title: Vaginal microbiota in CVD
design: Microbiome analysis was conducted using next-generation sequencing (NGS) of bacterial 16S rDNA (V4/V5). DNA was extracted from vaginal fluid pellets using the QIAamp DNA Mini Kit (QIAGEN, Netherlands). Target regions were amplified with Q5 High-Fidelity polymerase (New England BioLabs, UK) using specific primers: F519/R926 for 16S. For library preparation, sample-specific barcodes and Illumina P5/P7 adapters were added in a second PCR using Q5 polymerase. Final PCR products were gel-purified, quantified with Qubit dsDNA HS Assay (Thermo Fisher), pooled equimolarly, and quality-checked using Agilent 2100 Bioanalyzer (High Sensitivity DNA Kit). Library quantification was performed with the KAPA Library Quantification Kit (Kapa Biosystems). Libraries were denatured, spiked with 1020% PhiX control, diluted to 8.5 pM, and sequenced on an Illumina MiSeq using MiSeq Reagent Kit v2 (500 cycles), following the manufacturer's protocol.