Abstract
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.
Highlights
Despite being responsible for several million cases of clinical malaria every year, we still know very little about the biology of Plasmodium vivax
This observation suggested that the novel P. vivax predicted Erythrocyte-Binding Protein (EBP) might be able to bind to human erythrocytes
By de novo assembly of a field isolate genome, we have shown that large genomic regions of the P. vivax genome had not been previously characterized because they were missing from the Salvador I strain used for generating the P. vivax reference genome
Summary
Despite being responsible for several million cases of clinical malaria every year, we still know very little about the biology of Plasmodium vivax. An important limitation of P. vivax research is the lack of continuous in vitro propagation that hampers development of functional assays and collection of sufficient amounts of biological material for studying this parasite To circumvent these constraints, researchers often rely on materials derived from P. vivax strains that have been adapted to non-human primates (typically Saimiri and Aotus monkeys). Recent whole genome sequencing studies of monkey-adapted strains [5] and field isolates [6,7] have enabled genome-wide characterization of single nucleotide polymorphisms (SNPs) These studies have so far relied on mapping short reads generated by massively parallel sequencing onto the P. vivax reference genome sequence that was generated using DNA from a single strain, the monkeyadapted Salvador I strain [8]. These previous studies were only able to analyze variations at genomic loci present
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