Initial analysis demonstrated that deep 16S rRNA sequencing enhances the definition of microbial composition and diversity in the vaginal and rectal compartments during pregnancy,21 enhancing “resolution” of composition and diversity differences previously reported by Ravel and others. The number of sequences acquired per specimen is inversely associated with the number of samples multiplexed in a single sequencing reaction. Therefore, our initial objective was to determine the number

of 16S rRNA sequences required per sample for adequate definition of microbial communities in the oral, vaginal, and rectal compartments during pregnancy. Methodology included standard DNA extraction from swab specimens #click here keyword# of the oral cavity, vaginal mucosa, and rectal surface of a cohort of 29 third-trimester

women. Microbial identity was determined by polymerase chain reaction (PCR) amplification and sequencing of the 16S rRNA gene. We acquired 1,000–22,000 sequences per specimen (SPS), and the QIIME pipeline22 was used to assign sequences to the Inhibitors,research,lifescience,medical respective specimen and establish diversity parameters. Diversity in each anatomic site was defined by the number of unique operational taxonomic units (OTU >97% identity) that correlated Inhibitors,research,lifescience,medical with the simulated number of sequences obtained by pyrosequencing (Figure 2). A total of 1.3 million 16S rRNA sequences were sorted into 6,174 OTUs, representing unique genera. In the oral compartment, <2,500 sequences were required to detect the maximum of 220 genera per specimen. Microbial complexity Inhibitors,research,lifescience,medical was limited to 220 distinct genera even at a sequencing depth of 6,500 SPS, defining a diversity ceiling which is similar to the non-pregnant state. As expected, in the rectal compartment, diversity exceeded 650 genera per specimen with consistent linear increases through 6,500 SPS indicating a highly complex microbial environment. Surprisingly, in the vaginal compartment linear increases in the number of genera were also detected

through the collection of >6,500 16S rRNA SPS, similar to the rectal compartment, and >400 genera were identified per specimen in 21/29 Inhibitors,research,lifescience,medical subjects (Figure 1). Microbial composition in the vaginal compartment was complex in the majority of women, exceeding 600 distinct genera, which requires a sequencing depth of 6,500 SPS in a subset of pregnant women. At Linifanib (ABT-869) a sequencing depth of 6,500 SPS, this study represents the most extensive characterization of the vaginal microbiome, since no analysis beyond 2,200 SPS is currently available for the vaginal compartment in the pregnant or non-pregnant state. We conclude that in term pregnancy, a high level of microbial composition complexity exists in the vaginal and rectal compartments, which would require deep sequencing of 16S rRNA genes to define composition and diversity. Figure 2 Phylogenetic tree demonstrating successful cloning of a diverse library of microorganisms.