Fig. 1: Pooled screens of P. falciparum piggyBac mutants allow robust identification of heat-shock phenotypes.

a Experimental design for pooled heat shock (HS) phenotypic screens. The pilot library of pB-mutant clones (n = 128) was exposed to three rounds of temperature-cycling (41 °C for 8 h) to simulate malarial fever. A pilot-library control concurrently grown continuously at 37 °C established the inherent growth of each pB mutant. HS screens of the pilot library were conducted in biological duplicate and technical triplicate and were highly correlated, indicating high accuracy and reproducibility (see Supplementary Fig. 5a; “Methods, Pooled-screen assay-design, HS-Screen” section). b QIseq quantifies each pB-mutant in the pilot library from sequence-reads of the 5′ and 3′ ends of each pB insertion-site. Colored lines represent genes. Black boxes indicate transposon location (Supplementary Fig. 5b; “Methods, QIseq” section). c HS- and Growth-phenotypes of the pilot-library mutants. HS-phenotype of each mutant (displayed as line-graph) is superimposed on its corresponding Growth-phenotype (bar graph). Mutants are ordered by fold-change in response to HS (FC-HS) from Sensitive (red) to Tolerant (green). Mutants with inherently slower or faster growth under ideal conditions (FC-Growth) are shown in gray and blue, respectively. *Known HS-Sensitive and **HS-Tolerant pB-mutant clones served as benchmarks in the pilot-library HS-Screen for identifying sensitive/tolerant mutants¹⁰. See Supplementary Data File 1. d Phenotype comparison between mutants characterized in both individual HS-assays¹⁰ and pooled HS-screening (n = 20). Mutant clones without an observed phenotype in individual HS-assay as determined by above-average growth via flow cytometry (green) also had significantly higher Phenotypic Fitness Scores in response to HS (PFS_HS) in pooled screening, while mutant clones characterized as HS-Sensitive in individual assays (red) also had significantly lower PFS_HS in pooled screening (**p-value < 0.01, two-tailed Wilcoxon test). Boxplots are drawn to present an interquartile range of values (IQR). Lower bound of each box = 1st quartile, middle line = median, upper bound = 3rd quartile, and whiskers extend to at most 1.5× IQR.