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Sample records for clinical metagenomic samples

  1. Statistical methods for detecting differentially abundant features in clinical metagenomic samples.

    Directory of Open Access Journals (Sweden)

    James Robert White

    2009-04-01

    Full Text Available Numerous studies are currently underway to characterize the microbial communities inhabiting our world. These studies aim to dramatically expand our understanding of the microbial biosphere and, more importantly, hope to reveal the secrets of the complex symbiotic relationship between us and our commensal bacterial microflora. An important prerequisite for such discoveries are computational tools that are able to rapidly and accurately compare large datasets generated from complex bacterial communities to identify features that distinguish them.We present a statistical method for comparing clinical metagenomic samples from two treatment populations on the basis of count data (e.g. as obtained through sequencing to detect differentially abundant features. Our method, Metastats, employs the false discovery rate to improve specificity in high-complexity environments, and separately handles sparsely-sampled features using Fisher's exact test. Under a variety of simulations, we show that Metastats performs well compared to previously used methods, and significantly outperforms other methods for features with sparse counts. We demonstrate the utility of our method on several datasets including a 16S rRNA survey of obese and lean human gut microbiomes, COG functional profiles of infant and mature gut microbiomes, and bacterial and viral metabolic subsystem data inferred from random sequencing of 85 metagenomes. The application of our method to the obesity dataset reveals differences between obese and lean subjects not reported in the original study. For the COG and subsystem datasets, we provide the first statistically rigorous assessment of the differences between these populations. The methods described in this paper are the first to address clinical metagenomic datasets comprising samples from multiple subjects. Our methods are robust across datasets of varied complexity and sampling level. While designed for metagenomic applications, our software

  2. Comparative Viral Metagenomics of Environmental Samples from Korea

    OpenAIRE

    Kim, Min-Soo; Whon, Tae Woong; Bae, Jin-Woo

    2013-01-01

    The introduction of metagenomics into the field of virology has facilitated the exploration of viral communities in various natural habitats. Understanding the viral ecology of a variety of sample types throughout the biosphere is important per se, but it also has potential applications in clinical and diagnostic virology. However, the procedures used by viral metagenomics may produce technical errors, such as amplification bias, while public viral databases are very limited, which may hamper...

  3. Unbiased Parallel Detection of Viral Pathogens in Clinical Samples by Use of a Metagenomic Approach▿‡

    OpenAIRE

    Yang, Jian; Yang, Fan; Ren, Lili; Xiong, Zhaohui; Wu, Zhiqiang; Dong, Jie; Sun, Lilian; Zhang, Ting; Hu, Yongfeng; Du, Jiang; Wang, Jianwei; Jin, Qi

    2011-01-01

    Viral infectious diseases represent a major threat to public health and are among the greatest disease burdens worldwide. Rapid and accurate identification of viral agents is crucial for both outbreak control and estimating regional disease burdens. Recently developed metagenomic methods have proven to be powerful tools for simultaneous pathogen detection. Here, we performed a systematic study of the capability of the short-read-based metagenomic approach in the molecular detection of viral p...

  4. Longitudinal Metagenomic Analysis of Hospital Air Identifies Clinically Relevant Microbes

    Science.gov (United States)

    King, Paula; Pham, Long K.; Waltz, Shannon; Sphar, Dan; Yamamoto, Robert T.; Conrad, Douglas; Taplitz, Randy; Torriani, Francesca

    2016-01-01

    We describe the sampling of sixty-three uncultured hospital air samples collected over a six-month period and analysis using shotgun metagenomic sequencing. Our primary goals were to determine the longitudinal metagenomic variability of this environment, identify and characterize genomes of potential pathogens and determine whether they are atypical to the hospital airborne metagenome. Air samples were collected from eight locations which included patient wards, the main lobby and outside. The resulting DNA libraries produced 972 million sequences representing 51 gigabases. Hierarchical clustering of samples by the most abundant 50 microbial orders generated three major nodes which primarily clustered by type of location. Because the indoor locations were longitudinally consistent, episodic relative increases in microbial genomic signatures related to the opportunistic pathogens Aspergillus, Penicillium and Stenotrophomonas were identified as outliers at specific locations. Further analysis of microbial reads specific for Stenotrophomonas maltophilia indicated homology to a sequenced multi-drug resistant clinical strain and we observed broad sequence coverage of resistance genes. We demonstrate that a shotgun metagenomic sequencing approach can be used to characterize the resistance determinants of pathogen genomes that are uncharacteristic for an otherwise consistent hospital air microbial metagenomic profile. PMID:27482891

  5. [Bacterial genomics and metagenomics: clinical applications and medical relevance].

