16S rRNA sequencing and analysis

DNA extractions for microbiota analysis were performed as previously described^{22, 23} on aliquots of the same CVM samples used for microscopy studies and other characterizations described. The method of Fadrosh et al.⁵² was used to analyze the vaginal microbiota composition and structure and relied on amplification and sequencing on an Illumina MiSeq instrument (300-bp paired-end reads) of the V3-to-V4 regions of the 16S rRNA gene. Sequence analyses and taxonomic assignments were performed using a custom pipeline, freely available on GitHub (https://github.com/cwzkevin/MiSeq16S). The resulting taxonomic assignments are shown in Table S1. To assess whether the vaginal microbiota affected barrier properties and Ab-mediated trapping in CVM, we grouped samples into Community State Types (CSTs) I-V according to the most dominant bacterial species within a sample. Samples were assigned to CSTs according to those identified by Ravel et.al.²³: L. crispatus-dominated (CST I), L. gasseri dominated (CST II), L. iners-dominated (CST III), a diverse set of strict or facultative anaerobic bacteria such as G. vaginalis (CST IV), L. jensenii-dominated (CST V). A sample grouped into a particular CST contained an average of ~80% of the class-defining species (Figure S2). However, microbial populations between samples can be extremely diverse. In this sample set, percent of dominant bacterial species used to classify CST ranged from 34.26% to 99.9% of total bacteria.