A novel computational pipeline forvargene expression augments the discovery of changes in thePlasmodium falciparumtranscriptome during transition fromin vivoto short-termin vitroculture

The pathogenesis of severePlasmodium falciparummalaria involves cytoadhesive microvascular sequestration of infected erythrocytes, mediated byP. falciparumerythrocyte membrane protein 1 (PfEMP1). PfEMP1 variants are encoded by the highly polymorphic family ofvargenes, the sequences of which are largely unknown in clinical samples. Previously, we published new approaches forvargene profiling and classification of predicted binding phenotypes in clinicalP. falciparumisolates (Wicherset al., 2021), which represented a major technical advance. Building on this, we report here a novel method forvargene assembly and multidimensional quantification from RNA-sequencing that outperforms the earlier approach of Wicherset al., 2021 on both laboratory and clinical isolates across a combination of metrics. Importantly, the tool can interrogate thevartranscriptome in context with the rest of the transcriptome and can be applied to enhance our understanding of the role ofvargenes in malaria pathogenesis. We applied this new method to investigate changes invargene expression through early transition of parasite isolates toin vitroculture, using paired sets ofex vivosamples from our previous study, cultured for up to three generations. In parallel, changes in non-polymorphic core gene expression were investigated. Modest but unpredictablevargene switching and convergence towardsvar2csawere observed in culture, along with differential expression of 19% of the core transcriptome between pairedex vivoand generation 1 samples. Our results cast doubt on the validity of the common practice of using short-term cultured parasites to make inferences aboutin vivophenotype and behaviour.