Abstract
Plasmodium vivax is a major public health burden, responsible for the majority of malaria infections outside Africa. We explored the impact of demographic history and selective pressures on the P. vivax genome by sequencing 182 clinical isolates sampled from 11 countries across the globe, using hybrid selection to overcome human DNA contamination. We confirmed previous reports of high genomic diversity in P. vivax relative to the more virulent Plasmodium falciparum species; regional populations of P. vivax exhibited greater diversity than the global P. falciparum population, indicating a large and/or stable population. Signals of natural selection suggest that P. vivax is evolving in response to antimalarial drugs and is adapting to regional differences in the human host and the mosquito vector. These findings underline the variable epidemiology of this parasite species and highlight the breadth of approaches that may be required to eliminate P. vivax globally.
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Acknowledgements
We acknowledge J. Bochicchio and S. Chapman for project management, A. Gnirke for technical support, and members of the Broad Institute Genomics Platform and NYU's Genomics Core for data generation. We thank F. Santillan and P. Michon for technical assistance and MR4 for providing us with malaria parasites deposited by W.E. Collins. The following grants supported this work: National Institute of Allergy and Infectious Diseases (NIAID)/National Institutes of Health (NIH) International Centers of Excellence for Malaria Research U19AI089676 to J.M.C.; U19AI089681, K24AI068903 and D43TW007120 to J.M.V.; U19AI089672 to L.C.; São Paulo Research Foundation 2009/52729-9 to M.U.F.; National Council for Science and Technology Mexico 29005-M SALUD-2004-119 and National Institute of Public Health Mexico project 476191 to L.G.-C.; Victorian State Government Operational Infrastructure Support and Australian Government National Health and Medical Research Council Independent Medical Research Institutes Infrastructure Support Scheme (NHMRC IRIISS) to A.B. and I.M.; 5U19AI089702 to S.H. and M.A.-H.; Armed Forces Health Surveillance Center, Global Emerging Infections Surveillance and Response System and US NIH grant D43TW007393 to A.G.L.; NIH U19AI089686 to J.W.K.; and Bill and Melinda Gates Foundation grant to J.S. Sequencing and analysis work at the Broad Institute was supported by federal funds from the NIAID, NIH, US Department of Health and Human Services, under contract HHSN272200900018C. M.U.F. is supported by a senior research scholarship from the Conselho Nacional de Desenvolvimento Científico e Tecnológico of Brazil, I.M. is supported by NHMRC senior research fellowship 1043345 and D.N.H. is supported by NIH training grant T32AI007180. The content of this manuscript is solely the responsibility of the authors and does not necessarily represent the official policy or position of the US Department of the Navy, the US Department of Defense, the US government or the National Institutes of Health.
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J.M.C., I.M. and D.E.N. conceived and conducted the study. A.M., P.L.S., P.R., A.F.V., Q.F., Y.W., C.M.L., S.D., J.F.S., M.L., C.B., D.K., W.R., W.N. and M.K. undertook field and/or wet-lab work and sequencing of the samples. D.N.H., Z.L., J.M.C. and D.E.N. analyzed data. D.N.H., J.M.C., Z.L. and D.E.N. wrote the manuscript, and A.E., S.H., M.A.-H., L.C., G.C.B., A.G.L., A.B., I.M., J.W.K., A.E., N.V., M.U.F., J.S., D.G., J.M.V., L.G.-C. and B.W.B. revised the manuscript and made comments.
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Integrated supplementary information
Supplementary Figure 1 Evaluation of the performance of different hybrid selection baits on a sample with 0.27% initial P. vivax mappable reads.
(a) Enrichment of P. vivax reads using six different baits, including synthetic oligonucleotides (far left column), ‘whole-genome baits’ (WGB) constructed from individual sources (Brazil I, India VII, N. Korean and Mauritania) of genomic DNA, and a WGB mixture (far right column). The vertical axis indicates percent mappable reads following hybrid selection, and numerals above bars indicate fold enrichment in mappable reads. (b) The fold enrichment of P. vivax DNA using WGB (vertical axis) was compromised by contamination of genomic DNA with host (in this case, monkey) material (horizontal axis).
Supplementary Figure 3 Determination of complexity of infection using variant calls from 195 P. vivax isolates.
Each column along the x axis represents one isolate, and the scale along the y axis represents the number of high-quality variants annotated as heterozygous within that isolate. Isolates exhibiting more than 1,236 heterozygous calls, which represents twice the median observed in the population (vertical dotted line), were classified as complex infections containing more than one haploid parasite lineage.
