We cultured Legionella spp. isolates in a microaerophilic and humid environment at 37°C on BCYE (buffered charcoal yeast extract) agar plates for 48 h. We then picked individual colonies from the plates and grew them in ACES-buffered yeast extract broth containing Legionella BCYE Growth Supplement (Oxoid Ltd., Basingstoke, UK) with shaking at 37°C for 24–48 h. We extracted genomic DNA from fresh cultures by using the QIAGEN DNeasy Blood and Tissue Kit (QIAGEN Benelux B.V., Venlo, the Netherlands).
We prepared sequencing libraries by using the Nextera XT kit for MiSeq or HiSeq (all from Illumina, San Diego, CA, USA) sequencing at Edinburgh Genomics, University of Edinburgh (Edinburgh, Scotland, UK). For each isolate, one 2 × 250–bp or two 2 × 200–bp paired-end sequencing runs were carried out using the MiSeq and HiSeq technologies, respectively. Raw reads were quality checked using FastQC v0.10.1 (22 ), and primers were trimmed by using Cutadapt (23 ). We used wgsim software (24 ) to simulate sequence reads for publicly available, complete whole-genome sequences.
We prepared sequencing libraries by using the Nextera XT kit for MiSeq or HiSeq (all from Illumina, San Diego, CA, USA) sequencing at Edinburgh Genomics, University of Edinburgh (Edinburgh, Scotland, UK). For each isolate, one 2 × 250–bp or two 2 × 200–bp paired-end sequencing runs were carried out using the MiSeq and HiSeq technologies, respectively. Raw reads were quality checked using FastQC v0.10.1 (22 ), and primers were trimmed by using Cutadapt (23 ). We used wgsim software (24 ) to simulate sequence reads for publicly available, complete whole-genome sequences.