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PfEMP1 variants are encoded by the highly polymorphic family of\n                    <jats:italic>var<\/jats:italic>\n                    genes, the sequences of which are largely unknown in clinical samples. Previously, we published new approaches for\n                    <jats:italic>var<\/jats:italic>\n                    gene profiling and classification of predicted binding phenotypes in clinical\n                    <jats:italic>P. falciparum<\/jats:italic>\n                    isolates (Wichers et al., 2021), which represented a major technical advance. Building on this, we report here a novel method for\n                    <jats:italic>var<\/jats:italic>\n                    gene assembly and multidimensional quantification from RNA-sequencing that outperforms the earlier approach of Wichers et al., 2021, on both laboratory and clinical isolates across a combination of metrics. Importantly, the tool can interrogate the\n                    <jats:italic>var<\/jats:italic>\n                    transcriptome in context with the rest of the transcriptome and can be applied to enhance our understanding of the role of\n                    <jats:italic>var<\/jats:italic>\n                    genes in malaria pathogenesis. We applied this new method to investigate changes in\n                    <jats:italic>var<\/jats:italic>\n                    gene expression through early transition of parasite isolates to in vitro culture, using paired sets of ex vivo samples from our previous study, cultured for up to three generations. In parallel, changes in non-polymorphic core gene expression were investigated. Modest but unpredictable\n                    <jats:italic>var<\/jats:italic>\n                    gene switching and convergence towards\n                    <jats:italic>var2csa<\/jats:italic>\n                    were observed in culture, along with differential expression of 19% of the core transcriptome between paired ex vivo and generation 1 samples. Our results cast doubt on the validity of the common practice of using short-term cultured parasites to make inferences about in vivo phenotype and behaviour.\n                  <\/jats:p>","DOI":"10.7554\/elife.87726","type":"journal-article","created":{"date-parts":[[2023,6,8]],"date-time":"2023-06-08T10:13:49Z","timestamp":1686219229000},"update-policy":"https:\/\/doi.org\/10.7554\/elife.87726","source":"Crossref","is-referenced-by-count":8,"title":["A novel computational pipeline for var gene expression augments the discovery of changes in the Plasmodium falciparum transcriptome during transition from in vivo to short-term in vitro culture"],"prefix":"10.7554","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2461-5256","authenticated-orcid":true,"given":"Clare","family":"Andradi-Brown","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/041kmwe10","id-type":"ROR","asserted-by":"publisher"}],"name":"Section of Paediatric Infectious Disease, Department 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