16S rRNAseq of vaginal swab: 23321_Hnvaginal16
Vaginal samples were transferred to a 2 ml tube containing 200 mg of 106/500m glass beads (Sigma, St. Louis, MO) and 0.5 ml of Qiagen PM1 buffer (Valencia, CA). Mechanical lysis was performed for 40 minutes on a Digital Vortex Mixer. After a 5 minute centrifugation, 0.45 ml of supernatants was aspirated and transferred to a new tube containing 0.15 ml of Qiagen IRS solution. The suspension was incubated at 4C overnight. After a 5 minute centrifugation, supernatant was aspirated and transferred to deep well plates containing 0.45 ml of Qiagen binding buffer supplemented with Qiagen ClearMag Beads. DNA was purified using the automated KingFisher Flex Purification System and eluted in DNase free water.  12.5 ng of total DNA were amplified using universal primers targeting the V4 region of the bacterial 16S rRNA gene. Primer sequences contained overhang adapters appended to the 5 end of each primer for compatibility with Illumina sequencing platform. The primers used were F515/R806. Master mixes contained 12.5 ng of total DNA, 0.5 M of each primer and 2x KAPA HiFi HotStart ReadyMix (KAPA Biosystems, Wilmington, MA). The thermal profile for the amplification of each sample had an initial denaturing step at 95C for 3 minutes, followed by a cycling of denaturing of 95C for 30 seconds, annealing at 55C for 30 seconds and a 30 second extension at 72C (25 cycles), a 5 minute extension at 72C and a final hold at 4C. Each 16S amplicon was purified using the AMPure XP reagent (Beckman Coulter, Indianapolis, IN). In the next step each sample was amplified using a limited cycle PCR program, adding Illumina sequencing adapters and dualindex barcodes (index 1(i7) and index 2(i5)) (Illumina, San Diego, CA) to the amplicon target. The thermal profile for the amplification of each sample had an initial denaturing step at 95C for 3 minutes, followed by a denaturing cycle of 95C for 30 seconds, annealing at 55C for 30 seconds and a 30 second extension at 72C (8 cycles), a 5 minute extension at 72C and a final hold at 4C. The final libraries were again purified using the AMPure XP reagent (Beckman Coulter), quantified and normalized prior to pooling. The DNA library pool was then denatured with NaOH, diluted with hybridization buffer and heat denatured.