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The incidence of preterm birth exceeds 10% worldwide. There are significant disparities in the frequency of preterm birth among populations within countries, and women of African ancestry disproportionately bear the burden of risk in the United States. In the present study, we report a community resource that includes 'omics' data from approximately 12,000 samples as part of the integrative Human Microbiome Project. Longitudinal analyses of 16S ribosomal RNA, metagenomic, metatranscriptomic and cytokine profiles from 45 preterm and 90 term birth controls identified harbingers of preterm birth in this cohort of women predominantly of African ancestry. Women who delivered preterm exhibited significantly lower vaginal levels of Lactobacillus crispatus and higher levels of BVAB1, Sneathia amnii, TM7-H1, a group of Prevotella species and nine additional taxa. The first representative genomes of BVAB1 and TM7-H1 are described. Preterm-birth-associated taxa were correlated with proinflammatory cytokines in vaginal fluid. These findings highlight new opportunities for assessment of the risk of preterm birth.

The microbiome of the female reproductive tract has implications for women’s reproductive health. We examined the vaginal microbiome in two cohorts of women who experienced normal term births: a cross-sectionally sampled cohort of 613 pregnant and 1,969 non-pregnant women, focusing on 300 pregnant and 300 non-pregnant women of African, Hispanic or European ancestry case-matched for race, gestational age and household income; and a longitudinally sampled cohort of 90 pregnant women of African or non-African ancestry. In these women, the vaginal microbiome shifted during pregnancy toward Lactobacillus-dominated profiles at the expense of taxa often associated with vaginal dysbiosis. The shifts occurred early in pregnancy, followed predictable patterns, were associated with simplification of the metabolic capacity of the microbiome and were significant only in women of African or Hispanic ancestry. Both genomic and environmental factors are likely contributors to these trends, with socioeconomic status as a likely environmental influence. Ancestry and socioeconomic factors influence predictable changes in the vaginal microbiome that occur early in pregnancy in women who experience normal term birth.

The incidence of preterm birth exceeds 10% worldwide. There are significant disparities in the frequency of preterm birth among populations within countries, and women of African ancestry disproportionately bear the burden of risk in the United States. In the present study, we report a community resource that includes 'omics' data from approximately 12,000 samples as part of the integrative Human Microbiome Project. Longitudinal analyses of 16S ribosomal RNA, metagenomic, metatranscriptomic and cytokine profiles from 45 preterm and 90 term birth controls identified harbingers of preterm birth in this cohort of women predominantly of African ancestry. Women who delivered preterm exhibited significantly lower vaginal levels of Lactobacillus crispatus and higher levels of BVAB1, Sneathia amnii, TM7-H1, a group of Prevotella species and nine additional taxa. The first representative genomes of BVAB1 and TM7-H1 are described. Preterm-birth-associated taxa were correlated with proinflammatory cytokines in vaginal fluid. These findings highlight new opportunities for assessment of the risk of preterm birth.

Preterm premature rupture of membranes (PPROM) is the leading identifiable cause of preterm birth with ~ 40% of preterm births being associated with PPROM and occurs in 1% - 2% of all pregnancies. We hypothesized that multiple rare variants in fetal genes involved in extracellular matrix synthesis would associate with PPROM, based on the assumption that impaired elaboration of matrix proteins would reduce fetal membrane tensile strength, predisposing to unscheduled rupture. We performed whole exome sequencing (WES) on neonatal DNA derived from pregnancies complicated by PPROM (49 cases) and healthy term deliveries (20 controls) to identify candidate mutations/variants. Genotyping for selected variants from the WES study was carried out on an additional 188 PPROM cases and 175 controls. All mothers were self-reported African Americans, and a panel of ancestry informative markers was used to control for genetic ancestry in all genetic association tests. In support of the primary hypothesis, a statistically significant genetic burden (all samples combined, SKAT-O p-value = 0.0225) of damaging/potentially damaging rare variants was identified in the genes of interest-fibrillar collagen genes, which contribute to fetal membrane strength and integrity. These findings suggest that the fetal contribution to PPROM is polygenic, and driven by an increased burden of rare variants that may also contribute to the disparities in rates of preterm birth among African Americans.

