Malaria parasite genes can exhibit variation in sequence or transcriptional activity. A wealth of information is available on sequence polymorphism in clinical malaria isolates, but there are few quantitative whole-transcriptome studies of natural variation at specific developmental stages. It is challenging to obtain adequately precise and well-replicated transcriptome measurements in order to account for technical and biological variation among preparations, which can otherwise introduce noise or bias in analyses. We address the issue by obtaining of RNA-seq profiles of multiple independently cultured replicates of mature schizont-stage malaria parasites from a panel of clinical isolates and laboratory-adapted lines. With a goal of robustly identifying variably expressed genes, we show that increasing the biological sample replication improves the true positive discovery rate, and that six independent replicates of each isolate is significantly superior to lower numbers. Focusing on genes that are more highly expressed on average improves the discovery rate when fewer biological replicates are available. We identify genes encoding transcription factors and proteins implicated in gametocytogenesis that differ in expression between cultured clinical and laboratory adapted lines.We confirm the variable expression of known merozoite invasion ligands, and identify previously uncharacterised genes as highly differentially expressed among isolates. RT-qPCR assays confirm the variation, and extend quantitation of expression of these genes to a wider panel of ex vivo clinical isolate samples. This highlights new candidates for investigation as potential markers of alternative developmental pathways or targets of immunity. Author summaryWe analyse the transcriptomes of mature Plasmodium falciparum schizonts using RNA-sequencing.We use large numbers of replicates per sample to minimise the impact of inter-replicate biological variation on observed patterns of differential expression. We identify genes that are differentially expressed between isolates. We extensively validate our findings for novel putative targets of immunity. For a panel of ex vivo clinical isolates, we show that expression levels of these candidate genes in fall within the ranges observed for the cultured isolates. These genes constitute novel targets for characterisation for merozoite-stage intervention.
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