vsgseq2: an updated pipeline for analysis of the diversity and abundance of population-wide Trypanosoma brucei VSG expression

<i>Trypanosoma brucei</i> is an extracellular eukaryotic parasite that causes sleeping sickness in humans and Nagana, Surra and Dourine in livestock, game animals and horses. The parasite displays an extensive immune evasion mechanism, utilising the expression and ability to switch antigenically distinct variant surface glycoprotein (VSG) coats. VSG encoding genes account for ~10% of the <i>T. brucei</i> genome, and mosaic VSGs, assembled from distinct incomplete VSG gene copies, can be produced from this VSG library, generating an almost infinite VSG repertoire, which enables chronic infections. Each parasite expresses just one VSG at a time, but within a host, many VSGs can be expressed simultaneously. Understanding patterns of VSG expression is therefore central to studying parasite dynamics, tissue tropism, and infection persistence. VSGSeq is an amplicon sequencing approach that enables surveillance of the population-wide diversity and abundance of expressed VSGs. We present vsgseq2, an updated and fully reproducible workflow for analysing VSGSeq data. Implemented in Nextflow, vsgseq2 integrates modern tools for transcript assembly and quantification, improves computational efficiency. Benchmarking against defined <i>T. brucei</i> VSG expression datasets demonstrated that vsgseq2 accurately reconstructs population-wide VSG repertoires and better recapitulates VSG expression proportions. Analyses of in vivo infection data further confirmed that vsgseq2 enhances reproducibility and improves data utilisation, and improves computational efficiency. vsgseq2 enables researchers to efficiently and reproducibly analyse complex VSG expression data and the mechanisms driving immune evasion in <i>T. brucei</i>.