H fragment using the primer combinations PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28151467 shown in Table 2. Positive and negative controls (healthy donor PMNs) were included in each assay. Strict laboratory precautions were taken to avoid cross contamination. Specimens that had a clear amplification in each duplicate reaction were considered to be positive.Sequencing and phylogenetic analysisSequencher program 4.7 (Gene Code Corp., Ann Arbor, MI). The alignment of multiple sequences, including the reference sequences for Oxaliplatin chemical information subtypes A , F , J and K (http://hiv-web.lanl.gov), was performed using the CLUSTAL X program [30] and followed by manual editing using the BioEdit Sequence Alignment Editor program version 5.0.7 [31]. PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28549975 Gaps and ambiguous positions were removed from the final alignment. The phylogenetic relationships were determined by two methods: the neighbor-joining (NJ) algorithm of MEGA version 5.0 software [32] and maximum likelihood (ML) using PHYML v.2.4.4 [33]. For the NJ method, trees were constructed under the maximum composite likelihood substitution model and bootstrap re-sampling was carried out 1000 times. For the ML method, phylogentic trees were constructed using the GTR + I + G substitution model and a BIONJ starting tree. Heuristic tree searches under the ML optimality criterions were performed using the nearest-neighbor interchange (NNI) branch-swapping algorithm. The approximate likelihood ratio test (aLRT) based on a Shimodaira-Hasegawa-like procedure was used as a statistical test to calculate branch support. Trees were displayed using the MEGA v.5 package.Nucleotide sequence accession numbersThe sequences described here have been deposited (accession numbers pending)The amplified DNA fragments were purified using a QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and directly sequenced using second-round primers and the PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems/ Perkin-Elmer, Foster City, CA) on an automated sequencer (ABI 3130, Applied Biosystems). After excluding the primer regions, each amplicon was assembled into a contiguous sequence alignment and edited with theResults Blood samples were obtained from 41 MSM study participants who had been previously characterized as being infected with HIV-1 subtype B virus (Figure 1) [27]. The median VL in this cohort was 4.3 ?104 copies/ml (range, <400-39.3 ?104). The median baseline CD4positive T cell count was 564 cells/mm3 (range, 198?2449 cells/mm3). The age of the subjects ranged from 18 to 56 years, and the median age was 30.6 years. All patients were treatment-naive at the time of sampleSoares de Oliveira et al. Virology Journal 2012, 9:223 http://www.virologyj.com/content/9/1/Page 4 ofFigure 1 Phylogenetic tree constructed using a maximumlikelihood method from partial pol region (1279 bp; nt 3822?5101 of HXB2) of 41 samples from MSM that have previously been determined to be infected with HIV-1 subtype B (indicated by black circles) and 37 HIV-1 reference sequences from the Los Alamos HIV-1 database representing 11 genetic subtypes. Samples that were identified in this study to host subclade F1 DNA are indicated with star symbol. For purposes of clarity, the tree was midpoint rooted. The approximate likelihood ratio test (aLRT) values of 90 are indicated at nodes. The scale bar represents 0.05 nucleotide substitutions per site.collection. The main characteristics of the study population are shown in Table 1. Before processing the patient samples, we wanted to deter.
