Rprotksol ro

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).

Nasal samples from drivers and their spouses were subjected to characterization of the nasal microbiota by 16S rRNA gene sequencing. Before DNA extraction, a 200-μl sample volume (out of 1 ml eSwab liquid amies) was used for enzymatic prelysis by incubation for 30 min at 37°C with enzyme solution containing 2.5 U lysostaphine [SAE0091], 25 U mutanolysin [sae0092], and 3 mg lysozyme ([L4919]) (Sigma-Aldrich, St. Louis, MO, USA), followed by a 30-min incubation at 56°C with 20 μl protein kinase K (RPROTKSOL-RO; Sigma-Aldrich). DNA from samples and blank controls was then extracted on a MagNA Pure 96 robot using a DNA and viral nucleic acid (NA) small volume kit (Roche, Mannheim, Germany). A mock community was used as positive controls (ZymoBiomics microbial community standard, D6300; Zymo Research).
The V3-V4 region of the 16S rRNA gene was amplified in a PCR using the following barcoded primers: 341F, 5′-CCTACGGGNGGCWGCAG-3′; and 805R, 5′-GACTACHVGGGTATCTAATCC-3′ (30 (link)). The primers were preceded by heterogeneity spacers. Library preparation was done using a Nextera XT DNA library preparation kit (Illumina Inc., San Diego, CA, USA). A MiSeq instrument (Illumina Inc.) was used for sequencing with a 600-cycle V3 kit.