Genome Sequences of 14 Firmicutes Strains Isolated from the Human Vagina

Research on vaginal infections is currently limited by a lack of available fully sequenced bacterial reference strains. Here, we present strains (now available through BEI Resources) and genome sequences for a set of 14 vaginal isolates from the phylum Firmicutes. These genome sequences provide a valuable resource for future research in understanding the role of Gram-positive bacteria in vaginal health and disease.

bacteria or bacterial patterns colonizing the human vagina. For example, bacterial vaginosis (BV) is a common dysbiosis of the vaginal microbiota that is associated with increased risks of sexually transmitted infections and serious complications during pregnancy, such as intrauterine infections and preterm labor (1)(2)(3)(4)(5)(6). BV is generally characterized by the absence of vaginal Lactobacillus and overgrowth of diverse microbes, including Firmicutes, such as Anaerococcus, Finegoldia, Megasphaera, Peptoniphilus, and Veillonella species. However, little is known about the strategies these bacteria use to colonize the vagina or whether they are involved in the causes and complications associated with BV. We isolated 15 Firmicutes from vaginal swabs of healthy and BVaffected pregnant and nonpregnant women. While some of these bacterial species may have sequenced reference strains, there are very few strains from the human vagina whose genomes have been previously sequenced.
Vaginal swabs were collected from nonpregnant and pregnant women according to Washington University institutional review board (IRB)-approved protocols 201108155 and 20110382, respectively. Organisms were isolated from these swabs anaerobically on solid medium. Isolation procedures and clinical information will be described in a future publication. The culture conditions for each strain have been provided to BEI Resources. Sequencing of 16s rRNA genes Ͼ1,400 bp were used to initially assign bacteria genus and species identifications by comparison with the NCBI ribosomal database. Genomic DNA was obtained using the Wizard genomic DNA purification kit (Promega).
De novo assembly of genomes were conducted using the One Button Velvet assembly pipeline (version 1.1.06) (7), with hash sizes of 31, 33, and 35 after downsizing the sample input data to 100ϫ coverage. A minimum length for contigs was set (postassembly) at 200 bp. We performed a screen for core genes (as defined by the HMP [8]) on many of the assemblies to test for com- crossmark pleteness of the genome. Gene annotation was performed using both ab initio and evidence-based (BLAST) predictions. Coding sequences were predicted using GeneMark and Glimmer3 (9, 10). Intergenic regions not identified by GeneMark and Glimmer3 were searched by BLAST in NCBI's nonredundant bacterial (NR) database. The best prediction for each open reading frame was selected by evaluating all predictions against the best evidence (nonredundant bacterial, NR, and Pfam [11]) and resolving overlaps between adjacent coding genes. tRNA genes were determined using tRNAscan-SE (12) and noncoding RNA genes by RNAmmer (13) and Rfam (14). Metabolic pathways and subcellular localization were predicted using KEGG (15) and PSORTb (16), respectively, and functional domains were evaluated using InterProScan (17). Accession number(s). These whole-genome shotgun projects have been deposited in GenBank under the accession numbers listed in Table 1. The sequences described in this paper are the first versions. We have also made the strains available to the research community by depositing them with the Biodefense and Emerging Infections (BEI) Research Resource Repository (see BEI numbers in Table 1).

ACKNOWLEDGMENTS
We thank Justin Perry for technical assistance, Deborah Frank for editorial assistance, and the Women's and Infant's Health Specimen Consortium (especially Michele Landeau) for assistance with the collection of vaginal swabs from pregnant women. We also thank Jenifer Allsworth, Jeff Peipert, and Jennifer Bick of the Contraceptive CHOICE project for providing specimens from nonpregnant women. We also appreciate the generosity of the women who participated in this research.

FUNDING INFORMATION
This work, including the efforts of Makedonka Mitreva, was funded by HHS | National Institutes of Health (NIH) (5U54HG004968). This work, including the efforts of Grace E. Deitzler, was funded by HHS | National Institutes of Health (NIH) (P20DK097798). This work, including the efforts of Amanda L. Lewis and Warren G. Lewis, was funded by HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID) (NIH R01 AI114635). This work, including the efforts of Amanda L. Lewis, was funded by Burroughs Wellcome Fund (BWF).