5% agarose gel. Bioinformatics methods Illumina order Everolimus MiSeq reads from each isolate were adapter and quality trimmed before use with Trimmomatic.27 Phylogenetic reconstruction of isolates sequenced in this study were combined with data from a global collection of 55 P. aeruginosa strains collected world-wide which have been previously analysed by Stewart et al.28 For each of

the published strains, 600 000 paired-end reads of length 250 bases were simulated using wgsim (https://github.com/lh3/wgsim) from the complete or draft genome assembly deposited in Genbank. Read sets were mapped against the P. aeruginosa PAO1 reference genome using BWA-MEM 0.7.5a-r405 using default settings.29 Single nucleotide polymorphisms were called using VarScan 2.3.6 and filtered for regions with an excessive number of variants. These may represent regions of recombination, misalignments or strong Darwinian selection.30 FastTree (V2.1.7) was used for phylogenetic reconstruction. This software estimates an approximate maximum-likelihood tree

under the Jukes-Cantor model of nucleotide evolution with a single rate for each site (CAT).31 Trees were drawn in FigTree (http://tree.bio.ed.ac.uk/software/figtree/). For in silico MLST prediction, trimmed reads were assembled de novo using Velvet 32 with a k-mer size of 81 and searched using nucleotide BLAST against the multilocus sequence database downloaded from the pubMLST website on 5 August 2013 (http://pubmlst.org/paeruginosa/).33 For Clade E isolates, in order

to exhaustively search for discriminatory mutations, a nearly complete reference genome was generated by de novo assembly using Pacific Biosciences sequencing data. Reads were assembled using the ‘RS_HGAP_Assembly.3’ pipeline within SMRT Portal V2.2.0. Illumina reads from the same sample were mapped to this draft genome assembly in order to correct remaining indel errors in the assembly using Pilon (http://www.broadinstitute.org/software/pilon/). Isolates belonging to each clade were mapped individually against either the PacBio reference (Clade E) or P. aeruginosa PAO1 (“type”:”entrez-nucleotide”,”attrs”:”text”:”NC_002516″,”term_id”:”110645304″,”term_text”:”NC_002516″NC_002516; Clades C, D and G). Variants (single nucleotide polymorphisms and short insertion-deletions) were called using SAMtools mpileup and VarScan with an allele frequency threshold of 80%.30 Non-informative positions and regions of putative recombination were removed, the later with Cilengitide a variant density filter of more than 3 SNPs every 1000 nucleotides. Analysing samples in each clade individually maximised the number of variants detected by reducing the likelihood of the position being uncovered by a subset of samples. From these variants fine-grained phylogenetic trees were reconstructed for each clade using FastTree. The scripts used to perform this analysis are available at http://www.github.com/joshquick/snp_calling_scripts.