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Vcf file (bgzip compression) containing variants from patient sample 1, 2 and 3, which were sequenced with the Nanopore adaptive sampling mode selecting for reads aligning to the P. falciparum 3D7 reference genome. Variants were called using Clair3, and combined with GATK. No variant filtration was applied.

Malaria is among the most impactful infectious diseases globally, and is caused by protozoan parasites of the genus Plasmodium. Plasmodium vivax is the second most important cause of human malaria, mainly affecting non-African countries. Despite the global health impact of P. vivax, our understanding of its unique biology is severely limited due to the lack of a long-term in vitro culture system. The goal of this thesis was to gain a better understanding of the P. vivax reticulocyte invasion process and the involved receptors and ligands, which are interesting targets for drug and vaccine development. To overcome the absence of a culture system, we combined several wet and dry lab approaches, and placed a particular focus on next-generation sequencing as a new avenue to study the invasion process. First, we demonstrated that the red blood cell surface protein band 3 is a novel invasion receptor, through short-term culturing of field isolates from P. vivax patients in presence of band 3-blocking agents or Southeast Asian ovalocytosis red blood cells (mutated band 3). Transcriptome analysis of those same P. vivax isolates resulted in a list of potential band 3 ligand candidates, which was highly enriched in PvTRAg genes. In addition, we showed that presence of a PvTRAG38 peptide partially inhibited P. vivax invasion into reticulocytes. To further investigate the role of the PvTRAG38 in invasion, we made use of P. knowlesi models, as this species can be long-term cultured and is closely related to P. vivax. Using CRISPR/Cas9 genome editing, the PvTRAg38 ortholog (PkTRAg38.3) was tagged and knocked out to characterise its transcriptional timing and function. We found that PkTRAg38.3 was schizont-expressed, and that disruption of this gene resulted in an average growth defect of 15.5% over 2 blood cycles, supporting a role as invasion ligand. Finally, we developed tools and protocols to facilitate the generation and analysis of P. vivax sequencing data in future studies. Through low-input PacBio sequencing of a Peruvian P. vivax field isolate, the first high-quality reference genome for South-American isolates was assembled, called PvPAM. In addition, we successfully used the Nanopore adaptive sampling feature to enrich parasite DNA 4- to 6-fold during the sequencing run, thereby circumventing time-consuming and complex lab procedures to remove the predominant human DNA.

Supplementary Figures 1-2 and Supplementary Tables 1-6 and 8-10

Total sequencing depth and depth of the resistance allele for drug-resistance associated markers in P. falciparum (1) in patient 1, 2 and 3. The higher multiplicity of infection (MOI) of patient 1 and 3 explains the presence of both resistant and non-resistant alleles at some loci. For 38 of the resistance associated markers, a PCR fragment was Sanger sequenced to assess whether the SNP was called correctly with Nanopore sequencing. (1) WHO. 2020. Report on antimalarial drug efficacy, resistance and response: 10 years of surveillance (2010-2019). WHO, Geneva.

Total sequencing depth and depth of the resistance allele for drug-resistance associated markers in P. falciparum (1) in patient 1, 2 and 3. The higher multiplicity of infection (MOI) of patient 1 and 3 explains the presence of both resistant and non-resistant alleles at some loci. For 38 of the resistance associated markers, a PCR fragment was Sanger sequenced to assess whether the SNP was called correctly with Nanopore sequencing. (1) WHO. 2020. Report on antimalarial drug efficacy, resistance and response: 10 years of surveillance (2010-2019). WHO, Geneva.

Vcf file (bgzip compression) containing variants from patient sample 1, 2 and 3, which were sequenced with the Nanopore adaptive sampling mode selecting for reads aligning to the P. falciparum 3D7 reference genome. Variants were called using Clair3, and combined with GATK. No variant filtration was applied.