Draft sequencing and assembly of the genome of the world's largest fish, the whale shark: Rhincodon typus Smith 1828.

BACKGROUND: The whale shark (Rhincodon typus) has by far the largest body size of any elasmobranch (shark or ray) species. Therefore, it is also the largest extant species of the paraphyletic assemblage commonly referred to as fishes. As both a phenotypic extreme and a member of the group Chondrichthyes - the sister group to the remaining gnathostomes, which includes all tetrapods and therefore also humans - its genome is of substantial comparative interest. Whale sharks are also listed as an endangered species on the International Union for Conservation of Nature's Red List of threatened species and are of growing popularity as both a target of ecotourism and as a charismatic conservation ambassador for the pelagic ecosystem. A genome map for this species would aid in defining effective conservation units and understanding global population structure. RESULTS: We characterised the nuclear genome of the whale shark using next generation sequencing (454, Illumina) and de novo assembly and annotation methods, based on material collected from the Georgia Aquarium. The data set consisted of 878,654,233 reads, which yielded a draft assembly of 1,213,200 contigs and 997,976 scaffolds. The estimated genome size was 3.44Gb. As expected, the proteome of the whale shark was most closely related to the only other complete genome of a cartilaginous fish, the holocephalan elephant shark. The whale shark contained a novel Toll-like-receptor (TLR) protein with sequence similarity to both the TLR4 and TLR13 proteins of mammals and TLR21 of teleosts. The data are publicly available on GenBank, FigShare, and from the NCBI Short Read Archive under accession number SRP044374. CONCLUSIONS: This represents the first shotgun elasmobranch genome and will aid studies of molecular systematics, biogeography, genetic differentiation, and conservation genetics in this and other shark species, as well as providing comparative data for studies of evolutionary biology and immunology across the jawed vertebrate lineages.

Chlamydia psittaci comparative genomics reveals intraspecies variations in the putative outer membrane and type III secretion system genes.

Chlamydia psittaci is an obligate intracellular bacterium that can cause significant disease among a broad range of hosts. In humans, this organism may cause psittacosis, a respiratory disease that can spread to involve multiple organs, and in rare untreated cases may be fatal. There are ten known genotypes based on sequencing the major outer-membrane protein gene, ompA, of C. psittaci. Each genotype has overlapping host preferences and virulence characteristics. Recent studies have compared C. psittaci among other members of the Chlamydiaceae family and showed that this species frequently switches hosts and has undergone multiple genomic rearrangements. In this study, we sequenced five genomes of C. psittaci strains representing four genotypes, A, B, D and E. Due to the known association of the type III secretion system (T3SS) and polymorphic outer-membrane proteins (Pmps) with host tropism and virulence potential, we performed a comparative analysis of these elements among these five strains along with a representative genome from each of the remaining six genotypes previously sequenced. We found significant genetic variation in the Pmps and tbl3SS genes that may partially explain differences noted in C. psittaci host infection and disease.

Microbial biofilms on needleless connectors for central venous catheters: comparison of standard and silver-coated devices collected from patients in an acute care hospital.

Microorganisms may colonize needleless connectors (NCs) on intravascular catheters, forming biofilms and predisposing patients to catheter-associated infection (CAI). Standard and silver-coated NCs were collected from catheterized intensive care unit patients to characterize biofilm formation using culture-dependent and culture-independent methods and to investigate the associations between NC usage and biofilm characteristics. Viable microorganisms were detected by plate counts from 46% of standard NCs and 59% of silver-coated NCs (P=0.11). There were no significant associations (P>0.05, chi-square test) between catheter type, side of catheter placement, number of catheter lumens, site of catheter placement, or NC placement duration and positive NC findings. There was an association (P=0.04, chi-square test) between infusion type and positive findings for standard NCs. Viable microorganisms exhibiting intracellular esterase activity were detected on >90% of both NC types (P=0.751), suggesting that a large percentage of organisms were not culturable using the conditions provided in this study. Amplification of the 16S rRNA gene from selected NCs provided a substantially larger number of operational taxonomic units per NC than did plate counts (26 to 43 versus 1 to 4 operational taxonomic units/NC, respectively), suggesting that culture-dependent methods may substantially underestimate microbial diversity on NCs. NC bacterial communities were clustered by patient and venous access type and may reflect the composition of the patient's local microbiome but also may contain organisms from the health care environment. NCs provide a portal of entry for a wide diversity of opportunistic pathogens to colonize the catheter lumen, forming a biofilm and increasing the potential for CAI, highlighting the importance of catheter maintenance practices to reduce microbial contamination.

Whole-genome analysis of Exserohilum rostratum from an outbreak of fungal meningitis and other infections.