    Science.gov (United States)

    Diene, S M; Bertelli, C; Pillonel, T; Schrenzel, J; Greub, G

    2014-11-12

    New sequencing technologies provide in a short time and at low cost high amount of genomic sequences useful for applications such as: a) development of diagnostic PCRs and/or serological tests; b) detection of virulence factors (virulome) or genes/SNPs associated with resistance to antibiotics (resistome) and c) investigation of transmission and dissemination of bacterial pathogens. Thus, bacterial genomics of medical importance is useful to clinical microbiologists, to infectious diseases specialists as well as to epidemiologists. Determining the microbial composition of a sample by metagenomics is another application of new sequencing technologies, useful to understand the impact of bacteria on various non-infectious diseases such as obesity, asthma, or diabetes. Genomics and metagenomics will likely become a specialized diagnostic analysis.

  6. Challenges of the Unknown: Clinical Application of Microbial Metagenomics

    Directory of Open Access Journals (Sweden)

    Graham Rose

    2015-01-01

    Full Text Available Availability of fast, high throughput and low cost whole genome sequencing holds great promise within public health microbiology, with applications ranging from outbreak detection and tracking transmission events to understanding the role played by microbial communities in health and disease. Within clinical metagenomics, identifying microorganisms from a complex and host enriched background remains a central computational challenge. As proof of principle, we sequenced two metagenomic samples, a known viral mixture of 25 human pathogens and an unknown complex biological model using benchtop technology. The datasets were then analysed using a bioinformatic pipeline developed around recent fast classification methods. A targeted approach was able to detect 20 of the viruses against a background of host contamination from multiple sources and bacterial contamination. An alternative untargeted identification method was highly correlated with these classifications, and over 1,600 species were identified when applied to the complex biological model, including several species captured at over 50% genome coverage. In summary, this study demonstrates the great potential of applying metagenomics within the clinical laboratory setting and that this can be achieved using infrastructure available to nondedicated sequencing centres.

  7. In silico analyses of metagenomes from human atherosclerotic plaque samples

    DEFF Research Database (Denmark)

    Mitra, Suparna; Drautz-Moses, Daniela I; Alhede, Morten;

    2015-01-01

    BACKGROUND: Through several observational and mechanistic studies, microbial infection is known to promote cardiovascular disease. Direct infection of the vessel wall, along with the cardiovascular risk factors, is hypothesized to play a key role in the atherogenesis by promoting an inflammatory...... response leading to endothelial dysfunction and generating a proatherogenic and prothrombotic environment ultimately leading to clinical manifestations of cardiovascular disease, e.g., acute myocardial infarction or stroke. There are many reports of microbial DNA isolation and even a few studies of viable...... a challenge. RESULTS: To investigate microbiome diversity within human atherosclerotic tissue samples, we employed high-throughput metagenomic analysis on: (1) atherosclerotic plaques obtained from a group of patients who underwent endarterectomy due to recent transient cerebral ischemia or stroke. (2...

  8. The effects of variable sample biomass on comparative metagenomics.

    Science.gov (United States)

    Chafee, Meghan; Maignien, Loïs; Simmons, Sheri L

    2015-07-01

    Longitudinal studies that integrate samples with variable biomass are essential to understand microbial community dynamics across space or time. Shotgun metagenomics is widely used to investigate these communities at the functional level, but little is known about the effects of combining low and high biomass samples on downstream analysis. We investigated the interacting effects of DNA input and library amplification by polymerase chain reaction on comparative metagenomic analysis using dilutions of a single complex template from an Arabidopsis thaliana-associated microbial community. We modified the Illumina Nextera kit to generate high-quality large-insert (680 bp) paired-end libraries using a range of 50 pg to 50 ng of input DNA. Using assembly-based metagenomic analysis, we demonstrate that DNA input level has a significant impact on community structure due to overrepresentation of low-GC genomic regions following library amplification. In our system, these differences were largely superseded by variations between biological replicates, but our results advocate verifying the influence of library amplification on a case-by-case basis. Overall, this study provides recommendations for quality filtering and de-replication prior to analysis, as well as a practical framework to address the issue of low biomass or biomass heterogeneity in longitudinal metagenomic surveys.

  9. Metataxonomic and Metagenomic Approaches Versus Culture-Based Techniques For Clinical Pathology

    Directory of Open Access Journals (Sweden)

    Sarah K Hilton

    2016-04-01

    Full Text Available Diagnoses that are both timely and accurate are critically important for patients with life-threatening or drug resistant infections. Technological improvements in High-Throughput Sequencing (HTS have led to its use in pathogen detection and its application in clinical diagnoses of infectious diseases. The present study compares two HTS methods, 16S rRNA marker gene sequencing (metataxonomics and whole metagenomic shotgun sequencing (metagenomics, in their respective abilities to match the same diagnosis as traditional culture methods (culture inference for patients with ventilator associated pneumonia (VAP. The metagenomic analysis was able to produce the same diagnosis as culture methods at the species-level for five of the six samples, while the metataxonomic analysis was only able to produce results with the same species-level identification as culture for two of the six samples. These results indicate that metagenomic analyses have the accuracy needed for a clinical diagnostic tool, but full integration in diagnostic protocols is contingent on technological improvements to decrease turnaround time and lower costs.

  10. Metagenomics - a guide from sampling to data analysis

    OpenAIRE

    2012-01-01

    Metagenomics applies a suite of genomic technologies and bioinformatics tools to directly access the genetic content of entire communities of organisms. The field of metagenomics has been responsible for substantial advances in microbial ecology, evolution, and diversity over the past 5 to 10 years, and many research laboratories are actively engaged in it now. With the growing numbers of activities also comes a plethora of methodological knowledge and expertise that should guide future devel...

  11. Identification and assembly of genomes and genetic elements in complex metagenomic samples without using reference genomes

    DEFF Research Database (Denmark)

    Nielsen, Henrik Bjørn; Almeida, Mathieu; Juncker, Agnieszka;

    2014-01-01

    Most current approaches for analyzing metagenomic data rely on comparisons to reference genomes, but the microbial diversity of many environments extends far beyond what is covered by reference databases. De novo segregation of complex metagenomic data into specific biological entities......, such as particular bacterial strains or viruses, remains a largely unsolved problem. Here we present a method, based on binning co-abundant genes across a series of metagenomic samples, that enables comprehensive discovery of new microbial organisms, viruses and co-inherited genetic entities and aids assembly...... of microbial genomes without the need for reference sequences. We demonstrate the method on data from 396 human gut microbiome samples and identify 7,381 co-abundance gene groups (CAGs), including 741 metagenomic species (MGS). We use these to assemble 238 high-quality microbial genomes and identify...

  12. Beyond research: a primer for considerations on using viral metagenomics in the field and clinic

    NARCIS (Netherlands)

    Hall, Richard J; Draper, Jenny L; Nielsen, Fiona G G; Dutilh, Bas E

    2015-01-01

    Powered by recent advances in next-generation sequencing technologies, metagenomics has already unveiled vast microbial biodiversity in a range of environments, and is increasingly being applied in clinics for difficult-to-diagnose cases. It can be tempting to suggest that metagenomics could be used

  13. Quantitative field testing Rotylenchulus reniformis DNA from metagenomic samples isolated directly from soil.

    Directory of Open Access Journals (Sweden)

    Kurt Showmaker

    Full Text Available A quantitative PCR procedure targeting the β-tubulin gene determined the number of Rotylenchulus reniformis Linford & Oliveira 1940 in metagenomic DNA samples isolated from soil. Of note, this outcome was in the presence of other soil-dwelling plant parasitic nematodes including its sister genus Helicotylenchus Steiner, 1945. The methodology provides a framework for molecular diagnostics of nematodes from metagenomic DNA isolated directly from soil.

  14. Filtration and Normalization of Sequencing Read Data in Whole-Metagenome Shotgun Samples

    Science.gov (United States)

    Chouvarine, Philippe; Wiehlmann, Lutz; Moran Losada, Patricia; DeLuca, David S.; Tümmler, Burkhard

    2016-01-01

    Ever-increasing affordability of next-generation sequencing makes whole-metagenome sequencing an attractive alternative to traditional 16S rDNA, RFLP, or culturing approaches for the analysis of microbiome samples. The advantage of whole-metagenome sequencing is that it allows direct inference of the metabolic capacity and physiological features of the studied metagenome without reliance on the knowledge of genotypes and phenotypes of the members of the bacterial community. It also makes it possible to overcome problems of 16S rDNA sequencing, such as unknown copy number of the 16S gene and lack of sufficient sequence similarity of the “universal” 16S primers to some of the target 16S genes. On the other hand, next-generation sequencing suffers from biases resulting in non-uniform coverage of the sequenced genomes. To overcome this difficulty, we present a model of GC-bias in sequencing metagenomic samples as well as filtration and normalization techniques necessary for accurate quantification of microbial organisms. While there has been substantial research in normalization and filtration of read-count data in such techniques as RNA-seq or Chip-seq, to our knowledge, this has not been the case for the field of whole-metagenome shotgun sequencing. The presented methods assume that complete genome references are available for most microorganisms of interest present in metagenomic samples. This is often a valid assumption in such fields as medical diagnostics of patient microbiota. Testing the model on two validation datasets showed four-fold reduction in root-mean-square error compared to non-normalized data in both cases. The presented methods can be applied to any pipeline for whole metagenome sequencing analysis relying on complete microbial genome references. We demonstrate that such pre-processing reduces the number of false positive hits and increases accuracy of abundance estimates. PMID:27760173

  15. A Physicians' Wish List for the Clinical Application of Intestinal Metagenomics

    OpenAIRE

    Klymiuk, Ingeborg; Högenauer, Christoph; Halwachs, Bettina; Thallinger, Gerhard G.; Fricke, W. Florian; Steininger, Christoph

    2014-01-01

    Christoph Steininger and colleagues explore how multiple infectious, autoimmune, metabolic, and neoplastic diseases have been associated with changes in the intestinal microbiome, although a cause-effect relationship is often difficult to establish. Integration of metagenomics into clinical medicine is a challenge, and the authors highlight clinical approaches that are of high priority for the useful medical application of metagenomics. Please see later in the article for the Editors' Summary

  16. A Physicians' Wish List for the Clinical Application of Intestinal Metagenomics

    OpenAIRE

    Ingeborg Klymiuk; Christoph Högenauer; Bettina Halwachs; Thallinger, Gerhard G; W Florian Fricke; Christoph Steininger

    2014-01-01

    Christoph Steininger and colleagues explore how multiple infectious, autoimmune, metabolic, and neoplastic diseases have been associated with changes in the intestinal microbiome, although a cause-effect relationship is often difficult to establish. Integration of metagenomics into clinical medicine is a challenge, and the authors highlight clinical approaches that are of high priority for the useful medical application of metagenomics. Please see later in the article for the Editors' Summary.

  17. A physicians' wish list for the clinical application of intestinal metagenomics.

    Science.gov (United States)

    Klymiuk, Ingeborg; Högenauer, Christoph; Halwachs, Bettina; Thallinger, Gerhard G; Fricke, W Florian; Steininger, Christoph

    2014-04-01

    Christoph Steininger and colleagues explore how multiple infectious, autoimmune, metabolic, and neoplastic diseases have been associated with changes in the intestinal microbiome, although a cause-effect relationship is often difficult to establish. Integration of metagenomics into clinical medicine is a challenge, and the authors highlight clinical approaches that are of high priority for the useful medical application of metagenomics. Please see later in the article for the Editors' Summary.

  18. Quantitative field testing Heterodera glycines from metagenomic DNA samples isolated directly from soil under agronomic production.

    Directory of Open Access Journals (Sweden)

    Yan Li

    Full Text Available A quantitative PCR procedure targeting the Heterodera glycines ortholog of the Caenorhabditis elegans uncoordinated-78 gene was developed. The procedure estimated the quantity of H. glycines from metagenomic DNA samples isolated directly from field soil under agronomic production. The estimation of H. glycines quantity was determined in soil samples having other soil dwelling plant parasitic nematodes including Hoplolaimus, predatory nematodes including Mononchus, free-living nematodes and biomass. The methodology provides a framework for molecular diagnostics of nematodes from metagenomic DNA isolated directly from field soil.

  19. Beyond research: a primer for considerations on using viral metagenomics in the field and clinic

    OpenAIRE

    Hall, Richard J.; Draper, Jenny L.; Nielsen, Fiona G.G.; Dutilh, Bas E.

    2015-01-01

    Powered by recent advances in next-generation sequencing technologies, metagenomics has already unveiled vast microbial biodiversity in a range of environments, and is increasingly being applied in clinics for difficult-to-diagnose cases. It can be tempting to suggest that metagenomics could be used as a “universal test” for all pathogens without the need to conduct lengthy serial testing using specific assays. While this is an exciting prospect, there are issues that need to be addressed bef...

  20. Evaluation of convenient pretreatment protocols for RNA virus metagenomics in serum and tissue samples.

    Science.gov (United States)

    Rosseel, Toon; Ozhelvaci, Orkun; Freimanis, Graham; Van Borm, Steven

    2015-09-15

    Viral metagenomic approaches are increasingly being used for viral discovery. Various strategies are applied to enrich viral sequences, but there is often a lack of knowledge about their effective influence on the viral discovery sensitivity. We evaluate some convenient and widely used approaches for RNA virus discovery in clinical samples in order to reveal their sensitivity and potential bias introduced by the enrichment or amplifications steps. An RNA virus was artificially spiked at a fixed titer in serum and lung tissue, respectively, low and high nucleic acid content matrices. For serum, a simple DNase treatment on the RNA extract gave the maximum gain in proportion of viral sequences (83×), and a subsequent ribosomal RNA removal nearly doubled once more the proportion of viral sequences. For lung tissue, a ribosomal RNA depletion step on the RNA extract had the biggest gain in proportion of viral sequences (32×). We show also that direct sequencing of cDNA is recommended above an extra random PCR amplification step, and a that the virion enrichment strategy (filtration and nuclease treatment) has a beneficial effect for sequencing-based virus discovery. Our findings provide sample-dependent guidelines for targeted virus discovery strategies.

  1. Single sample resolution of rare microbial dark matter in a marine invertebrate metagenome

    Science.gov (United States)

    Miller, Ian J.; Weyna, Theodore R.; Fong, Stephen S.; Lim-Fong, Grace E.; Kwan, Jason C.

    2016-01-01

    Direct, untargeted sequencing of environmental samples (metagenomics) and de novo genome assembly enable the study of uncultured and phylogenetically divergent organisms. However, separating individual genomes from a mixed community has often relied on the differential-coverage analysis of multiple, deeply sequenced samples. In the metagenomic investigation of the marine bryozoan Bugula neritina, we uncovered seven bacterial genomes associated with a single B. neritina individual that appeared to be transient associates, two of which were unique to one individual and undetectable using certain “universal” 16S rRNA primers and probes. We recovered high quality genome assemblies for several rare instances of “microbial dark matter,” or phylogenetically divergent bacteria lacking genomes in reference databases, from a single tissue sample that was not subjected to any physical or chemical pre-treatment. One of these rare, divergent organisms has a small (593 kbp), poorly annotated genome with low GC content (20.9%) and a 16S rRNA gene with just 65% sequence similarity to the closest reference sequence. Our findings illustrate the importance of sampling strategy and de novo assembly of metagenomic reads to understand the extent and function of bacterial biodiversity. PMID:27681823

  2. Beyond research: a primer for considerations on using viral metagenomics in the field and clinic

    Directory of Open Access Journals (Sweden)

    Richard J Hall

    2015-03-01

    Full Text Available Powered by recent advances in next-generation sequencing technologies, metagenomics has already unveiled vast microbial biodiversity in a range of environments, and is increasingly being applied in clinics for difficult-to-diagnose cases. It can be tempting to suggest that metagenomics could be used as a universal test for all pathogens without the need to conduct lengthy serial testing using specific assays. While this is an exciting prospect, there are issues that need to be addressed before metagenomic methods can be applied with rigour as a diagnostic tool, including the potential for incidental findings, unforeseen consequences for trade and regulatory authorities, privacy and cultural issues, data sharing, and appropriate reporting of results to end-users. These issues will require consideration and discussion across a range of disciplines, including scientists, ethicists, clinicians, diagnosticians, health practitioners, and ultimately the public. Here, we provide a primer for consideration on some of these issues.

  3. Computational tools for viral metagenomics and their application in clinical research.

    Science.gov (United States)

    Fancello, L; Raoult, D; Desnues, C

    2012-12-20

    There are 100 times more virions than eukaryotic cells in a healthy human body. The characterization of human-associated viral communities in a non-pathological state and the detection of viral pathogens in cases of infection are essential for medical care and epidemic surveillance. Viral metagenomics, the sequenced-based analysis of the complete collection of viral genomes directly isolated from an organism or an ecosystem, bypasses the "single-organism-level" point of view of clinical diagnostics and thus the need to isolate and culture the targeted organism. The first part of this review is dedicated to a presentation of past research in viral metagenomics with an emphasis on human-associated viral communities (eukaryotic viruses and bacteriophages). In the second part, we review more precisely the computational challenges posed by the analysis of viral metagenomes, and we illustrate the problem of sequences that do not have homologs in public databases and the possible approaches to characterize them.

  4. The Sorcerer II Global Ocean Sampling Expedition: metagenomic characterization of viruses within aquatic microbial samples.

    Directory of Open Access Journals (Sweden)

    Shannon J Williamson

    Full Text Available Viruses are the most abundant biological entities on our planet. Interactions between viruses and their hosts impact several important biological processes in the world's oceans such as horizontal gene transfer, microbial diversity and biogeochemical cycling. Interrogation of microbial metagenomic sequence data collected as part of the Sorcerer II Global Ocean Expedition (GOS revealed a high abundance of viral sequences, representing approximately 3% of the total predicted proteins. Cluster analyses of the viral sequences revealed hundreds to thousands of viral genes encoding various metabolic and cellular functions. Quantitative analyses of viral genes of host origin performed on the viral fraction of aquatic samples confirmed the viral nature of these sequences and suggested that significant portions of aquatic viral communities behave as reservoirs of such genetic material. Distributional and phylogenetic analyses of these host-derived viral sequences also suggested that viral acquisition of environmentally relevant genes of host origin is a more abundant and widespread phenomenon than previously appreciated. The predominant viral sequences identified within microbial fractions originated from tailed bacteriophages and exhibited varying global distributions according to viral family. Recruitment of GOS viral sequence fragments against 27 complete aquatic viral genomes revealed that only one reference bacteriophage genome was highly abundant and was closely related, but not identical, to the cyanomyovirus P-SSM4. The co-distribution across all sampling sites of P-SSM4-like sequences with the dominant ecotype of its host, Prochlorococcus supports the classification of the viral sequences as P-SSM4-like and suggests that this virus may influence the abundance, distribution and diversity of one of the most dominant components of picophytoplankton in oligotrophic oceans. In summary, the abundance and broad geographical distribution of viral

  5. An Alignment-Free "Metapeptide" Strategy for Metaproteomic Characterization of Microbiome Samples Using Shotgun Metagenomic Sequencing.

    Science.gov (United States)

    May, Damon H; Timmins-Schiffman, Emma; Mikan, Molly P; Harvey, H Rodger; Borenstein, Elhanan; Nunn, Brook L; Noble, William S

    2016-08-01

    In principle, tandem mass spectrometry can be used to detect and quantify the peptides present in a microbiome sample, enabling functional and taxonomic insight into microbiome metabolic activity. However, the phylogenetic diversity constituting a particular microbiome is often unknown, and many of the organisms present may not have assembled genomes. In ocean microbiome samples, with particularly diverse and uncultured bacterial communities, it is difficult to construct protein databases that contain the bulk of the peptides in the sample without losing detection sensitivity due to the overwhelming number of candidate peptides for each tandem mass spectrum. We describe a method for deriving "metapeptides" (short amino acid sequences that may be represented in multiple organisms) from shotgun metagenomic sequencing of microbiome samples. In two ocean microbiome samples, we constructed site-specific metapeptide databases to detect more than one and a half times as many peptides as by searching against predicted genes from an assembled metagenome and roughly three times as many peptides as by searching against the NCBI environmental proteome database. The increased peptide yield has the potential to enrich the taxonomic and functional characterization of sample metaproteomes. PMID:27396978

  6. MetaCluster 5.0: a two-round binning approach for metagenomic data for low-abundance species in a noisy sample

    OpenAIRE

    Wang, Yi(Centre for Theoretical Cosmology, DAMTP, University of Cambridge, Wilberforce Road, Cambridge, CB3 0WA U.K.); Leung, Henry C. M.; Yiu, S. M.; Chin, Francis Y. L.

    2012-01-01

    Motivation: Metagenomic binning remains an important topic in metagenomic analysis. Existing unsupervised binning methods for next-generation sequencing (NGS) reads do not perform well on (i) samples with low-abundance species or (ii) samples (even with high abundance) when there are many extremely low-abundance species. These two problems are common for real metagenomic datasets. Binning methods that can solve these problems are desirable. Results: We proposed a two-round binning method (Met...

  7. Identification of a novel human papillomavirus by metagenomic analysis of samples from patients with febrile respiratory illness

    NARCIS (Netherlands)

    Mokili, J.L.; Dutilh, B.E.; Lim, Y.W.; Schneider, B.S.; Taylor, T.; Haynes, M.R.; Metzgar, D.; Myers, C.A.; Blair, P.J.; Nosrat, B.; Wolfe, N.D.; Rohwer, F.

    2013-01-01

    As part of a virus discovery investigation using a metagenomic approach, a highly divergent novel Human papillomavirus type was identified in pooled convenience nasal/oropharyngeal swab samples collected from patients with febrile respiratory illness. Phylogenetic analysis of the whole genome and th

  8. Identification and assembly of genomes and genetic elements in complex metagenomic samples without using reference genomes

    NARCIS (Netherlands)

    Nielsen, H.B.; Almeida, M.; Sierakowska Juncker, A.; Rasmussen, S.; Li, J.; Sunagawa, S.; Plichta, D.R.; Gautier, L.; Pedersen, A.G.; Chatelier, Le E.; Pelletier, E.; Bonde, I.; Nielsen, T.; Manichanh, C.; Arumugam, M.; Batto, J.M.; Quintanilha dos Santos, M.B.; Blom, N.; Borruel, N.; Burgdorf, K.S.; Boumezbeur, F.; Casellas, F.; Doré, J.; Dworzynski, P.; Guarner, F.; Hansen, T.; Hildebrand, F.; Kaas, R.S.; Kennedy, S.; Kristiansen, K.; Kultima, J.R.; Leonard, P.; Levenez, F.; Lund, O.; Moumen, B.; Paslier, Le D.; Pons, N.; Pedersen, O.; Prifti, E.; Qin, J.; Raes, J.; Sørensen, S.; Tap, J.; Tims, S.; Ussery, D.W.; Yamada, T.; Jamet, A.; Mérieux, A.; Cultrone, A.; Torrejon, A.; Quinquis, B.; Brechot, C.; Delorme, C.; M'Rini, C.; Vos, de W.M.; Maguin, E.; Varela, E.; Guedon, E.; Gwen, F.; Haimet, F.; Artiguenave, F.; Vandemeulebrouck, G.; Denariaz, G.; Khaci, G.; Blottière, H.; Knol, J.; Weissenbach, J.; Hylckama Vlieg, van J.E.; Torben, J.; Parkhil, J.; Turner, K.; Guchte, van de M.; Antolin, M.; Rescigno, M.; Kleerebezem, M.; Derrien, M.; Galleron, N.; Sanchez, N.; