Stage-predictive gene sets are enriched for specific biological processes but show no signature of selection by diversity/divergence measures.

<p>(A) Top 15 model <i>β_g,s</i> parameters specific to each stage; values indicate for each gene the degree of its expression attributed to each stage. (B) Gene set enrichments of GO and KEGG processes by stage (Supplementary <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003392#pcbi.1003392.s004" target="_blank">Table S2</a>). (C) Genetic diversity (within patient) vs. divergence (between isolate) of the <i>P. falciparum</i> genome (see Methods for data sources), highlighting genes identified as stage-specific. Several known markers are labeled for reference.</p

<i>In silico</i> dissection approach developing a linear regression model to identify stage-specific gene expression profiles within bulk parasite population gene expression.

<p>(A) Definition of physiologically relevant stage categories within <i>P. falciparum</i> development for which we will identify stage-specific expression signatures. Stages are as follows: R: asexual ring, T: asexual trophozoite and schizont, YG: young gametocyte ring and stage I, DG: developing gametocyte stages II, III, and IV, IG: all immature gametocytes (YG+DG), MG: mature gametocyte stage V, and U: unexpected profile not captured by our defined stages. (B) Linear regression model for the deconvolution of bulk gene expression data from mixed stage samples. Terms are as follows: <i>y_g</i>: total expression of gene g, β<i>_g,s</i>: expression of gene g attributed to stage s, <i>X_s</i>: proportion of the sample that is stage s. (C) Marker Selection. Filters used to narrow down gene sets to our set of sentinel markers for field-applicable qRT-PCR assay. As we chose markers for ring and trophozoite/schizont stages <i>a priori</i> based on published stage-specific gene expression data for asexual development <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003392#pcbi.1003392-Bozdech1" target="_blank">[4]</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003392#pcbi.1003392-LeRoch1" target="_blank">[5]</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003392#pcbi.1003392-Merrick1" target="_blank">[46]</a>, we used this selection method to identify markers for the remaining gametocyte stage categories. (D) Overall stage prediction schematic.</p

qRT-PCR assay optimization.

<p>(A) We collected and analyzed a range of <i>in vitro</i> time points with varying contributions of asexual and sexual stages, from both gametocyte-producing and non-producing lines of 3D7. Absolute number of parasites stages that went into each qRT-PCR reaction well is plotted. (B) Relative qRT-PCR-based gene expression of stage-specific markers for R, T, IG and MG are shown for time points corresponding vertically to those in part A. (C) Inferred proportion of each stage in the total parasite load (model predictions) are shown corresponding vertically to the time points in A and B, plotted as a percentage of total parasites in that sample. (D) <i>In vivo</i> peripheral blood samples from severe malaria patients in Blantyre, Malawi were collected and analyzed. Absolute numbers of parasites stages per µL of blood, as determined by microscopy, are plotted. (E) Relative qRT-PCR-based gene expression of stage-specific markers for T, IG and MG (normalized to <i>SBP1</i>) is shown for time points corresponding vertically to those in part D. (F) Inferred proportion of each stage (model predictions) are shown corresponding vertically to the time points in D and E. Stars indicate subjects in which gametocytes were observed by highly sensitive thick smear examination (one or more gametocytes in 100 high power fields).</p

Genes used in qRT-PCR assay.

<p>Details of the qRT-PCR compatible marker set selected by our combined filtering process. PCR efficiency was calculated based on the slope of the line after running a series of 10-fold dilutions of mixed-stage cDNA (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003392#pcbi.1003392.s002" target="_blank">Figure S2A</a>). Limit of detection was calculated based on the number of parasite stages estimated to be present in the last dilution where the marker was detected. Detection limit ranged from 10¹–10³ pg cDNA, corresponding to approximately 20–200 cells, depending on the stage.</p

Admission characteristics of severe malaria patients tested by qRT-PCR, Blantyre, Malawi, 2011.

<p>Blood was sampled from participants who met the clinical case description of cerebral malaria during the malaria transmission season in 2011. All patients were from Blantyre, Malawi and surrounding areas. Parasitemia was measured by microscopy and qRT-PCR was performed on an RNA sample stored in trizol.</p