Application of barcode sequencing to increase the throughput and complexity of Plasmodium falciparum genetic screening

Abstract

All the pathology and symptoms associated with malaria are caused by the growth of Plasmodium parasites inside human red blood cells. This process, which in the case of the major human malaria pathogen Plasmodium falciparum takes place over a 48-hour period, involves multiple tightly regulated developmental transitions. Understanding the P. falciparum genes that regulate these key processes could lead to the identification of targets for new drugs. However, while large-scale sequencing efforts have led to a good understanding of the P. falciparum genome and how it evolves over time and space, a disconnect remains between the amount of genome sequence data available and the amount of data describing what exactly the genes contained within it do - the phenotype. We have generated a panel of 66 P. falciparum lines carrying individual gene knockouts tagged with unique DNA barcodes. We then used these lines in a series of assays that combine flow cytometry, cell sorting and DNA barcode quantification using next generation sequencing (Barcode Sequencing or BarSeq) to phenotype key aspects of the parasite life cycle such as growth, replication capacity and cell cycle progression. This approach both yields new data about individual gene function, and outlines a new approach where barcoded P. falciparum lines are used in pooled BarSeq-based assays to generate more precise phenotype data at scale.