title: Quantitative insertion-site sequencing (QIseq) for high throughput phenotyping of transposon mutants.
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 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.
authors.affiliation: Malaria Programme, Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridgeshire CB10 1SA, United Kingdom;
authors.forename: Iraad F
authors.initials: IF
authors.lastname: Bronner
authors.affiliation: Malaria Programme, Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridgeshire CB10 1SA, United Kingdom;
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Otto
authors.affiliation: Center for Global Health and Infectious Diseases, Department of Global Health, University of South Florida, Tampa, Florida 33612, USA.
authors.forename: Min
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Center for Global Health and Infectious Diseases, Department of Global Health, University of South Florida, Tampa, Florida 33612, USA.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Udenze
authors.affiliation: Center for Global Health and Infectious Diseases, Department of Global Health, University of South Florida, Tampa, Florida 33612, USA.
authors.forename: Chengqi
authors.initials: C
authors.lastname: Wang
authors.affiliation: Malaria Programme, Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridgeshire CB10 1SA, United Kingdom;
authors.forename: Michael A
authors.initials: MA
authors.lastname: Quail
authors.affiliation: Center for Global Health and Infectious Diseases, Department of Global Health, University of South Florida, Tampa, Florida 33612, USA.
authors.forename: Rays H Y
authors.initials: RH
authors.lastname: Jiang
authors.affiliation: Center for Global Health and Infectious Diseases, Department of Global Health, University of South Florida, Tampa, Florida 33612, USA.
authors.forename: John H
authors.initials: JH
authors.lastname: Adams
authors.affiliation: Malaria Programme, Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridgeshire CB10 1SA, United Kingdom;
authors.forename: Julian C
authors.identifier: 0000-0002-9835-1014
authors.initials: JC
authors.lastname: Rayner
mesh_terms: D001483:Base Sequence; D004251:DNA Transposable Elements; D015723:Gene Library; D063990:Gene Ontology; D059014:High-Throughput Nucleotide Sequencing; D016254:Mutagenesis, Insertional; D010641:Phenotype; D010963:Plasmodium falciparum / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D004251:DNA Transposable Elements