Exserohilum rostratum was the cause of most cases of fungal meningitis and other infections associated with the injection of contaminated methylprednisolone acetate produced by the New England Compounding Center (NECC). Until this outbreak, very few human cases of Exserohilum infection had been reported, and very little was known about this dematiaceous fungus, which usually infects plants. Here, we report using whole-genome sequencing (WGS) for the detection of single nucleotide polymorphisms (SNPs) and phylogenetic analysis to investigate the molecular origin of the outbreak using 22 isolates of E. rostratum retrieved from 19 case patients with meningitis or epidural/spinal abscesses, 6 isolates from contaminated NECC vials, and 7 isolates unrelated to the outbreak. Our analysis indicates that all 28 isolates associated with the outbreak had nearly identical genomes of 33.8 Mb. A total of 8 SNPs were detected among the outbreak genomes, with no more than 2 SNPs separating any 2 of the 28 genomes. The outbreak genomes were separated from the next most closely related control strain by ∼136,000 SNPs. We also observed significant genomic variability among strains unrelated to the outbreak, which may suggest the possibility of cryptic speciation in E. rostratum.

Isolation and molecular characterization of Fikirini rhabdovirus, a novel virus from a Kenyan bat.

Zoonotic and vector-borne pathogens have comprised a significant component of emerging human infections in recent decades, and bats are increasingly recognized as reservoirs for many of these disease agents. To identify novel pathogens associated with bats, we screened tissues of bats collected in Kenya. Virus isolates were identified by next generation sequencing of viral nucleic acid preparations from the infected cell culture supernatant and characterized. Here we report the identification of Fikirini rhabdovirus, a novel rhabdovirus isolated from a bat, Hipposideros vittatus, captured along the Kenyan coast.

Investigation of the first laboratory-acquired human cowpox virus infection in the United States.

BACKGROUND: Cowpox virus is an Orthopoxvirus that can cause infections in humans and a variety of animals. Infections occur in Eurasia; infections in humans and animals have not been reported in the United States. This report describes the occurrence of the first known human case of laboratory-acquired cowpox virus infection in the United States and the ensuing investigation. METHODS: The patient and laboratory personnel were interviewed, and laboratory activities were reviewed. Real-time polymerase chain reaction (PCR) and serologic assays were used to test the patient's specimens. PCR assays were used to test specimens obtained during the investigation. RESULTS: A specimen from the patient's lesion tested positive for cowpox virus DNA. Genome sequencing revealed a recombinant region consistent with a strain of cowpox virus stored in the research laboratory's freezer. Cowpox virus contamination was detected in 6 additional laboratory stocks of viruses. Orthopoxvirus DNA was present in 3 of 20 environmental swabs taken from laboratory surfaces. CONCLUSIONS: The handling of contaminated reagents or contact with contaminated surfaces was likely the mode of transmission. Delays in recognition and diagnosis of this infection in a laboratory researcher underscore the importance of a thorough patient history-including occupational information-and laboratory testing in facilitating a prompt investigation and application of control and remediation measures.

RNA-Seq and RNA immunoprecipitation analyses of the transcriptome of Streptomyces coelicolor identify substrates for RNase III.

RNase III is a key enzyme in the pathways of RNA degradation and processing in bacteria and has been suggested as a global regulator of antibiotic production in Streptomyces coelicolor. Using RNA-Seq, we have examined the transcriptomes of S. coelicolor M145 and an RNase III (rnc)-null mutant of that strain. RNA preparations with reduced levels of structural RNAs were prepared by subtractive hybridization prior to RNA-Seq analysis. We initially identified 7,800 transcripts of known and putative protein-coding genes in M145 and the null mutant, JSE1880, along with transcripts of 21 rRNA genes and 65 tRNA genes. Approximately 3,100 of the protein-coding transcripts were categorized as low-abundance transcripts. For further analysis, we selected those transcripts of known and putative protein-coding genes whose levels changed by ≥ 2-fold between the two S. coelicolor strains and organized those transcripts into 16 functional categories. We refined our analysis by performing RNA immunoprecipitation of the mRNA preparation from JSE1880 using a mutant RNase III protein that binds to transcripts but does not cleave them. This analysis identified ca. 800 transcripts that were enriched in the RNA immunoprecipitates, including 28 transcripts whose levels also changed by ≥ 2-fold in the RNA-Seq analysis. We compare our results with those obtained by microarray analysis of the S. coelicolor transcriptome and with studies describing the characterization of small noncoding RNAs. We have also used the RNA immunoprecipitation results to identify new substrates for RNase III cleavage.

TULIP: An RNA-seq-based Primary Tumor Type Prediction Tool Using Convolutional Neural Networks.

