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

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

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 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

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