Proportion of monoclonal infected detected as a function of the number of markers genotyped.

<p>The black circles indicate the percentage of infections determined to be monoclonal using only single SNPs (i.e., the initially targeted SNPs) while the grey diamonds show the results using the entire locus sequences.</p

Population stratification analyses showing assignment of the parasites into <i>K</i> = 2, 3, and 4 populations based solely on their genotypes.

<p>Each vertical line displays the ancestry of one parasite (organized along x-axis) in each of the ancestral populations (represented by the different colors). Thin black lines separate districts in Cambodia. Individuals from Thailand and non-Southeast Asia are divided by thick vertical lines.</p

Association between clinical and demographic parameters and complexity of infection.

<p>The table shows the significance of the association (<i>p</i>-values) when comparing monoclonal to polyclonal infections (% monoclonal) or testing the correlation with the most likely numbers of clones of the infections (# clones). The association of the same parameters with parasitemia is also indicated.</p

Complexity of infection according to time of collection.

<p>The infections are separated in monoclonal and polyclonal infections <i>(a)</i> or according to the most likely number of clones <i>(b)</i>. Numbers within the bars indicate sampling size for each category.</p

Novel <i>P. vivax</i> Reticulocyte-binding protein gene.

<p>(<b>A</b>) Next-generation sequencing read coverage along the ∼30 kb contig assembled from the C127 sample. The samples displayed are, from top to bottom: C127, C08, M08, M15, Brazil I, Mauritania I, North Korea, Belem, Chesson and Salvador I. The bottom track shows, in grey, predicted protein coding genes and in black, the position of the predicted reticulocyte-binding protein gene (also highlighted by the red box). Note that there is no coverage of the contig in Salvador I. (<b>B</b>) Phylogenetic tree showing the relationships among protein sequences of <i>P. vivax</i>, <i>P. cynomolgi</i>, <i>P. simiovale</i> and <i>P. knowlesi</i> RBP genes. The position of the predicted <i>P. vivax</i> RBP2e gene is highlighted by the red arrow.</p

Analysis of <i>vir</i> genes in the C127 contigs.

<p>(<b>A</b>) Phylogenetic tree showing the relationships between the protein coding sequences of <i>vir</i> genes from the Salvador I reference genome (solid dots) and those predicted from the C127 contigs (branches without dots at the tips). Annotated <i>vir</i> genes (solid dots) are colored according to their subfamilies. Nodes used to assign predicted C127 <i>vir</i> genes into subfamilies are shown by the colored branches derived from them. (<b>B</b>) Proportion of genes assigned to each major <i>vir</i> subfamily for Salvador I (empty bars) and C127 (black bars).</p

Genomic location of all targeted SNPs.

<p>The SNPs included in the final analyses are highlighted in black.</p

Overview of the genotyping assay.

<p>The grey bar represents the targeted DNA sequence containing a known SNP. A first PCR <i>(a)</i> amplifies 100–300 bp of target DNA sequence using locus-specific primers (in red) including a 5’end tail (in blue). Note that this first PCR is typically performed in multiplex with 8–10 primer pairs in the same amplification reaction. After pooling the amplified products from 13 independent multiplex PCRs, the molecules are amplified by a second PCR <i>(b)</i> targeting the 5’end tail. This PCR adds to each amplified molecule a oligonucleotide index specific to each sample (grey box) and the Illumina sequencing primers. The pools are then sequenced <i>(c)</i> to generate an index sequence and two overlapping reads.</p

Predicted genes in novel <i>P. vivax</i> DNA sequences.

*<p>best blast hit corresponds to a <i>Plasmodium</i> gene</p

Novel <i>P. vivax</i> Erythrocyte-binding protein gene.

<p>(<b>A</b>) Sequence coverage along the 80 kb contig containing a novel predicted <i>P. vivax</i> Erythrocyte-Binding Protein gene. The bottom track shows, in grey, predicted protein coding genes and, in black, the position of the predicted EBP gene (also highlighted by the red box). The upper panels display next-generation sequencing read coverage along the contig sequence. The samples are from top to bottom: C127, C08, M08, M15, Brazil I, Mauritania I, North Korea, Belem, Chesson and Salvador I. Note that in the Salvador I sample, there are essentially no reads mapping to this contig. (<b>B</b>) Phylogenetic tree showing the relationships among EBP protein sequences from <i>P. vivax</i>, <i>P. cynomolgi</i>, <i>P. simiovale</i> and <i>P. knowlesi</i>. The position of the novel predicted <i>P. vivax</i> EBP gene is highlighted by the red arrow. (<b>C</b>) Comparison of the protein domain annotations for the novel predicted EBP gene (top) and the known <i>P. vivax</i> DBP gene. The red box indicates the signal sequence; the blue box the Duffy-binding like domain; the yellow box the C-terminus cysteine-rich domain and the green box the transmembrane domain. (<b>D</b>) Amino acid alignment of the Duffy-binding like domain for different <i>Plasmodium</i> DBP genes and the novel <i>P. vivax</i> and <i>P. cynomolgi</i> EBP genes. The alignment shows the amino acid positions 215–509 of PvDBP (the section of the alignment not displayed corresponds to amino acid 303–371). The grey boxes indicate conserved cysteine positions. The red boxes indicate the positions of two additional cysteines present in the novel EBP genes. (<b>E</b>) The novel <i>P. vivax</i> EBP gene is expressed in blood-stage parasites. The gel picture shows the PCR products for the novel EBP gene (left) and known DBP gene (right) amplified from cDNA of the Belem strain. Note that, for both genes, the primers are located on either side of an intron and that the product size is consistent with amplification of cDNA molecules and excludes DNA contamination.</p