16S metagenomic from sputum
DNA was extracted from sputum using the ZymoBIOMICS DNA Miniprep Kit (Zymo Research) following the manufacturer instructions. DNA was amplified using universal bacterial primers flanking the V4 region (515F (GTGCCAGCMGCCGCGGTAA) - 806R (GGACTACHVGGGTWTCTAAT)), each tagged with a spacer of 2-4 nucleotides and the sequencing adapter. PCR was performed in 25 ul volumes composed of Q5 High-Fidelity 1X Master Mix (New England Biolabs GmbH), 25 pmol of each primer and 2 uL of DNA under the following conditions (FlexCycler2, Analytik Jena AG): 94C for 3 min; 25 cycles (94C for 45 s, 50C for 1 min and 72C for 1 min 30 s); 72C for 10 min. PCR products were purified using magnetic beads MagSi-NGSprep Plus (Steinbrenner Laborsystem GmbH) and re-amplified with primers targeting the sequencing adapter region and tagged with unique barcodes (differing by at least 3 nucleotides) and the illumina i5/i7 sequences. Amplicons were purified with magnetic beads MagSi-NGSprep Plus, quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific), pooled equimolarly and sequenced on a Miseq system (Illumina) (600 cycles).