MOESM2 of MinorityReport, software for generalized analysis of causal genetic variants

Additional file 2: Table S2. Nonsynonymous variant data for a Plasmodium falciparum mutant strain raised for resistance to a dihydroorotate dehydrogenase inhibitor, versus the susceptible parent strain. Multiple mutations arise for all resistant strains, following the copy number variation and amplification shown in Fig. 5

<i>Crithidia mellificae</i> genome assembly statistics.

<p><i>Crithidia mellificae</i> genome assembly statistics.</p

A Draft Genome of the Honey Bee Trypanosomatid Parasite <i>Crithidia mellificae</i>

<div><p>Since 2006, honey bee colonies in North America and Europe have experienced increased annual mortality. These losses correlate with increased pathogen incidence and abundance, though no single etiologic agent has been identified. <i>Crithidia mellificae</i> is a unicellular eukaryotic honey bee parasite that has been associated with colony losses in the USA and Belgium. <i>C. mellificae</i> is a member of the family Trypanosomatidae, which primarily includes other insect-infecting species (<i>e.g</i>., the bumble bee pathogen <i>Crithidia bombi</i>), as well as species that infect both invertebrate and vertebrate hosts including human pathogens (<i>e.g.,Trypanosoma cruzi</i>, <i>T. brucei</i>, and <i>Leishmania spp.</i>). To better characterize <i>C. mellificae</i>, we sequenced the genome and transcriptome of strain SF, which was isolated and cultured in 2010. The 32 megabase draft genome, presented herein, shares a high degree of conservation with the related species <i>Leishmania major.</i> We estimate that <i>C. mellificae</i> encodes over 8,300 genes, the majority of which are orthologs of genes encoded by <i>L. major</i> and other <i>Leishmania</i> or <i>Trypanosoma</i> species. Genes unique to <i>C. mellificae,</i> including those of possible bacterial origin, were annotated based on function and include genes putatively involved in carbohydrate metabolism. This draft genome will facilitate additional investigations of the impact of <i>C. mellificae</i> infection on honey bee health and provide insight into the evolution of this unique family.</p></div

<i>Crithidia mellificae</i>, a trypanosomatid parasite of honey bees.

<p>(A) Majority consensus tree of select members of the Trypanosomatidae derived from Bayesian analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095057#pone.0095057-Yang2" target="_blank">[55]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095057#pone.0095057-Lewis1" target="_blank">[56]</a> (<i>i.e.,</i> MrBayes v3.1.2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095057#pone.0095057-Ronquist1" target="_blank">[57]</a>) of a <i>glyceraldehyde 3-phosphate dehydrogenase</i> (<i>GAPDH</i>) nucleotide alignment (799 nt). <i>T. cruzi</i> was selected as the outgroup based on results from previous phylogenetic analyses <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095057#pone.0095057-Maori1" target="_blank">[15]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095057#pone.0095057-Baer1" target="_blank">[39]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095057#pone.0095057-Simpson3" target="_blank">[50]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095057#pone.0095057-Drummond1" target="_blank">[52]</a>. Numbers on branches are Bayesian posterior probabilities (0–1); scale bar corresponds to the proportion of nucleotide change. The genus and species names are consistent with the GenBank accession numbers in the figure; we note that <i>Crithidia deanei</i> was renamed <i>Angomonas deanei</i>. (B) Composite of light and fluorescent microscope images of <i>C. mellificae</i> illustrate the flagellum, kinetoplast (smaller, brighter DAPI stained organelle; yellow arrow) and nucleus (white arrow) of the crithidial stage and (C) additional life-stages in culture.</p

Predicted genes unique to <i>Crithidia mellificae.</i>

<p>*tblastx of each predicted gene was performed using all Trypanosomatidae sequences in the nr database, a tblastx threshold of an E-value ≤10⁻⁶ was selected and alignments that scored within this threshold and had an associated accession number and annotation are reported above. Significant alignments are reported as follows, “<i>C. mellificae</i> only” indicates that no other annotated sequence aligned with this gene, “Trypanosomatidae chromosome” indicates additional unannotated chromosomal sequences from trypanosomes, often very large data files, have a region within them that aligns with the annotated sequence from <i>C. mellificae</i>.</p

The gene catalogues of <i>Leishmania major</i> and <i>Crithidia mellificae</i> are compared after ortholog analysis by INPARANOID [60].

<p>*Truncated genes at contig ends were included in this analysis for a total of 9,971 ORFs. Approximately 17% of these ORFs are incomplete ends of the same presumed gene, resulting in ∼8,300 actual genes (see Results).</p

Figure 3

<p>The change of gene expression in Fzf1p response clusters, <i>FZF1</i> and galactose utilization genes in response to galactose. (a) Wild type and gal promoter driven <i>FZF1</i> over-expression strains in response to the change from glucose to galactose (experiment E2). (b) Wild type and <i>fzf1</i>Δ strains in response to the change from glucose to galactose (experiment E3). (c) Wild type and <i>fzf1</i>Δ strains in response to the change from raffinose to galactose (experiment E4). Color unit is fold change of gene expression. Gene expression too low to be detected is colored in blue.</p

Figure 1

<p>Illustration of the iterative network learning and experimental feedback algorithm.</p

Figure 4

<p>Glucose repression and derepression of Yhb1p-GFP measured by flow cytometry. (a) wild type strain. (b) <i>tup1</i> deletion strain. To calculate a mean GFP intensity, a minimum 100,000 cells were measured for each time point.</p

Figure 2

<p>The Bayesian average network representation of the models. (a, b) initial model. (c, d) second model. (e, f) third model. (a, c, e) network graphic representation. The green nodes represent gene expression clusters. Representative genes of each cluster are shown in the box below each node. ESR: environmental stress response cluster. energy: glucose metabolism cluster. oxidative stress: the application of H₂O₂ or menadione. Nitric oxide: the duration of NO· exposure. galactose: galactose utilization. diauxic shift: shift between anaerobic growth and aerobic respiration. Nodes with missing values are colored in gray. The CPD table shows the conditional probability distribution of Fzf1p activity. The red edges represent novel predictions from the first network model. (b, d, f) edge confidence score histogram. The dot-filled columns represent edges excluded from a model by structural constraints.</p