Computational protocol: Evidence of at Least Two Introductions of HIV-1 in the Amerindian Warao Population from Venezuela

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Protocol publication

[…] The HIV-1 pol (n = 16), env (n = 15), nef and vif (n = 4) sequences obtained from the Waraos were aligned against a set of Venezuelan sequences and related sequences obtained from BLAST analysis, for phylogenetic analysis by Neighbor Joining Method, and against 141 pol and 112 env subtype B “background” sequences isolated from the general population in Venezuela for maximum likelihood (ML) and Bayesian analysis. Subtype B “background” Venezuelan sequences were gathered from the Los Alamos HIV Database ( Sequences were excluded if they originated from the same patient or displayed a multidrug resistance profile. In order to avoid any bias on the phylogenetic analyses, a few sites with major antiretroviral drug resistance mutations and other substitutions associated to drug resistance in PR (46) and RT (70, 90, 103, 106, 138, 179, 184), in at least two sequences, were also excluded. Nucleotide sequences were aligned using CLUSTAL X program . The first alignment of approximately 1000-nt covered the entire protease and part of the reverse transcriptase (PR/RT) region of pol gene (positions 2253–3520 relative to HXB2). The second alignment of approximately 310-nt spanned the V3 region of the Env-gp120 gene (positions 7023 to 7330 relative to HXB2). For nef and vif, alignements spanned almost complete genes (positions 4980 to 5520 and 8760 to 9450, respectively). Alignments are available from the authors upon request. [...] Phylogenetic tree was inferred by Neighbor Joining method using a computerized program DNAman 5.2.2. (Lynnon Bio Soft, Canadá), and the ML method under the GTR+I+Γ4 nucleotide substitution model, selected using the jModeltest program . ML tree was reconstructed with program PhyML using an online web server . Heuristic tree search was performed using the SPR branch-swapping algorithm and the reliability of the obtained topology was estimated with the approximate likelihood-ratio test (aLRT) based on a Shimodaira-Hasegawa-like procedure. Trees were visualized using the FigTree v1.3.1 program . [...] Estimates of the evolutionary rate (μ, units are nucleotide substitutions per site per year, subst./site/year) and the time of the most recent common ancestor (Tmrca, years) were obtained using a Bayesian Markov Chain Monte Carlo (MCMC) approach as implemented in BEAST v1.6.2 , . The temporal structure of the pol and env datasets was not sufficient to reliably estimate the evolutionary rate under a chronological time-scale employing the dates of the sequences. Therefore, the 95% Highest Posterior Density (HPD) intervals of rates of evolution previously estimated for subtype B at PR/RT (1.5×10−3–2.5×10−3 subst./site/year) and gp120-V3 (4×10−3–8×10−3 subst./site/year) genomic regions were incorporated as a prior probability distribution in our analyses. Analyses were performed with a Bayesian Skyline coalescent tree prior , under the GTR+I+Γ nucleotide substitution model and using a relaxed (uncorrelated Lognormal model) molecular clock . Three separate MCMC chains were run for 20×106 generations with a burn-in of 2×106. BEAST output was analyzed using TRACER v1.4 , with uncertainty in parameter estimates reflected in the 95% HPD intervals. All Bayesian MCMC independent runs converged to almost identical values for all parameters, and the ESS values for parameter estimates were >100. The results reported are the combined estimates of the three independent runs. The programs TreeAnnotator v1.6.2 (available from and FigTree v1.3.1 were used to summarize the posterior trees samples from BEAST runs and to visualize the annotated time-scaled maximum clade credibility (MCC) trees. […]

Pipeline specifications

Software tools Clustal W, DNAMAN, jModelTest, PhyML, FigTree, BEAST
Application Phylogenetics
Organisms Human immunodeficiency virus 1
Diseases Communicable Diseases, Hepatitis B, Tuberculosis, HIV Infections