De Novo Assembly of a Field Isolate Genome Reveals Novel Plasmodium vivax Erythrocyte Invasion Genes

Recent sequencing of Plasmodium vivax field isolates and monkey-adapted strains enabled characterization of SNPs throughout the genome. These analyses relied on mapping short reads onto the P. vivax reference genome that was generated using DNA from the monkey-adapted strain Salvador I. Any genomic locus deleted in this strain would be lacking in the reference genome sequence and missed in previous analyses. Here, we report de novo assembly of a P. vivax field isolate genome. Out of 2,857 assembled contigs, we identify 362 contigs, each containing more than 5 kb of contiguous DNA sequences absent from the reference genome sequence. These novel P. vivax DNA sequences account for 3.8 million nucleotides and contain 792 predicted genes. Most of these contigs contain members of multigene families and likely originate from telomeric regions. Interestingly, we identify two contigs containing predicted protein coding genes similar to known Plasmodium red blood cell invasion proteins. One gene encodes the reticulocyte-binding protein gene orthologous to P. cynomolgi RBP2e and P. knowlesi NBPXb. The second gene harbors all the hallmarks of a Plasmodium erythrocyte-binding protein, including conserved Duffy-binding like and C-terminus cysteine-rich domains. Phylogenetic analysis shows that this novel gene clusters separately from all known Plasmodium Duffy-binding protein genes. Additional analyses showing that this gene is present in most P. vivax genomes and transcribed in blood-stage parasites suggest that P. vivax red blood cell invasion mechanisms may be more complex than currently understood. The strategy employed here complements previous genomic analyses and takes full advantage of next-generation sequencing data to provide a comprehensive characterization of genetic variations in this important malaria parasite. Further analyses of the novel protein coding genes discovered through de novo assembly have the potential to identify genes that influence key aspects of P. vivax biology, including alternative mechanisms of human erythrocyte invasion. Author SummaryPlasmodium vivax is responsible for most malaria cases outside Africa, but is poorly understood, as the parasite is difficult to study in vitro. Genome sequencing studies offer a novel and exciting opportunity to better understand this parasite but, so far, have directly mapped reads onto the reference genome sequence generated from a single P. vivax strain. Here, we use sequence data generated from a field isolate to reconstruct long DNA sequences without relying on the reference genome. Our analyses reveal many P. vivax DNA sequences that are absent from the reference genome and contain 792 predicted genes. One of these novel genes encodes a predicted protein similar to known Plasmodium proteins involved in red blood cell invasion. This new gene is present in all P. vivax strains sequenced so far, except for the strain used to generate the reference genome, and is transcribed in blood-stage parasites. Overall, our analyses show that the catalogue of P. vivax genes was incomplete and that potentially important genes have been missed. We notably identified one putative invasion gene that seems functional and could dramatically change our understanding of the mechanisms determining red blood cell invasion by this important malaria parasite.