DNA extraction and 16S rRNA amplicon sequencing
DNA was extracted from the vaginal samples using the Magnetic Soil and
Stool DNA Kit (Tiangen Biotech, Beijing), following the manufacturer’s
instructions. The DNA samples were stored at −80°C. Then, the V1-V2
region of the 16S rRNA gene was amplified using the 27F and 338R
primers. Processing of raw sequencing data was performed on a Illumina
Novaseq platform. A coverage depth of at least 50,000 reads was
generated in each sample. The median sequencing read depth was 58,457
reads. Data was processed using
USEARCH (version 11). Reads of an inferior quality (MaxEE less than
1.00) and length less than 300 bp were removed before further analysis.
We detected Chimeras using the gold database of
UCHIME220. The taxonomy of each amplicon sequence
variant (ASV) was generated using UNOSIE3. Representative sequences were
annotated using the Ribosomal Database Project (RDP) classifier and the
RDP database (rdp_16s_v16_sp.fa)21 with a
confidence threshold of 0.8. Overall, 126,966,654 reads and 4,250 ASVs
were obtained.