Background Bacterial vaginosis (BV) is the leading dysbiosis of the vaginal microbiome. The pathways leading towards the development of BV are not well understood. Gardnerella vaginalis is frequently associated with BV. G. vaginalis produces the cholesterol-dependent cytolysin (CDC), vaginolysin, which can lyse a variety of human cells and is thought to play a role in pathogenesis. Because membrane cholesterol is required for vaginolysin to function, and because HMG-CoA reductase inhibitors (statins) affect not only serum levels of cholesterol but membrane levels as well, we hypothesized that statins might affect the vaginal microbiome. Methods To investigate the relationship between use of the statins and the vaginal microbiome, we analyzed 16S rRNA gene taxonomic surveys performed on vaginal samples from 133 women who participated in the Vaginal Human Microbiome Project and who were taking statins at the time of sampling, 152 women who reported high cholesterol levels but were not taking statins, and 316 women who did not report high cholesterol. To examine the effect of statins on the cytolytic effect of vaginolysin, the cholesterol-dependent cytolysin (CDC) produced by Gardnerella vaginalis, we assessed the effect of simvastatin pretreatment of VK2E6/E7 vaginal epithelial cells on vaginolysin-mediated cytotoxicity. Results The mean proportion of G. vaginalis among women taking statins was significantly lower relative to women not using statins. Women using statins had higher mean proportions of Lactobacillus crispatus relative to women with normal cholesterol levels, and higher levels of Lactobacillus jensenii relative to women with high cholesterol but not taking statins. In vitro, vaginal epithelial cells pretreated with simvastatin were relatively resistant to vaginolysin and this effect was inhibited by cholesterol. Conclusions In this cross-sectional study, statin use was associated with reduced proportions of G. vaginalis and greater proportions of beneficial lactobacilli within the vaginal microbiome. The negative association between statin use and G. vaginalis may be related to inhibition of vaginolysin function.

Background Recent advances in next-generation sequencing have revolutionized genomic research. 16S rRNA amplicon sequencing using paired-end sequencing on the MiSeq platform from Illumina, Inc., is being used to characterize the composition and dynamics of extremely complex/diverse microbial communities. For this analysis on the Illumina platform, merging and quality filtering of paired-end reads are essential first steps in data analysis to ensure the accuracy and reliability of downstream analysis. Results We have developed the Merging and Filtering Tool (MeFiT) to combine these pre-processing steps into one simple, intuitive pipeline. MeFiT invokes CASPER (context-aware scheme for paired-end reads) for merging paired-end reads and provides users the option to quality filter the reads using the traditional average Q-score metric or using a maximum expected error cut-off threshold. Conclusions MeFiT provides an open-source solution that permits users to merge and filter paired end illumina reads. The tool has been implemented in python and the source-code is freely available at https://github.com/nisheth/MeFiT. Electronic supplementary material The online version of this article (doi:10.1186/s12859-016-1358-1) contains supplementary material, which is available to authorized users.

Quantitative relationship between the magnitude of variation in minor histocompatibility antigens (mHA) and graft versus host disease (GVHD) pathophysiology in stem cell transplant (SCT) donor-recipient pairs (DRP) is not established. In order to elucidate this relationship, whole exome sequencing (WES) was performed on 27 HLA matched related (MRD), & 50 unrelated donors (URD), to identify nonsynonymous single nucleotide polymorphisms (SNPs). An average 2,463 SNPs were identified in MRD, and 4,287 in URD DRP (p

Vaginal lactobacilli can inhibit colonization by and growth of other bacteria, thereby preventing development of bacterial vaginosis (BV). Amongst the lactobacilli, Lactobacillus crispatus appears to be particularly effective at inhibiting growth of BV-associated bacteria. Nonetheless, some women who are colonized with this species can still develop clinical BV. Therefore, we sought to determine whether strains of L. crispatus that colonize women with lactobacilli-dominated vaginal microbiomes are distinct from strains that colonize women who develop BV. The genomes of L. crispatus isolates from four women with lactobacilli-dominated vaginal microbiomes ( 12 % 16S rRNA reads from bacterial taxa associated with BV) were sequenced and compared. Lactic acid production by the different strains was quantified. Phage induction in the strains was also analysed. There was considerable genetic diversity between strains, and several genes were exclusive to either the strains from Lactobacillus-dominated microbiomes or those containing BV-associated bacteria. Overall, strains from microbiomes dominated by lactobacilli did not differ from strains from microbiomes containing BV-associated bacteria with respect to lactic acid production. All of the strains contained multiple phage, but there was no clear distinction between the presence or absence of BV-associated bacteria with respect to phage-induced lysis. Genes found to be exclusive to the Lactobacillus-dominated versus BV-associated bacteria-containing microbiomes could play a role in the maintenance of vaginal health and the development of BV, respectively.

Stem cell transplants may be considered as dynamical systems to allow sequence differences across the exomes of the transplant donors and recipients to be used to simulate an alloreactive T cell response. Whole exome sequencing was performed on HLA matched stem cell transplant donor-recipient pairs, and the nucleotide sequence differences translated to peptides. The binding affinity of the peptides to the relevant HLA in each pair was determined. The resulting array of peptide-HLA binding affinity values in each patient was used to simulate an alloreactive donor derived T cell repertoire. This simulated T cell repertoire reproduces a number of features of clinically observed T cell repertoire. The simulated, alloreactive T cell repertoire was markedly different in HLA matched stem cell transplant donors and recipients and demonstrates a possible correlation with survival., 50 pages (including references and supplementary materials), 12 figures, 3 supplementary figures

