Whole genome sequencing of field isolates provides robust characterization of genetic diversity in Plasmodium vivax

Abstract

Background: An estimated 2.85 billion people live at risk of Plasmodium vivax transmission. In endemic countries vivax malaria causes significant morbidity and its mortality is becoming more widely appreciated, drug-resistant strains are increasing in prevalence, and an increasing number of reports indicate that P. vivax is capable of breaking through the Duffy-negative barrier long considered to confer resistance to blood stage infection. Absence of robust in vitro propagation limits our understanding of fundamental aspects of the parasite's biology, including the determinants of its dormant hypnozoite phase, its virulence and drug susceptibility, and the molecular mechanisms underlying red blood cell invasion.

Methodology/principal findings: Here, we report results from whole genome sequencing of five P. vivax isolates obtained from Malagasy and Cambodian patients, and of the monkey-adapted Belem strain. We obtained an average 70-400 X coverage of each genome, resulting in more than 93% of the Sal I reference sequence covered by 20 reads or more. Our study identifies more than 80,000 SNPs distributed throughout the genome which will allow designing association studies and population surveys. Analysis of the genome-wide genetic diversity in P. vivax also reveals considerable allele sharing among isolates from different continents. This observation could be consistent with a high level of gene flow among parasite strains distributed throughout the world.

Conclusions: Our study shows that it is feasible to perform whole genome sequencing of P. vivax field isolates and rigorously characterize the genetic diversity of this parasite. The catalogue of polymorphisms generated here will enable large-scale genotyping studies and contribute to a better understanding of P. vivax traits such as drug resistance or erythrocyte invasion, partially circumventing the lack of laboratory culture that has hampered vivax research for years.

Figure 1. Overview of the genetic diversity in P. vivax genomes.

The 14 P. vivax chromosomes are organized circularly. The red histograms show the number of SNPs per 5 kb (varying from 0 to 100); the vertical black bars indicate regions masked for SNP analysis as putative paralogous sequences. The concentric black histograms represent, from outside to inside, the sequence coverage for Belem, C127 and M08 respectively.

Figure 2. Reference Allele Frequency in (A) monkey-adapted strains and (B) field isolates.

Each variable nucleotide position observed in a sample is displayed according to the proportion of reads carrying the reference allele (x-axis). The y-axis shows the number of variable positions with a given RAF.

Figure 3. Haplotype reconstruction at the Duffy binding protein locus for C15 and C127.

The figure displays, from top to bottom, the Duffy binding protein structure, its chromosomal location, the polymorphic positions in C15 and C127 (non-synonymous polymorphisms are shown in red) and the haplotype reconstruction for C15 and C127. The numbers in boxes represent for each sample the number of reads carrying the reference (top row) or alternative (bottom row) alleles, while the numbers above the lines joining boxes indicate the number of reads carrying two consecutive alleles. The reconstructed haplotypes are shown by the green and blue lines. Note that for C127, a few reads support the presence of a third haplotype (dashed lines).

Figure 4. Allele sharing among P. vivax parasites.

The ternary plot shows the sample carrying the closest haplotype (i.e. the haploid sequence with the smallest number of nucleotide differences) to C08 (left) and Belem (right) for all annotated genes. The number in the red box indicates the number of genes for which the closest sequence is the C127 haplotype, while the green and purple boxes indicate the numbers of genes for which the closest sequence are, respectively, the M15 and Sal I haplotypes. The numbers of the edges represent genes for which two or more haplotypes are equally distant from the sample considered.