Computational protocol: Plasmodium falciparum Maf1 Confers Survival upon Amino Acid Starvation

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Protocol publication

[…] For each sample to be analyzed, 1 ml of parasite culture was pelleted and resuspended in 1 ml of phosphate-buffered saline (PBS) containing 4% (wt/vol) paraformaldehyde. Samples were rocked at 4°C for 20 to 30 h of fixation. Samples were then pelleted and resuspended in PBS containing 0.1% (vol/vol) Triton X-100 and rocked at room temperature for 1 h for permeabilization. This process was then repeated three additional times with PBS (without Triton X-100) to remove as much hemoglobin as possible. Samples were then diluted approximately 100-fold into normal culture medium containing 1× SYBR green I (Thermo Fisher Scientific; catalog no. S7563) and analyzed on a FACSCalibur (BD Biosciences) flow cytometer. The resulting data were further analyzed using FlowJo 10.0.7 analysis software. [...] The analysis of the sequencing data largely followed the protocol described by Balu et al. (), who had previously sequenced the NF54 clone that was used as the parental line in the transposon screen which produced the PB-11 mutant (). Two sets of paired-end reads from the parental NF54 clone (ERS038926 and ERS184445), produced by two different sequencing platforms, were obtained from the European Nucleotide Archive. These reads were used to “update” the PlasmoDB-26 P. falciparum 3D7 genome sequence using ICORN as part of the PAGIT software package (). Seven iterations of ICORN generated 561 1-bp substitutions and 774 small insertions or deletions across the entire reference genome. The PB-11 reads were aligned to this updated reference sequence using bwa () with the default parameters. Approximately 82% of the reads aligned to the reference sequence. The resulting sam file was sorted, indexed, and filtered of duplicates using Picard-tools v 1.119. Reads were realigned around indels using the Genome Analysis Toolkit (GATK) (), and raw variants were called using the GATK HaploTypeCaller, with ploidy set to 1. The raw variants were then filtered using vcftools v0.1.13 () to include only calls with a quality score greater than 60 and a minimum depth of 10 reads. The program snpEff v3 () was then used to filter only those variants found within open reading frames. This resulted in 32 potential variants within open reading frames. However, upon visual inspection using the Integrated Genomics Viewer v2.3.9 (), every call was found in either a low-complexity region (e.g., an extended mononucleotide tract) or a repetitive region. In each case, there was read support for the reference sequence, leaving us to conclude that these variants more than likely represented false positives. To verify that our pipeline was indeed capable of detecting variants, we reran the pipeline analysis using the reads from the C9 piggyBac insertion mutant sequenced by Balu et al. (ENA ERS038913) (). We were able to easily detect the same two SNPs reported previously. [...] All statistical analyses were performed using R version 3.2.3 (10 December 2015) (). t tests, linear and logistic regressions, and some figures were generated using R base functions. The drc package () was used for determining the 50% survival point of PB-11, and the ggplot2 package () was used for the production of several figures. […]

Pipeline specifications

Software tools FlowJo, iCORN, PAGIT, BWA, Picard, GATK, VCFtools, SnpEff, Ggplot2
Databases ENA PlasmoDB
Applications Miscellaneous, Flow cytometry
Organisms Plasmodium falciparum, Toxoplasma gondii, Homo sapiens
Diseases Malaria
Chemicals Amino Acids, Sirolimus