300 bp reagent kit

Total genomic DNA was extracted from vaginal swabs using a PowerSoil^® DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA, USA) according to the manufacturer’s instructions with minor modifications^{39 (link)}. Extracted nucleic acids were stored at −80 °C until use. The V4 region of the 16S rRNA gene was amplified using Illumina adaptor universal primers (515F/806R) and the 16S rRNA Amplification Protocol from the Earth Microbiome Project^{40 (link)}. The PCR amplicon was purified using an UltraClean^® PCR Clean-Up Kit (MO BIO Laboratory, Inc., Carlsbad, CA, USA) and quantified using a Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies, Carlsbad, CA, USA). The samples were pooled and sequenced on a MiSeq platform with a 2 × 300 bp reagent kit (Illumina, San Diego, CA, USA)^{41 (link)}. The generated reads underwent quality filtering and trimming using the FASTX-Toolkit. Sequence data were analysed using Quantitative Insights Into Microbial Ecology (QIIME) 1.5.0 (http://qiime.sourceforge.net)^{42 (link)}. Open-reference operational taxonomic unit picking was performed at the 97% sequence similarity level with reference to the Greengene database (gg_12_10). Taxonomic composition analysis was performed to compare taxonomic abundance between the groups.

Extracted DNA was amplified and sequenced using barcoded universal primers and protocol modified to reduce amplification of host DNA^{31 –33} . The variable 4 (V4) region of the 16S rRNA gene was amplified in triplicate with the following PCR reaction components: 1 × 5Prime Hotstart mastermix, 1X Evagreen, 500 nM forward and reverse primers. Input template concentration varied. Amplification was monitored in a CFX96 RT-PCR machine (Bio-Rad) and samples were removed once fluorescence measurements reached ~10,000 RFU (late exponential phase). Cycling conditions were as follows: 94 °C for 3 min, up to 40 cycles of 94 °C for 45 s, 54 °C for 60 s, and 72 °C for 90 s. Triplicate reactions that amplified were pooled together and quantified with Kapa library quantification kit (Kapa Biosystems, KK4824, Wilmington, MA, USA) before equimolar sample mixing. Libraries were concentrated and cleaned using AMPureXP beads (Beckman Coulter, Brea, CA, USA). The final library was quantified using a High Sensitivity D1000 Tapestation Chip. Sequencing was performed by Fulgent Genetics (Temple City, CA, USA) using the Illumina MiSeq platform and 2×300 bp reagent kit for paired-end sequencing.