2.2 16S rRNA and ITS2 amplification and sequencing
Total DNA was extracted from 200 μL of lysate using Cador Pathogen 96
QIAcube HT Kit (Qiagen, Hilden, North Rhine-Westphalia, Germany)
according to the instructions provided by the manufacturer. The V3-V4
region of bacterial 16S rRNA was amplified using the primers Pro341F
(5′-CCTACGGGNBGCASCAG -3′) and Pro805R (Rev 5′-GACTACNVGGGTATCTAATCC
-3′)(26). ITS2 region of the fungal ribosomal small subunit RNA was
amplified using the primers ITS3f (5′- GCATCGATGAAGAACGCAGC -3′) and
ITS4r (5′- TCCTCCGCTTATTGATATGC -3′). PCR was performed with the
following conditions: 94°C for 1 minute, 35 cycles of 94°C for 30
seconds, 55°C for 30 seconds, and 68°C for 45 seconds, followed by final
extension at 68°C for 7 minutes. PCR products were analyzed by
electrophoresis on 1.5% agarose gel. Negative controls (PCR reagents
without template DNA and template from blank swabs) were included to
verify the possible presence of contaminating DNA. The PCR-amplified
amplicons were purified using Agencourt AMPure XP kit (Beckman Coulter,
Inc., Brea, California, USA), quantified with a Qubit 2.0 instrument
(Thermofisher, Waltham, Massachusetts, US) and sequenced with the
Illumina MiSeq platform (MiSeq ver. 3, 600 cycles, Illumina, Inc., San
Diego, California, USA) to generate paired-end reads with a nominal
length of 300 bp in each direction.