Computational protocol: Lysophosphatidylcholine Regulates Sexual Stage Differentiation in the Human Malaria Parasite Plasmodium falciparum

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Protocol publication

[…] RNA-seq reads from each sample were aligned to the P. falciparum reference genome (PlasmoDB version 28). A maximum of one mismatch per read was allowed. The mapped reads from TopHat () were used to assemble known transcripts from the reference and their abundances were estimated using Cufflinks (). The expression level of each gene was normalized as FPKM (fragments per kilobase of exon per million mapped reads). We developed an induction score (IS) to measure transcriptional responses of genes to different culture conditions. For two strains (Sϵ NF54, Pf2004) in different conditions (Cϵ +SerM, −SerM and −SerM/LysoPC), the transcription level (Exp) of each gene i in 4 time points (Tϵ TP1, TP2, TP3 and TP4) can be represented as Expi,TS,C. For each time points, we first paired condition C1 (C1ϵ -SerM ) with C2 (C2ϵ +SerM, −SerM/LysoPC ) and represented as Expi,TS,C1 vs. Expi,TS,C2 . 8 (4 time points × 2non-inducing) pairs can be obtained and the IS can be represented asIS=log2[∑T,C2Expi,TS,C2+1Expi,TS,C1+1]To identify deregulated genes, the group of non-induced parasites can be represented as vector(Expi,TP1NF54,+SerM,…,Expi,TP4Pf2004,+SerM,Expi,TP1NF54,−SerM/LysoPC,…,Expi,TP4Pf2004, −SerM/LysoPC), while the induction group is(Expi,TP1NF54,−SerM,…,Expi,TP4Pf2004,−SerM,Expi,TP1NF54,−SerM,…,Expi,TP4Pf2004, −SerM). Each vector has 16 elements (8 × 2 strains) and Paired Wilcoxon test and BH p value adjustment were implemented. Genes with an adjusted p value of less than 0.05 were identified as significantly differentially regulated. Gene Ontology (GO) annotations were obtained from PlasmoDB version 28. The Barnard test () was used to test enrichment of specific GO terms in each gene group against the genomic background. […]

Pipeline specifications

Software tools TopHat, Cufflinks
Databases PlasmoDB
Application RNA-seq analysis
Organisms Homo sapiens, Plasmodium falciparum
Chemicals Lysophosphatidylcholines, Phosphatidylcholines