Computational protocol: Environmental Heterogeneity Drives Within-Host Diversification and Evolution of Pseudomonas aeruginosa

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Protocol publication

[…] Genomic DNA was prepared from P. aeruginosa isolates using the Wizard genomic DNA purification kit (Promega). Genomes were sequenced to a coverage depth of 90-fold (range, 17- to 207-fold) on either an Illumina GAIIx platform generating 75-bp single-end reads (6 isolates) or a HiSeq2000 platform generating either 75-nt single-end reads (7 isolates) or 100-nt paired-end reads (9 isolates). The genome of DK1-P33F0 was de novo assembled into 6,156,016 bp (252 contigs, N50 = 49,984 bp) with the assistance of the genome sequences of P. aeruginosa PAO1, PA14, and LESB58 by using the Columbus module of Velvet, version 1.0.16 (). The genome assembly of DK1-P33F0 was then used as a reference for the alignment of sequence reads from each of the isolates to identify high-quality SNPs in the nonrepetitive parts of the genome.Mutation detection was performed as previously described (). Maximum-parsimony analysis () was used to reconstruct the phylogeny of DK1 clones, and the bootstrap support values for branches were calculated using PAUP 4.0 (). Calculation of the dN/dS ratio was based on the assumption that a random mutation in the total number of possible SNP mutations would cause a synonymous change 25% of the time (, ). Bayesian analysis of evolutionary rates was performed using BEAST, version 1.7.2 (), with a lognormal relaxed molecular clock model and a general time-reversible substitution model. Mutation rates were calculated from a chain length of 50 million steps, sampled every 5,000 steps. The first 5 million steps were discarded as a burn-in. A maximum clade credibility tree was generated using the TreeAnnotator program from the BEAST package, and the effective sample sizes (ESS) of all parameters were >1,000 as calculated by Tracer, version 1.5 (available from http://beast.bio.ed.ac.uk/Tracer), which was also used to calculate the 95% HPD confidence intervals of the divergence times (i.e., an interval within which the modeled parameter resides with 95% probability). […]

Pipeline specifications

Software tools Velvet, PAUP*, BEAST
Application Phylogenetics
Organisms Pseudomonas aeruginosa, Homo sapiens
Diseases Cystic Fibrosis, Infection