DNA was extracted from swabs using an enzymatic prelysis step (30 min incubation at 37 °C with an enzyme solution containing 4 U lysostaphine (SAE0091), 25 U mutanolysin (sae0092), and 3 mg lysozyme (L4919) (Sigma-Aldrich, St. Louis, MO, USA); then 30 min incubation at 56 °C with 20 µL protein kinase K (RPROTKSOL-RO, Sigma-Aldrich, St. Louis, MO, USA), followed by DNA extraction on a MagNa-Pure 96 robot using a DNA and Viral NA Small Volume Kit (Roche, Mannheim, Germany).
The V3-V4 region of the 16S rRNA gene and that of the tuf gene were amplified in two separate PCRs (95 °C for 3 min; 25 cycles of 98 °C for 20 s, 60 °C for 15 s, 72 °C for 45 s; 72 °C for 5 min), using primers (16SrRNA: 341F: 5′- CCTACGGGNGGCWGCAG -3′; 805R: 5′- GACTACHVGGGTATCTAATC-3′; tuf: F: 5′- CAGAAGAAAAAGAACGTGG-3′; R: 5′- GTCCTCAACWGGCATCA-3′) with preceding heterogeneity spacers [23 (link),24 (link)]. Amplicon libraries were constructed using nextera indexing primers (Illumina Inc., San Diego, CA, USA) (PCR program used: 95 °C for 3 min; 20 cycles of 98 °C for 20 s, 55 °C for 15 s, and 72 °C for 45 s; 72 °C for 5 min) and sequenced on a MiSeq instrument using a 600 cycle V3 kit (Illumina Inc., San Diego, CA, USA).
The V3-V4 region of the 16S rRNA gene and that of the tuf gene were amplified in two separate PCRs (95 °C for 3 min; 25 cycles of 98 °C for 20 s, 60 °C for 15 s, 72 °C for 45 s; 72 °C for 5 min), using primers (16SrRNA: 341F: 5′- CCTACGGGNGGCWGCAG -3′; 805R: 5′- GACTACHVGGGTATCTAATC-3′; tuf: F: 5′- CAGAAGAAAAAGAACGTGG-3′; R: 5′- GTCCTCAACWGGCATCA-3′) with preceding heterogeneity spacers [23 (link),24 (link)]. Amplicon libraries were constructed using nextera indexing primers (Illumina Inc., San Diego, CA, USA) (PCR program used: 95 °C for 3 min; 20 cycles of 98 °C for 20 s, 55 °C for 15 s, and 72 °C for 45 s; 72 °C for 5 min) and sequenced on a MiSeq instrument using a 600 cycle V3 kit (Illumina Inc., San Diego, CA, USA).