Diversity in gene expression profiles captured through highly resolved whole-transcriptome profiling of clinical and laboratory-adapted malaria parasite isolates

We present highly replicated whole transcriptome gene expression profiles of schizont-stage malaria parasites using RNA-seq analysis of multiple clinical isolates and laboratory-adapted lines Methods: Transcript profiles of schizont-stage laboratory-adapted and clinical malaria parasite isolates were generated by RNA sequencing. Five to ten replicates were sequenced per sample. Illumina stranded TruSeq libraries were sequenced using an Illumina MiSeq. Paired-end fastQ files were aligned using hisat2 and converted to indexed bam files using samtools. Bam files were filtered to exclude reads with MAPQ scores below 60. Reads were counted using SummarizeOverlaps feature of the GenomicAlignments package in R. Differential expression analysis was conducted using DESeq2 in R. qRT–PCR validation of differentially expressed genes was performed using SYBR Green assays for eight genes. Results: We show that increasing sample replication improves the true-positive discovery rate, and that when fewer replicates are available, focussing on the most highly expressed genes maintains the true-positive discovery rate. We identify schizont-stage genes that appear to alter in expression through the process of culture adaptation, as well as genes that show variable expression between isolates. We extend transcript quantitation for variably expressed genes to an even wider panel of ex vivo clinical isolate samples. Conclusions: Our study represents the first detailed analysis of replicated P. falciparum schizont-stage transcriptomes. Our data show that high levels of replication are necessary to capture gene expression differences among parasite isolates. We identify schizont-stage expressed genes that may be differentially expressed as a mechanism of immune evasion