring sites over a >10,000-fold dynamic range of sequence counts. Applying QIseq to uncloned parasites shortly after transfections revealed multiple insertions in mixed populations and suggests that >4000 independent mutants could be generated from relatively modest scales of transfection, providing a clear pathway to genome-scale screens in P. falciparum. QIseq was also used to monitor the growth of pools of previously cloned mutants and reproducibly differentiated between deleterious and neutral mutations in competitive growth. Among the mutants with fitness defects was a mutant with a piggyBac insertion immediately upstream of the kelch protein K13 gene associated with artemisinin resistance, implying mutants in this gene may have competitive fitness costs. QIseq has the potential to enable the scale-up of piggyBac-mediated genetics across multiple eukaryotic systems.