Supplementary Materials

Uncovering the essential genes of the human malaria parasite Plasmodium falciparum by saturation mutagenesis

Min Zhang, Chengqi Wang, Thomas D. Otto, Jenna Oberstaller, Xiangyun Liao, Swamy R. Adapa, Kenneth Udenze, Iraad F. Bronner, Deborah Casandra, Matthew Mayho, Jacqueline Brown, Suzanne Li, Justin Swanson, Julian C. Rayner, Rays H. Y. Jiang, John H. Adams

Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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  • Figs. S1 to S11
  • Tables S1 to S9
  • References
Table S1
piggyBac insertion sites identified from preliminary study.
Quantitative Insertion-site Sequencing (QIseq) identified 3651 piggyBac insertion sites distributed across all fourteen chromosomes, combined with 326 previous published mutagenesis results shown on this table.
Table S2
QC samples in each of QIseq run.
128 pooled mutants with known pB insertion sites identified in previous study were prepared in advance as QC samples for monitoring accuracy and depth of each QIseq run.
Table S3
piggyBac insertion sites identified from this study.
34,522 piggyBac insertion sites identified from this study. A total of 38,173 piggyBac insertion sites were used for determining saturation-level mutagenesis scores (MIS and MFS) including preliminary study data (Table S1).
Table S4
The non-mutable genes located in essential blocks.
We observed that some non-disrupted genes were completely devoid of insertions even in the surrounding intergenic regions. 143 non-mutable genes (representing 2.9% of genes) are located in essential blocks, which are defined as having an insertion-free gap > 10 kb.
Table S5
MIS and MFS identify essentiality of the genes.
Mutagenesis index score (MIS) was calculated based on the susceptibility of the ORF in each transcriptional unit to being disrupted (Fig. 2A). Mutagenesis Fitness Score (MFS) which is calculated by the normalized reads number (Fig. 2E). Two measuring models (MIS and MFS) were used to validate essentiality of the genes of Plasmodium falciparum genome.
Table S6
RNA metabolism, related to Fig. 2 C, D and fig. S6.
RNA metabolism genes by compartment or process as displayed in figure S7. MIS is indicated from 0 to 1. Genes in red text indicate those with TTAA density <7 or length <400bp (that are thus not scored) that have 0 recovered gene-body insertions. Translation and splicing-related genes of interest were identified via GO term. RNA granule-related genes of interest are as classified in Reddy et al. 2015 and as reviewed (41). Unknown, validated RNA-associated genes are proteins identified as being mRNA-bound as per (42) that have no or little functional annotation as per PlasmoDB.
Table S7
Phenotype screen of competitive growth assay, related to Fig. 3 A to C.
Phenotype screen of competitive growth assay identifies growth winners and losers from ~400 mutants in four individual pools which provides evidence to validate MIS and MFS scoring of each gene's essentiality.
Table S8
Genes corresponding to GO enrichment.
Functional annotation of biological processes, molecular function and cellular component are represented by the p-value and the fraction of the genes with MIS > 0.5. Each GO term is assigned a p-value to represent the tendency to be essential or dispensable.
Table S9
Sample pool ID and accession number for this study, related to Fig. 1, 2 and table S3.
Sample pool ID and accession number from all QIseq runs are shown on this table. Insertion sites identified from each pool are shown on table S3.