Thermal cycler system

Microbial genomic DNA from fecal samples was isolated using a QIAamp stool DNA mini kit (QIAGEN, Hilden, Germany) according to the kit manufacturer’s instructions. The PCR of the V1-V2 region of the 16S rRNA genes was performed using a thermal cycler system (Bio-Rad, Hercules, CA, United States), after which the amplicons were purified using a LaboPass PCR purification kit (COSMO GENTECH, Seoul, Republic of Korea). An equimolar concentration of each amplicon from the different samples was pooled to equal proportions based on their molecular weights and purified using Agencourt AMPure XP PCR purification beads (Agencourt Bioscience, Beverly, MA, United States). The DNA concentration and quality were checked using a BioAnalyzer 2100 microfluidic device (Agilent Technologies, Inc., Santa Clara, CA, United States) using a DNA 100 lab chip (Agilent Technologies, Inc.). The mixed amplicons were then amplified on sequencing beads by emulsion PCR (BioRad). The sequencing reactions were performed using an ion torrent personal genome machine (Ion PGM, Thermo Scientific) according to the manufacturer’s instructions.

To extract DNA from vaginal fluids, protocol (A) of G-spin Total DNA Extraction Kit (Intron Biotechnology, Korea) was followed, according to the manufacturer’s instructions. The concentration and purity of extracted DNA detected using the Nanodrop System (Thermo Scientific, United Kingdom) was at a mean of 45.7 ng/μL and absorbance (A260/A280) of 1.73, respectively. For targeting internal transcribed spacer (ITS) [4 ], we used AccuPower PCR Premix Kit (Bioneer, Korea) and one set of primers (TFR3: 5’-CGG GTC TTC CTA TAT GAG ACA GAA CC-3’ and TFR4: 5’-CGG GTC TTC CTA TAT GAG ACA GAA CCG GAG CTG AAT G-3’) was used to prepare the Mastermix tubes at a final volume of 20 μL (5 μL DNA template, 1 μL forward primer, 1 μL reverse primer, and 13 μL nuclease-free water). The Thermal Cycler System (BioRad, USA) was used for PCR, under the following conditions: 1 cycle of initial denaturation at 95°C for 5 min; 30 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 1 min; and 1 cycle for a final extension at 72°C for 5 min. Polymerase chain reaction products were electrophoresed using 1.5% agarose gel stained with ethidium bromide at 100 V and 80 Amp for 1 h. Gel bands were visualized using an ultraviolet-transilluminator (Clinex, China). Amplified PCR products were considered to be positive at approximately 347 bp.