The oral cavity represents an initial entry way for oral and gut indigenous colonization. Skin-to-skin (STS) care, in which the mother holds the diaper clad naked preterm (PT) infant between her breasts, is associated with improved digestive function, decreased stress, and improved survival. This study evaluated the development of oral microbial colonization repertoires and health characteristics in PT infants with or without STS exposure.Saliva from 42 PT infants (32 weeks of gestation at birth) was collected prospectively at 1 month and/or at discharge. High-throughput 16S rRNA sequencing identified microbial diversity and prevalence of bacterial signatures correlated with clinical STS or non-STS care.Corrected for gestational age (CGA) at sampling, bacterial taxa demonstrated increased Streptococcus as a signature of oral repertoire maturation. STS was associated with increased Streptococcus (p 0.024), while non-STS was associated with greater Corynebacterium (p 0.023) and Pseudomonas (p 0.019) in infants ≤ 32 weeks CGA. In infants 32 weeks CGA, Neisseria and Acinetobacter were more prevalent, 50 vs. 16.7% and 40 vs. 0%, respectively. STS care was associated with shorter hospitalization (p 0.039).STS care during earlier gestation was associated with a distinct microbial pattern and an accelerated pace of oral microbial repertoire maturity.

Background Characterizing microbial communities via next-generation sequencing is subject to a number of pitfalls involving sample processing. The observed community composition can be a severe distortion of the quantities of bacteria actually present in the microbiome, hampering analysis and threatening the validity of conclusions from metagenomic studies. We introduce an experimental protocol using mock communities for quantifying and characterizing bias introduced in the sample processing pipeline. We used 80 bacterial mock communities comprised of prescribed proportions of cells from seven vaginally-relevant bacterial strains to assess the bias introduced in the sample processing pipeline. We created two additional sets of 80 mock communities by mixing prescribed quantities of DNA and PCR product to quantify the relative contribution to bias of (1) DNA extraction, (2) PCR amplification, and (3) sequencing and taxonomic classification for particular choices of protocols for each step. We developed models to predict the “true” composition of environmental samples based on the observed proportions, and applied them to a set of clinical vaginal samples from a single subject during four visits. Results We observed that using different DNA extraction kits can produce dramatically different results but bias is introduced regardless of the choice of kit. We observed error rates from bias of over 85% in some samples, while technical variation was very low at less than 5% for most bacteria. The effects of DNA extraction and PCR amplification for our protocols were much larger than those due to sequencing and classification. The processing steps affected different bacteria in different ways, resulting in amplified and suppressed observed proportions of a community. When predictive models were applied to clinical samples from a subject, the predicted microbiome profiles were better reflections of the physiology and diagnosis of the subject at the visits than the observed community compositions. Conclusions Bias in 16S studies due to DNA extraction and PCR amplification will continue to require attention despite further advances in sequencing technology. Analysis of mock communities can help assess bias and facilitate the interpretation of results from environmental samples. Electronic supplementary material The online version of this article (doi:10.1186/s12866-015-0351-6) contains supplementary material, which is available to authorized users.

Quality-based sequence trimming of next generation sequencing (NGS) data is essential as it can potentially prevent missed or incorrect alignment downstream or prevent missassembly in a de novo assembly process by over-representing false k-mers. Trimming algorithms in tools like sickle, trimmomatic, etc. either depend on a running sum of quality of bases; or rely on average base quality of a sliding window. They consider PHRED quality, which is exponentially related to the probability of an erroneous base call. Here we present MEEPTOOLS, which is an open-source tool based on maximum expected error (MEE) as a percentage of read length (MEEP score) to filter, trim, truncate and assess NGS data in FASTQ file. By treating read trimming as a minimum subarray problem, MEEPTOOLS can simultaneously retain more reliable bases and remove unreliable bases than the traditional quality filtering strategies. MEEPTOOLS is ready available for download under the GNU GPLv3 at https://github.com/nisheth/meeptools.

Stem cell transplant (SCT) recipients who develop human cytomegalovirus (hCMV) reactivation are at risk of developing graft versus host disease (GVHD). This may stem from immune cross reactivity (Figure 1) towards both pathogen-derived peptides and nearly identical recipient-derived alloreactive peptide minor histocompatibility antigens from SCT donor-recipient pairs (DRP). Whole exome sequencing was performed on 9 SCT DRP, and the resulting nucleotide sequences aligned and compared, identifying all the single nucleotide polymorphisms (SNPs) present in the recipient and absent in the donor. Alloreactive peptide libraries were compiled for each DRP (9-mer peptides) and HLA binding affinity calculated for relevant HLA (Front Immunol 2014). A library representing the hCMV proteome was generated from the NCBI Protein database and directly compared to libraries of recipient specific peptide-HLA complexes from the SCT D-R pairs. BLAST Protein sequence alignment was performed to interrogate each pair's peptide-HLA complex library for matches with hCMV peptides, identifying CMV peptides with a 6/6-9/9 amino acid sequence matching to recipient peptides bound to HLA (IC50 Disclosures Buck: CHRB: Research Funding. Neale:CHRB: Research Funding.