Model of HIV copy-choice recombination.

<p>The reverse transcriptase (RT) complex is shown moving from the 3′ end of the donor RNA to the 5′ end. The RNAse activity of RT is indicated by the light grey nucleotides on the donor RNA. The nascent negative DNA strand can be observed to the right of the RT complex. A potential strand-transfer event by RT is indicated by the dashed arrow. The dashed boxes indicate windows of decreased probability of crossover that have been anchored to the 5′ side of each mismatch. The probability of a crossover occurring at each base on the acceptor strand is indicated by <i>p</i>₁, <i>p</i>₂, or <i>p</i>₃ as described in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1000178#s4" target="_blank">Methods</a>. The plot along the bottom is a representation of each of the probability values across the sequence. For this stretch of 16 nucleotides, the total probability of a crossover occurring is given by the equation shown.</p

The significance of local sequence identity to recombination.

<p>The main plot displays the normalized distribution of in vitro breakpoints falling within zones ranging from size 1 to 25 (vertical grey bars); see Baird et al. <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1000178#pcbi.1000178-Baird1" target="_blank">[21]</a> for further details. The horizontal lines indicate the expected random distribution of breakpoints for the zones. The inset plot shows the normalised frequency of both the in vitro breakpoints and randomly generated breakpoints for zones up to size 25 (arranged in groups of five). On the main plot, error bars on the random distributions (vertical lines) represent one standard error to include 68.3% of the distribution. On the inset, the error bars on the random distributions represent 1.96×standard error to include 95% of the distribution.</p

Implementation of the recombination model.

<p>Comparison of model-predicted breakpoints (horizontal lines) with breakpoint locations from HIV-1 recombinants (vertical bars) <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1000178#pcbi.1000178-Fan1" target="_blank">[26]</a> from the HIV Sequence Database. White bars indicate where the number of breakpoints for the global data is significantly higher than the prediction for the region, light grey bars indicate where the global data falls within the prediction, while dark grey indicates where the global data is significantly lower than the model prediction. The error bars on the model-predicted values represent 1.645×standard error to include 90% of the distribution. The normalised frequency data (<i>y</i>-axis) have been divided into bins of size 400 nucleotides (<i>x</i>-axis). Below the <i>x</i>-axis, the various genomic regions of the HIV-1 genome are displayed. Note, positioning of genes is relative to a gap-stripped sequence alignment.</p

Comparison of the frequency of variants across NGS platforms.

<p>The heights of the bars represent the combined frequency of V3 variants detected by the NGS platforms 454™, Illumina®, PacBio®, and Ion Torrent™ prior to filtering. The colors within each bar denote the proportional contribution made by each platform after normalization based on coverage. Insets show low frequency variants up to a maximum of 20 unique sequences.</p

HIV-1 coreceptor tropism determination using deep sequencing.

<p>(A) HIV-1 tropism determined at baseline using Trofile™ (Monogram Biosciences) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049602#pone.0049602-Whitcomb1" target="_blank">[11]</a>; R5, CCR5-tropic virus; D/M, dual mixed. (B) Virologic response at week 12 of a maraviroc-based antiretroviral regimen. Y or N corresponds to plasma viral load below or not 400 copies/ml at week 12, respectively. E.S., end of study (patient did no enter the study following the detection of non-R5 variants at baseline using Trofile™). (C) Quantification of non-R5 variants detected by deep sequencing as predicted using four HIV-1 tropism algorithms, i.e., 11/24/25 rule <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049602#pone.0049602-Archer1" target="_blank">[24]</a>, Geno2Pheno 3.5% FPR <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049602#pone.0049602-Swenson2" target="_blank">[25]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049602#pone.0049602-Swenson3" target="_blank">[28]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049602#pone.0049602-Harrigan1" target="_blank">[42]</a>, Geno2Pheno 10% FPR, and Web PSSM using the subtype B x4r5 matrix <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049602#pone.0049602-Jensen1" target="_blank">[43]</a>. Dotted line represents the ≥2% suggested cutoff for the minimal amount of non-R5 sequences to be present in the viral population in order to classify a given virus as non-R5 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049602#pone.0049602-Swenson2" target="_blank">[25]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049602#pone.0049602-Swenson3" target="_blank">[28]</a>.</p

Comparison of the clustering of variants across platforms.

<p>The ten most common nucleotide V3 sequences from samples 10–172, 10–176, and 10–180 -obtained with each of the four NGS platforms (454™, Illumina®, PacBio®, and Ion Torrent™)- were aligned against the respective population (sanger) sequence and analyzed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049602#pone-0049602-g004" target="_blank">Figure 4</a> legend.</p

Schema summarizing the strategy followed in this study to compare the use of the four next-generation sequencing platforms (454™, Illumina®, PacBio®, and Ion Torrent™) to determine HIV-1 coreceptor tropism (see text for full details).

<p>Schema summarizing the strategy followed in this study to compare the use of the four next-generation sequencing platforms (454™, Illumina®, PacBio®, and Ion Torrent™) to determine HIV-1 coreceptor tropism (see text for full details).</p

Comparison of data processing across NGS platforms.

<p>Number of sequencing errors, substitutions, deletions, and insertions (per read) for the NGS platforms: 454™, Illumina®, PacBio®, and Ion Torrent™. The mean and interquartile range (IQR) are indicated for each sample. Whiskers indicate 1.5 times the IQR as is the default value in the R-statistical package <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049602#pone.0049602-Team1" target="_blank">[75]</a>.</p

Additional file 1: of In silico screening for candidate chassis strains of free fatty acid-producing cyanobacteria

Table S1. Classification of orthologous groups. Table S2. Proteins used to construct Fig. 1. Table S3. Compilation of control dataset. Table S4. Constructed target species dataset. Table S5. Comparison of FFASC and Model SEED. Table S6. Free fatty acid (FFA) protein/enzyme domains. Table S7. Orthologous group hit number matrix of 49 OGs and 128 (cyanobacteria and diatom) strains. Table S8. Ranked list of 125 strains using FFASC. Table S9. 128 strains clustered using K-mean. Table S10. Ranked list of 125 cyanobacteria using FFASC without optimization. (XLSX 373 kb

Diversifying selection at individual nucleotide sites.

<p>A summary of the number of codon sites identified by the SLAC method implemented in by HyPhy <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004281#ppat.1004281-Pond1" target="_blank">[50]</a> that show positive or negative selection at the APOBEC3 and non-APOBEC3 motifs in the regions of the Gag and Vif genes of HIV-1 we sequenced (<i>P</i>-value<0.02). The Vif gene of HIV-1 was over-represented with G-to-A mutations in an APOBEC3 editing context at positively selected sites (<b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004281#ppat.1004281.s008" target="_blank">Table S5</a></b>).</p