Additional modules were included in PyroTrop to assess the frequency of each amino acid at each position

Technique 4: the complete established of UDPS sequences was interpreted with our in-home software PyroTrop. Briefly, the earlier explained PyroMute computer software [15] was modified to evaluate each and every validated V3 sequence by means of the eleven/twenty five/cost algorithm. PI4KIIIbeta-IN-9 customer reviews PyroTrop employs a few top quality filters to remove unreliable sequences, including sequences with a PHRED top quality rating <20 [18], sequences not spanning the full-length HIV V3 loop, and sequences that are too divergent from the HXB2 strain reference HIV sequence (GenBank accession number K03455). The latter filter was used to eliminate eventual contaminations by non-HIV sequences. Then, PyroTrop ascribes each valid V3 sequence to either CCR5 or CXCR4 by means of the 11/25/charge algorithm [19] and provides the respective percentages of these two populations within the patient's quasispecies (2% of CXCR4 variants was set as the detection threshold) [20]. The results are provided as the respective proportions of CCR5 and CXCR4 populations in the patient's viral quasispecies, with a predefined lower limit of detection of 2% [7]. Additional modules were included in PyroTrop to assess the frequency of each amino acid at each position, allowing for "machine learning" on the full data set.Sensitivity, specificity, positive predictive value (PPV, defined as the capacity of the test to correctly predict a significant viral level decrease), negative predictive value (NPV, defined as the capacity of the test to predict the lack of significant viral level decrease) and Receiver Operating Characteristic (ROC) curves were compared for the 4 methods by means of McNemar test and area under the curves (AUROC) comparisons. In addition, supervised classification using random forest, regression using lasso, and decision tree methods were used to assess the relationship between amino acid polymorphisms and the response to maraviroc. For each selected candidate polymorphism, ROC curves were plotted and the AUROCs were compared by means of bootstrap methods using an AUROC of 0.5 as non-predictive [21]. Analyses were made with packages randomForest, rpart, MASS, ROCR, cluster, pvclust, glmnet and pROC in R language software by means of RGui (64-bit) (v2.14.1) [22,23].HIV RNA could not be amplified in 11 patients at D0. Ten of them had an HIV RNA level below 1000 copies/ml the remaining one had an HIV RNA level of 4.6 log copies/ml but could not be amplified on three repeat tests. Pyrosequencing data of good quality were obtained in the remaining 102 patients at D0 85, 79 and 73 of them had samples available for HIV RNA level determination at M1, M3 and M6, respectively. Overall, 16%, 13% and 12% of patients were considered as non-responders (<1 log HIV RNA level decrease) at M1, M3 and M6, respectively. The HIV RNA kinetics in responders is shown in S1 Fig. UDPS generated a total of 626,874 sequences (mean coverage 614698 per sample), among which 539,096 were valid for22277057 geno2pheno[454] analysis (mean coverage 528590 per sample rejection rate: 14.3%) and 461,044 for PyroTrop analysis (mean coverage 452006 per sample rejection rate: 26,6%). Most of the removed sequences were rejected because the coverage of the V3 loop was partial (criterion used by all methods).