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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.

A variety of microbial communities and their genes (the microbiome) exist throughout the human body, with fundamental roles in human health and disease. The National Institutes of Health (NIH)-funded Human Microbiome Project Consortium has established a population-scale framework to develop metagenomic protocols, resulting in a broad range of quality-controlled resources and data including standardized methods for creating, processing and interpreting distinct types of high-throughput metagenomic data available to the scientific community. Here we present resources from a population of 242 healthy adults sampled at 15 or 18 body sites up to three times, which have generated 5,177 microbial taxonomic profiles from 16S ribosomal RNA genes and over 3.5 terabases of metagenomic sequence so far. In parallel, approximately 800 reference strains isolated from the human body have been sequenced. Collectively, these data represent the largest resource describing the abundance and variety of the human microbiome, while providing a framework for current and future studies.

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

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.

We have collected over half a million splice sites from five species—Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans and Arabidopsis thaliana—and classified them into four subtypes: U2-type GT–AG and GC–AG and U12-type GT–AG and AT–AC. We have also found new examples of rare splice-site categories, such as U12-type introns without canonical borders, and U2-dependent AT–AC introns. The splice-site sequences and several tools to explore them are available on a public website (SpliceRack). For the U12-type introns, we find several features conserved across species, as well as a clustering of these introns on genes. Using the information content of the splice-site motifs, and the phylogenetic distance between them, we identify: (i) a higher degree of conservation in the exonic portion of the U2-type splice sites in more complex organisms; (ii) conservation of exonic nucleotides for U12-type splice sites; (iii) divergent evolution of C.elegans 3′ splice sites (3′ss) and (iv) distinct evolutionary histories of 5′ and 3′ss. Our study proves that the identification of broad patterns in naturally-occurring splice sites, through the analysis of genomic datasets, provides mechanistic and evolutionary insights into pre-mRNA splicing. Published version

Use of RNA interference (RNAi) in forward genetic screens is proliferating. Currently, short-interfering RNAs (siRNAs) and short-hairpin RNAs (shRNAs) are being used to silence genes to tease out functional information. It is becoming easier to harness RNAi to silence specific genes, owing to the development of libraries of readymade shRNA and siRNA gene-silencing constructs by using a variety of sources. RNAi Codex, which consists of a database of shRNA related information and an associated website, has been developed as a portal for publicly available shRNA resources and is accessible at http://codex.cshl.org. RNAi Codex currently holds data from the Hannon-Elledge shRNA library and allows the use of biologist-friendly gene names to access information on shRNA constructs that can silence the gene of interest. It is designed to hold user-contributed annotations and publications for each construct, as and when such data become available. We will describe features of RNAi Codex and explain the use of the tool.

Loss-of-function phenotypes often hold the key to understanding the connections and biological functions of biochemical pathways. We and others previously constructed libraries of short hairpin RNAs that allow systematic analysis of RNA interference-induced phenotypes in mammalian cells. Here we report the construction and validation of second-generation short hairpin RNA expression libraries designed using an increased knowledge of RNA interference biochemistry. These constructs include silencing triggers designed to mimic a natural microRNA primary transcript, and each target sequence was selected on the basis of thermodynamic criteria for optimal small RNA performance. Biochemical and phenotypic assays indicate that the new libraries are substantially improved over first-generation reagents. We generated large-scale-arrayed, sequence-verified libraries comprising more than 140,000 second-generation short hairpin RNA expression plasmids, covering a substantial fraction of all predicted genes in the human and mouse genomes. These libraries are available to the scientific community.

Donor T cell mediated graft vs. host effects may result from the aggregate alloreactivity to minor histocompatibility antigens (mHA) presented by the HLA in each donor-recipient pair (DRP) undergoing stem cell transplantation (SCT). Whole exome sequencing has demonstrated extensive nucleotide sequence variation in HLA-matched DRP. Non-synonymous single nucleotide polymorphisms (nsSNPs) in the GVH direction (polymorphisms present in recipient and absent in donor) were identified in 4 HLA-matched related and 5 unrelated DRP. The nucleotide sequence flanking each SNP was obtained utilizing the ANNOVAR software package. All possible nonameric-peptides encoded by the non-synonymous SNP were then interrogated in-silico for their likelihood to be presented by the HLA class I molecules in individual DRP, using the Immune-Epitope Database (IEDB) SMM algorithm. The IEDB-SMM algorithm predicted a median 18,396 peptides/DRP which bound HLA with an IC50 of, 235, Words: 6422, Figures: 7, Tables: 3, Supplementary figures: 2, Supplementary tables: 2

Humans are colonized by thousands of bacterial species, but it is difficult to assess the metabolic and pathogenic potential of the majority of these because they have yet to be cultured. Here, we characterize an uncultivated vaginal mycoplasma tightly associated with trichomoniasis that was previously known by its 16S rRNA sequence as “Mnola.” In this study, the mycoplasma was found almost exclusively in women infected with the sexually transmitted pathogen Trichomonas vaginalis, but rarely observed in women with no diagnosed disease. The genomes of four strains of this species were reconstructed using metagenome sequencing and assembly of DNA from four discrete mid-vaginal samples, one of which was obtained from a pregnant woman with trichomoniasis who delivered prematurely. These bacteria harbor several putative virulence factors and display unique metabolic strategies. Genes encoding proteins with high similarity to potential virulence factors include two collagenases, a hemolysin, an O-sialoglycoprotein endopeptidase and a feoB-type ferrous iron transport system. We propose the name “Candidatus Mycoplasma girerdii” for this potential new pathogen.

