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.

Phenotypic Screens Identify Parasite Genetic Factors Associated with Malarial Fever Response in Plasmodium falciparum piggyBac Mutants

“…We used a random selection of 25 single-insertion P. falciparum piggyBac mutant clones from a previously described mutant library (8, 10), as well as the wild-type parent NF54 to associate altered phenotypic effects of febrile temperature with specific genotypes (Table 1). piggyBac insertions were first identified by thermal asymmetric interlaced (TAIL) PCR for transposon insertion identification (11) and verified by quantitative insertion site sequencing (QISeq) (10).…”

“…piggyBac insertions were first identified by thermal asymmetric interlaced (TAIL) PCR for transposon insertion identification (11) and verified by quantitative insertion site sequencing (QISeq) (10). Several mutants were additionally verified by whole-genome sequencing (WGS; Table 1) to ensure that no major genomic changes occurred aside from the piggyBac insertion.…”

“…piggyBac mutants are genetically identical, save a single genetic lesion where the transposon inserts itself randomly into the genome at TTAA tetranucleotide sites (9, 10). Mutants presenting significant phenotypes in febrile response screens compared to wild-type parasites implicate the disrupted gene’s involvement in this process.…”

Identification of essential genes and fluconazole resistance genes inCandida glabrataby profiling ofHermestransposon insertions

“…Next-generation DNA sequencing technology facilitates en masse analyses of very large pools of insertion mutants (14)(15)(16). Typically, genomic DNA adjacent to each insertion site is directly amplified by PCR and sequenced using Illumina technology, and then the reads are mapped to precise sites in the genome and tabulated.…”

Transposon Insertion Site Sequencing (TIS‐Seq): An Efficient and High‐Throughput Method for Determining Transposon Insertion Site(s) and Their Relative Abundances in a PiggyBac Transposon Mutant Pool by Next‐Generation Sequencing

“…Since PB transposons can integrate at more than one site in a single genome, the identification of individual/hand‐picked PB transposon mutants by the above methods requires extensive clone screening. Recently, the Splinkerette PCR method has been used for en masse identification of TIS in PB transposon mutant libraries by Illumina sequencing (Bronner et al., ; Choi et al., ; Friedel et al., ; Ni, Landrette, Bjornson, Bosenberg, & Xu, ). This approach consists of DNA fragmentation by restriction digestion or acoustic shearing, followed by end repair, A‐tailing, Splinkerette adaptor ligation, nested PCR, and Illumina sequencing.…”