Quantitative Insertion-site Sequencing (QIseq) for high throughput phenotyping of transposon mutants
- Iraad F. F Bronner1,
- Thomas D Ottoa1,
- Min Zhang2,
- Kenneth Udenze2,
- Chengqi C. Q Wang2,
- Michael A Quail1,
- Rays H Y Jiang2,
- John H Adams2 and
- Julian C Rayner1,3
- ↵* Corresponding author; email: jr9{at}sanger.ac.uk
Abstract
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 the methodological advance of 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 >4,000 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 up to 128 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 kelch13 (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.
- Received October 2, 2015.
- Accepted May 4, 2016.
- Published by Cold Spring Harbor Laboratory Press
This manuscript is Open Access.
This article, published in Genome Research, is available under a Creative Commons License (Attribution 4.0 International license), as described at http://creativecommons.org/licenses/by/4.0/.