Supplementary Figure 4 Region-specific projections of the variation data that use two principal components and are limited to Old World and New World populations.
(a) Two-eigenvector PCA limited to Old World isolates. (b) Two-eigenvector PCA limited to New World isolates. Both analyses show similar results to the PCA in Figure 3a that uses the global population of 195 P. vivax isolates.
Supplementary Figure 5 Analysis of the incidence and extent of genomic regions exhibiting identity by descent (IBD) within subpopulations.
This analysis was limited to populations with at least 12 single-infection isolates that were also classified as high quality as listed in Supplementary Table 1.
Supplementary Figure 6 Three maximum-likelihood trees computed by RAxML and REALPHY software that use three different reference genomes (Salvador I, Mauritania I and North Korean) and a subset of high-quality sequenced P. vivax isolates.
The tree is rooted at the node between the Old World and Indian/African isolates in each case. Highlighted in red is the reference genome used, and the bootstrap support is shown at each node (100 bootstraps).
Supplementary Figure 7 Admixture analysis of 195 isolates.
Admixture plots under six different K cluster values. Colors correspond to K clusters within each graph, whereas columns are consistent across graphs and correspond to geographical population. The tenfold cross-validation error associated with each admixture analysis was minimized at K = 5.
Supplementary Figure 8 A comparison of the per-gene fixation index (FST) between New World and Old World isolates and per-gene nucleotide diversity calculated in each case from 73 single-infection, high-quality samples.
In blue are genes annotated as antigens, including members of the P. vivax serine-repeat antigen (SERA) family; members of the merozoite surface protein (MSP) superfamily, including single-copy genes MSP1, MSP4, MSP5, MSP8, MSP9, MSP10 and several members of the MSP3 multigene family and the MSP7 multigene family; members of the variant interspersed repeat (vir) gene family (excluding the most highly conserved and potential founder gene PVX_113230); members of the Pv-fam-a (PvTRAG), Pv-fam-b, Pv-fam-c, Pv-fam-d (HYPB), Pv-fam-e (RAD), Pv-fam-g, Pv-fam-h (HYP16) and Pv-fam-i (HYP11) gene families; and any gene annotated as an antigen in the Salvador I reference genome annotation. In green are genes identified as being putatively involved in antimalarial drug resistance, including dihydrofolate reductase–thymidylate synthase (DHFR-TS), dihydropteroate synthase (DHPS), multidrug resistance 1 protein (MDR1) and chloroquine resistance transporter gene (PvCRT). Gray dots represent all other annotated genes for which the two statistics could be calculated.
Supplementary Figure 9 Results from the McDonald–Kreitman (MK) test using a subset of single-infection and high-quality isolates from Colombia, Peru, Mexico, Thailand, Myanmar and Papua New Guinea.
Codon gapped-nucleotide alignments for each gene in the P. vivax genome were created and aligned to the nearest P. cynomolgi ortholog where available; genes without a one-to-one ortholog were excluded. Plotted along the y axis is the χ2 value associated with the MK test for each gene within the P. vivax alignment set. From this χ2 value, a P value was calculated. A q-value FDR correction was added to account for the effects of multiple sampling. Genes that were found to be significant (q value < 0.05) are shown in red. α, an estimate of the proportion of bases fixed by positive selection, is plotted along the x axis and ranges from ∞ (infinity) to 1. Values above 0 are inferred to be under positive selection, whereas values below zero are subject to purifying selection and/or balancing selection. Significant results are also listed in Supplementary Table 3.
Supplementary Figure 10 Plots of five different population genetic values across chromosome 5 of P. vivax.
In each case, the statistics were calculated using high-quality, single-infection isolates. Values of linkage disequilibrium (LD) are collapsed into a mean value across a window of 50 kb for each nucleotide. In the case of nucleotide diversity (π), Tajima’s D and FST, values represent the mean value across all nucleotides within a 1-kb window. Plotted along the y axis in each figure is a trendline representing a moving average (period 10) for each population genetic statistic. The vertical red line indicates genes of interest identified through high FST values as labeled in Figure 5 or genes with significant McDonald–Kreitman test results as listed in Supplementary Table 3. The thickness of the red line is an approximation of gene length.
Supplementary Figure 11 Plots of five different population genetic values across chromosome 7 of P. vivax.
