16S RNA of Homo sapien: Vaginal
Metataxonomic profiling was carried out for both vaginal and semen samples through high-throughput sequencing of the 16S rRNA gene using the Illumina MiSeq platform at the Madrid Science Park. Universal primers S-D-Bact-0341-b-S-17 (CCTACGGGNGGCWGCAG) and S-D-Bact-0785-a-A-21 (GACTACHVGGGTATCTAATCC), targeting the V3-V4 hypervariable regions of the 16S rRNA gene, were utilized [24]. In the secondary PCR, forward and reverse sequences were demultiplexed using unique barcodes appended to the 3&#39; and 5&#39; termini of the PCR amplicons. DNA concentration for each sample was determined using the 2100 Bioanalyzer system (Agilent, Santa Clara, CA, USA). PCR products were pooled in equimolar concentrations, and the correct-sized fragments were excised from agarose gels, purified using the QIAEX II Gel Extraction Kit (Qiagen), and quantified using the PicoGreen assay (BMG Labtech, Jena, Germany). The pooled, purified, and barcoded amplicons were sequenced using the paired-end protocol on an Illumina MiSeq platform (Illumina Inc., San Diego, USA) following the manufacturer&#39;s protocols, with sequencing performed at the Madrid Science Park (Tres Cantos, Spain).