DNA extraction

Vaginal and stool samples were processed at UCSF. Two sub-samples (approximately 0.25 g each) per frozen stool (maintained on dry ice during sub-sampling) were obtained under aseptic conditions in a biosafety cabinet using a sterile punch biopsy prior to being pooled and added to 500 μL of cetyltrimethylammonium bromide (CTAB) extraction buffer (5% CTAB in 0.25 M phosphate buffer and 1M NaCl). Vaginal samples were vortexed with the swab remaining in the tube; 500 μL of transport medium was withdrawn from the tube and added to 500 uL of CTAB extraction buffer. DNA from all samples and several extraction blanks were extracted using a modified CTAB–polyethylene glycol (PEG) protocol as previously described.^4,56 Briefly, cells were lysed by bead-beating in Lysis Matrix E tubes (MB Biomedicals) at 5.5 m/s for 30 seconds, phases were separated by centrifugation at 16,000 g for 5 min, and the aqueous layer transferred to a new tube. To improve extraction efficiency, a secondary extraction was performed via addition of 500 μL CTAB to the original extraction tube, bead-beating and phase separation, after which the aqueous layer was mixed with that from the previous extraction. To remove excess phenol, the pooled aqueous layers from both extractions were extracted by vortexing in an equal volume of chloroform followed by centrifugation at 16,000 g for 5 min. The resulting aqueous phase was added to 1mL of PEG precipitating solution (30% PEG 6000 in 1.6M NaCl) and stored overnight at 4°C. Precipitated DNA was recovered by centrifugation for 60 min at 3,000g. DNA pellets were washed twice with 300μL 70% ethanol, air-dried for 10 minutes in a biosafety cabinet, and resuspended in 50 μL of molecular grade water. DNA concentrations were quantified using the Qubit dsDNA HS Assay Kit (ThermoFisher Scientific, MA), diluted to 10 ng/μL⁻¹ and stored at −20°C.