The Human Microbiome Project (HMP) aims to characterize the microbial communities of 18 body sites from healthy individuals. To accomplish this, the HMP generated two types of shotgun data: reference shotgun sequences isolated from different anatomical sites on the human body and shotgun metagenomic sequences from the microbial communities of each site. The alignment strategy for characterizing these metagenomic communities using available reference sequence is important to the success of HMP data analysis. Six next-generation aligners were used to align a community of known composition against a database comprising reference organisms known to be present in that community. All aligners report nearly complete genome coverage (>97%) for strains with over 6X depth of coverage, however they differ in speed, memory requirement and ease of use issues such as database size limitations and supported mapping strategies. The selected aligner was tested across a range of parameters to maximize sensitivity while maintaining a low false positive rate. We found that constraining alignment length had more impact on sensitivity than does constraining similarity in all cases tested. However, when reference species were replaced with phylogenetic neighbors, similarity begins to play a larger role in detection. We also show that choosing the top hit randomly when multiple, equally strong mappings are available increases overall sensitivity at the expense of taxonomic resolution. The results of this study identified a strategy that was used to map over 3 tera-bases of microbial sequence against a database of more than 5,000 reference genomes in just over a month.

Generation of complex libraries of defined nucleic acid sequences can greatly aid the functional analysis of protein and gene function. Previously, such studies relied either on individually synthesized oligonucleotides or on cellular nucleic acids as the starting material. As each method has disadvantages, we have developed a rapid and cost-effective alternative for construction of small-fragment DNA libraries of defined sequences. This approach uses in situ microarray DNA synthesis for generation of complex oligonucleotide populations. These populations can be recovered and either used directly or immortalized by cloning. From a single microarray, a library containing thousands of unique sequences can be generated. As an example of the potential applications of this technology, we have tested the approach for the production of plasmids encoding short hairpin RNAs (shRNAs) targeting numerous human and mouse genes. We achieved high-fidelity clone retrieval with a uniform representation of intended library sequences.

Whole exome sequencing was performed on HLA-matched stem cell donors and transplant recipients to measure sequence variation contributing to minor histocompatibility antigen differences between the two. A large number of nonsynonymous single nucleotide polymorphisms were identified in each of the nine unique donor-recipient pairs tested. This variation was greater in magnitude in unrelated donors as compared with matched related donors. Knowledge of the magnitude of exome variation between stem cell transplant recipients and donors may allow more accurate titration of immunosuppressive therapy following stem cell transplantation., 12 pages- main article, 29 pages total, 5 figures, 1 supplementary figure

Background The application of next-generation sequencing to the study of the vaginal microbiome is revealing the spectrum of microbial communities that inhabit the human vagina. High-resolution identification of bacterial taxa, minimally to the species level, is necessary to fully understand the association of the vaginal microbiome with bacterial vaginosis, sexually transmitted infections, pregnancy complications, menopause, and other physiological and infectious conditions. However, most current taxonomic assignment strategies based on metagenomic 16S rDNA sequence analysis provide at best a genus-level resolution. While surveys of 16S rRNA gene sequences are common in microbiome studies, few well-curated, body-site-specific reference databases of 16S rRNA gene sequences are available, and no such resource is available for vaginal microbiome studies. Results We constructed the Vaginal 16S rDNA Reference Database, a comprehensive and non-redundant database of 16S rDNA reference sequences for bacterial taxa likely to be associated with vaginal health, and we developed STIRRUPS, a new method that employs the USEARCH algorithm with a curated reference database for rapid species-level classification of 16S rDNA partial sequences. The method was applied to two datasets of V1-V3 16S rDNA reads: one generated from a mock community containing DNA from six bacterial strains associated with vaginal health, and a second generated from over 1,000 mid-vaginal samples collected as part of the Vaginal Human Microbiome Project at Virginia Commonwealth University. In both datasets, STIRRUPS, used in conjunction with the Vaginal 16S rDNA Reference Database, classified more than 95% of processed reads to a species-level taxon using a 97% global identity threshold for assignment. Conclusions This database and method provide accurate species-level classifications of metagenomic 16S rDNA sequence reads that will be useful for analysis and comparison of microbiome profiles from vaginal samples. STIRRUPS can be used to classify 16S rDNA sequence reads from other ecological niches if an appropriate reference database of 16S rDNA sequences is available.