Sequencing of bacterial microbiota in the vaginal samples
The metagenomic DNA in the collected vaginal secretions was extracted by QIAamp kit (Qiagen, Hilden, Germany), and the purity and concentration of the extracted DNA were detected by Nanodrop (Thermo Fisher Scientific, USA) and agarose gel electrophoresis. Qualified DNA samples were amplified, targeting to the 16S rRNA V4 region using barcoded region-specific primers, 515F (GTGYCAGCMGCCGCGGTA) and 806R (GGACTACHVGGGTWTCTAAT). The amplification conditions were pre-denaturation at 95°C for 1 min; denaturation at 95°C for 30 s; annealing at 60°C for 40 s; extension at 72°C for 1 min, for a total of 30 cycles; terminal extension at 72°C for 7 min and termination at 4°C. Amplified samples were checked by agarose gel electrophoresis for the amplicon product size and purity. Follow-up analysis was only performed on amplicon products appearing as a single and bright band.The samples that met the quality requirement were used for DNA library construction and sequenced using the Illumina novaseq PE250 platform. The original sequences were quality controlled and grouped by the sample nucleotide barcode. Microbial diversity analysis was performed using the QIIME platform (