Evaluation of EMIRGE, modQIIME and RTAX on different datasets.

<p>Precision and recall rates for the “Oral”, “Gut”, “Complex” and ABC33 datasets using EMIRGE, modQIIME and RTAX at a 0.1% relative abundance threshold. The percentage of sequences/OTUs removed because of the abundance threshold is given in parentheses for each method.</p

<i>In silico</i> evaluation of 16S rRNA PCR primers.

<p>A) Percentage of sequences matching individual primers, with the top two primers highlighted in boxes. B) Percentage of sequences amplifiable by various primer pairs (338F*/1061R is the best pair). Percentage of matched sequences is measured against the Greengenes 16S rRNA sequence database. See Table S4 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060811#pone.0060811.s001" target="_blank">File S1</a> for primer sequences and results measured against the RDP and SILVA databases. Primer numbering is based on the <i>E. coli</i> system of nomenclature as in Brosius <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060811#pone.0060811-Brosius1" target="_blank">[37]</a> and for simplicity the same name (say 784F) is used for both forward and reverse primers at a given position.</p

Species- and genus-level resolution of various sequencing approaches.

<p>Resolution was measured by the number of OTUs/clusters produced using UCLUST <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060811#pone.0060811-Edgar1" target="_blank">[21]</a> at the species (97% identity) and genus level (95% identity) for 16S rRNA sequences in the Greengenes database, based on various end-sequencing (76 bases in length from either the 5′ or 3′ end) and shotgun-sequencing approaches and primer combinations. A higher OTU/cluster number indicates a theoretical higher level of resolution for taxonomic classification. The numbers in parenthesis provide the purity of clusters as measured by the percentage of clusters with homogenous taxonomy assignments in Greengenes. Entries with the highest resolution and/or purity for each sequencing approach are marked in bold. The primer sequences can be found in <b>Table S4</b> in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060811#pone.0060811.s001" target="_blank">File S1</a></b>.</p

Community composition based on 16S rRNA sequence reconstruction using EMIRGE.

<p>A) Correlation between known and estimated relative abundances of predicted species on three <i>in silico</i> datasets. A log-scaled version of this plot can be seen in <b>Figure S1</b> in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060811#pone.0060811.s001" target="_blank">File S1</a></b>. B) Composition at the phylum level for the throat swab and stool sequencing datasets.</p

Measures of intra-host DENV2 diversity in human and mosquito hosts.

<p><b>(A)</b> Number of SNVs; <b>(B)</b> Sum of SNV frequencies; <b>(C)</b> Average SNV frequency; <b>(D)</b> Standard error of the mean SNV frequency; all calculated on a per sample basis. Abd, abdomen; SG, salivary gland.</p

Frequencies of maintained SNVs in the human, mosquito abdomen, and mosquito salivary gland.

<p>Red, non-synonymous SNVs; black, synonymous SNVs. SNV positions mentioned in the text are highlighted with arrows and drawn as dotted lines for ease of viewing.</p

SNVs maintained between EDEN transmission pairs.

<p>NS, non-synonymous; S, synonymous; aa, amino acid.</p><p>SNVs maintained between EDEN transmission pairs.</p

SNVs maintained from human to mosquito.

<p>NS, non-synonymous; S, synonymous; aa, amino acid.</p><p>SNVs maintained from human to mosquito.</p

Selection pressures on the DENV genome, analyzed per gene, at different stages of horizontal transmission.

<p>Ratios of the number of non-synonymous (NS) to synonymous (S) SNVs per gene are shown for <b>(A)</b> human- versus mosquito-derived (both abdomen and salivary gland) DENV populations, and <b>(B)</b> mosquito abdomen- versus salivary gland-derived DENV populations. Human-derived samples are a pool of DENV2 patient samples from this study and DENV1 and DENV3 patient samples from the EDEN study [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004052#pntd.0004052.ref019" target="_blank">19</a>]. Numbers of NS and S SNVs for each gene are indicated in tables below the graphs. *, p < 0.05; Fisher's exact test (M, p = 0.00439; E, p = 0.031; NS1, p = 0.002).</p

Analysis of Dengue Virus Genetic Diversity during Human and Mosquito Infection Reveals Genetic Constraints

<div><p>Dengue viruses (DENV) cause debilitating and potentially life-threatening acute disease throughout the tropical world. While drug development efforts are underway, there are concerns that resistant strains will emerge rapidly. Indeed, antiviral drugs that target even conserved regions in other RNA viruses lose efficacy over time as the virus mutates. Here, we sought to determine if there are regions in the DENV genome that are not only evolutionarily conserved but genetically constrained in their ability to mutate and could hence serve as better antiviral targets. High-throughput sequencing of DENV-1 genome directly from twelve, paired dengue patients’ sera and then passaging these sera into the two primary mosquito vectors showed consistent and distinct sequence changes during infection. In particular, two residues in the NS5 protein coding sequence appear to be specifically acquired during infection in <i>Ae</i>. <i>aegypti</i> but not <i>Ae</i>. <i>albopictus</i>. Importantly, we identified a region within the NS3 protein coding sequence that is refractory to mutation during human and mosquito infection. Collectively, these findings provide fresh insights into antiviral targets and could serve as an approach to defining evolutionarily constrained regions for therapeutic targeting in other RNA viruses.</p></div