Demographic characteristics, ancestry estimations and <i>GYPB</i>^*<i>S/s</i> genotype frequencies in cases and controls, tests for Hardy-Weinberg equilibrium and association between <i>GYPB</i>^*<i>S/s</i> genotype frequency and infection with malaria.

<p>*SD, standard deviation.</p><p>**Association persists (<i>P</i><0.02) if age, gender and European, African or Native American ancestry are included as covariates.</p

Primers designed to amplify <i>GYPB</i> exons 2, 4, 5 and 6 by PCR.

<p>Primers designed to amplify <i>GYPB</i> exons 2, 4, 5 and 6 by PCR.</p

Summary of <i>GYPB</i> diversity indexes and tests of neutrality based on re-sequencing data of a subset of cases and controls and their partitions in S and s alleles (rs7683365) of the Ss blood group antigens.

<p>*The samples of cases and controls were selected so the proportion of SS, Ss and ss genotypes observed in the total set of cases and controls was matching.</p>a<p><i>P</i><0.02,</p>b<p><i>P</i><0.01,</p>c<p><i>P</i><0.001.</p

Linkage disequilibrium among common SNPs in <i>GYPB</i>.

<p>Linkage disequilibrium among common SNPs in <i>GYPB</i> was estimated in both study groups: the controls (a) and in the cases, malaria infected individuals from Brazilian Amazon (b). Underlined SNPs are non-synonymous substitutions: rs7683365 is the SNP determining S/s antigens; rs1132783 is a Ser/Thr polymorphism (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016123#pone-0016123-t003" target="_blank">Table 3</a>).</p

<i>GYPB</i> haplotype frequencies determined on the re-sequencing panel on the basis of common SNPs (MAF>0.05).

a<p>SNP accounting for S (Thr) and s (Met) phenotypes.</p>b<p>Ser(G)/Thr(C).</p>c<p>The modal haplotype in each group is underlined.</p><p>Non-synonymous substitutions are underlined.</p

Estimation of admixture using Ancestry Informative Markers genotyping.

<p>Individual European, African and Native American ancestry were inferred from 60 ancestry informative markers in cases (magenta) and controls (yellow). Admixture was inferred by comparison with individuals from the putative parental populations: Europeans (red), African/African American (green) and Native Americans (blue). Admixture was estimated using the software Structure and average admixture over cases and controls is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016123#pone-0016123-t001" target="_blank">Table 1</a>.</p

Global statistical assessment of biological replicates.

<p>Heat-map <b>(A)</b> and Principal Component Analysis (PCA) plots <b>(B)</b> of RNA-Seq data generated from the libraries mapped to the <i>T</i>. <i>cruzi</i> genome following removal of rRNA/tRNA features. Once the outlier sample was removed and data passed through normalization and surrogate variable analysis, the strong clustering by condition became evident in both analyses. In both plots, each sample is color coded by developmental stage/strain (Tryp: trypomastigotes; A60: amastigotes collected 60 hpi; A96: amastigotes collected 96 hpi).</p

Differential gene expression across <i>T</i>. <i>cruzi</i> CL-14 and CL Brener developmental stages.

<p>Bar plots of the numbers of genes deemed significantly differentially expressed (adjusted P value <0.05) in (A) CL-14 and (B) CL Brener. The numbers of genes in each category are defined as: log2 fold changes |0–1| (light cyan for positive, light plum for negative), |1–2| (light sky blue for positive, orchid for negative), and |2+| (dodger blue and purple respectively). The diverging patterns of expression are shown by changing bar patterns.</p

Comparative global transcriptional expression patterns of <i>T</i>. <i>cruzi</i> genes encoding polymorphic cell surface proteins in CL Brener and CL-14.

<p>MA plots depicting the log2 fold change (logFC) of genes against the average expression level during the transition of CL Brener and CL-14 across developmental stages. Each dot represents one gene and colored dots represent members of the four of the six largest <i>T</i>. <i>cruzi</i> gene families analyzed: MASP (red), Mucin (blue), Trans-sialidase (purple), and GP63 (green). Dots above and below the red lines represent differentially expressed genes (logFC >1).</p

Constitutive expression of a TS gene in CL-14.

<p>The pROCKNeo vector used for transfection of CL-14 has the TS gene (Tc00.1047053509495.30) flanked by the ribosomal promoter and sequences containing signals for mRNA processing derived from the constitutively expressed housekeeping genes TcP2β (at the 5’ end) and gapdh (at the 3’ end) <b>(A)</b>. Total RNA purified from epimastigotes from WT and transgenic parasites were subjected to northern blot and hybridized with a ³²P-labelled probe that contains sequences corresponding to the C-terminal SAPA repeats present in the TS gene. Lower panel shows ethidium bromide staining of rRNAs in the same gel before transferring to the membrane <b>(B)</b>. Total protein extracts from epimastigotes from WT and transgenic parasites were evaluated for the expression of the transfected TS gene by western blotting with a monoclonal antibody anti-SAPA <b>(C)</b>. The infection profiles of four cloned cell lines derived from CL-14 parasites transfected with the TS gene or with the empty pROCKNeo vector, were compared to WT CL-14 and CL Brener in <i>in vitro</i> infection assays of Vero cells. Equal numbers of tissue culture derived trypomastigotes from each parasite cultures were added to Vero cell monolayers and the total number of trypomastigotes released in the supernatant <b>(D)</b> or the numbers of trypomastigotes released in the supernatant each day post-infection were evaluated over 8 days <b>(E)</b>. Five replicates for each infection experiment were performed.</p