In each case, the statistics were calculated using high-quality, single-infection isolates. Values of linkage disequilibrium (LD) are collapsed into a mean value across a window of 50 kb for each nucleotide. In the case of nucleotide diversity (π), Tajima’s D and FST, values represent the mean value across all nucleotides within a 1-kb window. Plotted along the y axis in each figure is a trendline representing a moving average (period 10) for each population genetic statistic. The vertical red line indicates genes of interest identified through high FST values as labeled in Figure 5 or genes with significant McDonald–Kreitman test results as listed in Supplementary Table 3. The thickness of the red line is an approximation of gene length.
Supplementary Figure 12 Plots of six different population genetic values across chromosome 9 of P. vivax.
In each case, the statistics were calculated using high-quality, single-infection isolates. Values of linkage disequilibrium (LD) are collapsed into a mean value across a window of 50 kb for each nucleotide. In the case of nucleotide diversity (π), Tajima’s D and FST, values represent the mean value across all nucleotides within a 1-kb window. Plotted along the y axis in each figure is a trendline representing a moving average (period 10) for each population genetic statistic. The vertical red line indicates genes of interest identified through high FST values as labeled in Figure 5 or genes with significant McDonald–Kreitman test results as listed in Supplementary Table 3. The thickness of the red line is an approximation of gene length.
Supplementary Figure 13 Plots of six different population genetic values across chromosome 11 of P. vivax.
In each case, the statistics were calculated using high-quality, single-infection isolates. Values of linkage disequilibrium (LD) are collapsed into a mean value across a window of 50 kb for each nucleotide. In the case of nucleotide diversity (π), Tajima’s D and FST, values represent the mean value across all nucleotides within a 1-kb window. Plotted along the y axis in each figure is a trendline representing a moving average (period 10) for each population genetic statistic. The vertical red line indicates genes of interest identified through high FST values as labeled in Figure 5 or genes with significant McDonald–Kreitman test results as listed in Supplementary Table 3. The thickness of the red line is an approximation of gene length.
Supplementary Figure 14 Plots of six different population genetic values across chromosome 12 of P. vivax.
In each case, the statistics were calculated using high-quality, single-infection isolates. Values of linkage disequilibrium (LD) are collapsed into a mean value across a window of 50 kb for each nucleotide. In the case of nucleotide diversity (π), Tajima’s D and FST, values represent the mean value across all nucleotides within a 1-kb window. Plotted along the y axis in each figure is a trendline representing a moving average (period 10) for each population genetic statistic. The vertical red line indicates genes of interest identified through high FST values as labeled in Figure 5 or genes with significant McDonald–Kreitman test results as listed in Supplementary Table 3. The thickness of the red line is an approximation of gene length.
Supplementary Figure 15 Plots of six different population genetic values across chromosome 14 of P. vivax.
In each case, the statistics were calculated using high-quality, single-infection isolates. Values of linkage disequilibrium (LD) are collapsed into a mean value across a window of 50 kb for each nucleotide. In the case of nucleotide diversity (π), Tajima’s D and FST, values represent the mean value across all nucleotides within a 1-kb window. Plotted along the y axis in each figure is a trendline representing a moving average (period 10) for each population genetic statistic. The vertical red line indicates genes of interest identified through high FST values as labeled in Figure 5 or genes with significant McDonald–Kreitman test results as listed in Supplementary Table 3. The thickness of the red line is an approximation of gene length.
Supplementary Figure 16 Haplotype map of three genes found to exhibit signals of positive selection within P. vivax.
Shown in red are nonsynonymous alternative alleles within each gene. In gray are sites that are identical to the reference genome nucleotide. Blank entries (white) indicate missing data.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–16, Supplementary Table 4 and Supplementary Note. (PDF 3319 kb)
Supplementary Table 1
Genome statistics and metadata. Country of origin, GenBank accession number, and various in silico and molecular biology assay results for the 195 P. vivax isolates analyzed in this study. (XLSX 151 kb)
Supplementary Table 2
Genes of interest. List of nonsynonymous SNPs, insertions, and deletions in 22 genes identified as, or being associated with, genes having high FST. (XLSX 71 kb)
Supplementary Table 3
Summary of results from the McDonald–Kreitman test. (XLSX 536 kb)
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Hupalo, D., Luo, Z., Melnikov, A. et al. Population genomics studies identify signatures of global dispersal and drug resistance in Plasmodium vivax. Nat Genet 48, 953–958 (2016). https://doi.org/10.1038/ng.3588
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DOI: https://doi.org/10.1038/ng.3588
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