16S-amplicon sequencing (V3V4) of Human vaginal microbiota: sample: 113, CST: III
The first swab (E-swab, Copan, Brescia, Italy) was used for diagnostic tests to exclude the presence of STIs by NAAT (Alinity m STI Assay; Abbott Molecular Diagnostics, Des Plaines, IL, USA). The second was collected with a sterile cotton bud, re-suspended in 1 mL of sterile saline, and stored at -80C until use. Frozen vaginal swabs were thawed, vortexed for 1 min and removed from the liquid. After centrifugation (10000  g for 15 min), the cell-free supernatants were used for metabolomic analysis, whereas bacterial pellets were employed for vaginal microbiome profiling. Nucleic acids were extracted from vaginal swabs by means of the DNeasy Blood & Tissue kit (QIAGEN GmbH, Hilden, Germany), following the manufacturers protocol. The hypervariable V3-V4 regions of the bacterial 16S rRNA gene were amplified, according to 16S metagenomic sequencing library preparation protocol (Illumina, San Diego, CA). Final indexed libraries were prepared by equimolar (4 nmol/L) pooling, denaturation and dilution to 6 pmol/L before loading onto the MiSeq flow cell. A 2  300 bp paired-end run was used.