Induction and initial sample handling
Sputum induction was undertaken and processed as previously described (15). Frozen sputum samples were sent to Second Genome (San Francisco, California, USA) for metagenomic sequencing. Genomic DNA was extracted using the MoBio Tissue and Cells DNA Isolation Kit (Qiagen, Germantown, USA) at 250μl input volume, performed in a BioSafety Cabinet in an ISO8 Cleanroom. Each batch included negative and positive controls, as described previously (17). Extracted DNA was stored at -20°C. Samples were prepared for sequencing with the Illumina Nextera kit (Illumina, San Diego, USA) and quantified with Quant-iT dsDNA High Sensitivity assays (ThermoFisher Scientific, Waltham, USA). Libraries were pooled and run with 100 base-pairs paired-end sequencing protocols on the Illumina HiSeq 2500 platform.