Computational protocol: Genetic Characterization of Continually Evolving Highly Pathogenic H5N6 Influenza Viruses in China, 2012–2016

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Protocol publication

[…] The phylogenetic analyses were performed using the listed gene sequences, which were acquired during a NCBI BLAST analysis. H5- and N6-subtype influenza virus sequences that were deposited from 2012 to 2016 were downloaded from the NCBI and/or the GISAID database. The reference sequences used for the H5-subtype influenza virus classifications were chosen based on guidance from OIE. All of the segmented sequence datasets were aligned using ClustalW 1.6. The alignment lengths for each gene segment dataset were as follows: PB2, 2,280 nt; PB1, 2,271 nt; PA, 2,151 nt; HA, 1692 nt; NP, 1482 nt; NA, 1,413 nt; M, 981 nt; and NS, 822 nt.Preliminary analyses were performed to estimate the best-fit nucleotide substitution model using jModeltest2 (Darriba et al., ). To estimate divergence times and rates of nucleotide substitutions, we used an uncorrelated relax-clock Bayesian Markov chain Monte Carlo method in BEAST v1.8.4 (Drummond et al., ). To determine which were the best-fit phylodynamic models, we performed different combinations of relaxed-clock models (i.e., exponential and log-normal models) and branch rate models (i.e., constant, exponential, expansion, and Bayersian SkyGrid models). The resultant *log files were imported into Tracer (v1.6) and then analyzed using model comparison analyses (AICM analysis; Drummond and Bouckaert, ). The best fit substitution models, relaxed-clock models, and tree models are listed in Supplementary Tables , . For each analysis, MCMC chains were run for 50 million iterations and sampled every 5 1000 iterations after a 10% burn-in to ensure an effective sample size was obtained for all posterior parameters, including the posterior, prior, likelihood, and meanRate (ESS > 200). For Clade 2.3.4.4 subtype H5 AIVs and N6 subtype AIVs analyses, the MCMC chains were run for 400 million iterations. Maximum clade credibility (MCC) trees were obtained from the MCMC tree samples using TreeAnnotator v1.8.4 (Drummond et al., ). The resulting trees were edited and illustrated using FigTree (v1.4.2; Morariu et al., ). […]

Pipeline specifications

Software tools BLASTN, Clustal W, jModelTest, BEAST, FigTree
Application Phylogenetics
Organisms Homo sapiens, Pavo cristatus, Gallus gallus, Mus musculus
Diseases Infection