title: 16S metagenomic from sputum
design: 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).