Background: With cancer as one of the leading causes of death worldwide, accurate primary tumor type prediction is critical in identifying genetic factors that can inhibit or slow tumor progression. There have been efforts to categorize primary tumor types with gene expression data using machine learning, and more recently with deep learning, in the last several years. Methods: In this paper, we developed four 1-dimensional (1D) Convolutional Neural Network (CNN) models to classify RNA-seq count data as one of 17 highly represented primary tumor types or 32 primary tumor types regardless of imbalanced representation. Additionally, we adapted the models to take as input either all Ensembl genes (60,483) or protein coding genes only (19,758). Unlike previous work, we avoided selection bias by not filtering genes based on expression values. RNA-seq count data expressed as FPKM-UQ of 9,025 and 10,940 samples from The Cancer Genome Atlas (TCGA) were downloaded from the Genomic Data Commons (GDC) corresponding to 17 and 32 primary tumor types respectively for training and validating the models. Results: All 4 1D-CNN models had an overall accuracy of 94.7% to 97.6% on the test dataset. Further evaluation indicates that the models with protein coding genes only as features performed with better accuracy compared to the models with all Ensembl genes for both 17 and 32 primary tumor types. For all models, the accuracy by primary tumor type was above 80% for most primary tumor types. Conclusions: We packaged all 4 models as a Python-based deep learning classification tool called TULIP (TUmor CLassIfication Predictor) for performing quality control on primary tumor samples and characterizing cancer samples of unknown tumor type. Further optimization of the models is needed to improve the accuracy of certain primary tumor types.

Differential activation profiles of multiple transcription factors during dendritic cell maturation.

Immature dendritic cells (DC) at the environmental interfaces, such as the skin, constantly survey the tissue for the emergence of microbial products and pro-inflammatory mediators. Upon recognition of such "danger" signals, they undergo dynamic reprogramming of gene expression and functions, the process known as DC maturation, which plays critical roles in both innate and adaptive immune responses. Although DC have been shown to discriminate different maturation stimuli by expressing stimulus-specific signature genes and unique phenotypic and functional properties, underlying mechanisms for this extraordinary plasticity remain relatively unclear. We hypothesized that DC might activate unique sets of transcription factors (TF) upon sensing different stimuli. To test this hypothesis, we transduced a mouse epidermal-derived DC line XS106 to express the luciferase reporter gene under the control of each of 15 different cis-enhancer elements. The resulting DC panels were then exposed to 14 different microbial, endogenous, environmental, and pharmacological agents that produced unique maturational changes. This approach allowed systematic determination of TF activation profiles in DC. Our results revealed striking diversity, with different classes of stimuli triggering preferential activation of distinct sets of TF. We propose that differential TF usage represents a previously unrecognized mechanism regulating the direction of DC maturation.

Global mRNA decay analysis at single nucleotide resolution reveals segmental and positional degradation patterns in a Gram-positive bacterium.

BACKGROUND: Recent years have shown a marked increase in the use of next-generation sequencing technologies for quantification of gene expression (RNA sequencing, RNA-Seq). The expression level of a gene is a function of both its rate of transcription and RNA decay, and the influence of mRNA decay rates on gene expression in genome-wide studies of Gram-positive bacteria is under-investigated. RESULTS: In this work, we employed RNA-Seq in a genome-wide determination of mRNA half-lives in the Gram-positive bacterium Bacillus cereus. By utilizing a newly developed normalization protocol, RNA-Seq was used successfully to determine global mRNA decay rates at the single nucleotide level. The analysis revealed positional degradation patterns, with mRNAs being degraded from both ends of the molecule, indicating that both 5' to 3' and 3' to 5' directions of RNA decay are present in B. cereus. Other operons showed segmental degradation patterns where specific ORFs within polycistrons were degraded at variable rates, underlining the importance of RNA processing in gene regulation. We determined the half-lives for more than 2,700 ORFs in B. cereus ATCC 10987, ranging from less than one minute to more than fifteen minutes, and showed that mRNA decay rate correlates globally with mRNA expression level, GC content, and functional class of the ORF. CONCLUSIONS: To our knowledge, this study presents the first global analysis of mRNA decay in a bacterium at single nucleotide resolution. We provide a proof of principle for using RNA-Seq in bacterial mRNA decay analysis, revealing RNA processing patterns at the single nucleotide level.

Global survey of chromatin accessibility using DNA microarrays.

An increasing number of studies indicate a central role for chromatin remodeling in the regulation of gene expression. Current methods for high-resolution studies of the relationship between chromatin accessibility and transcription are low throughput, making a genome-wide study impractical. To enable the simultaneous measurement of the global chromatin accessibility state at the resolution of single genes, we developed the Chromatin Array technique, in which chromatin is separated by its condensation state using either the solubility differences of mono- and oligonucleosomes in specific buffers or controlled DNase I digestion and selection of the large refractory (condensed) DNA fragments. By probing with a comparative genomic hybridization style microarray, we can determine the condensation state of thousands of individual loci and correlate this with transcriptional activity. Applying this technique to the breast tumor model cell line, MCF7, we found that when the condensation is homogeneous in the population of cells, expression is inversely proportional to the level of accessibility and the two methods of accessibility-based target selection correlate well. Using functional annotation and comparative genomic hybridization data, we have begun to decipher the possible biological implications of the relationship between chromatin accessibility and expression.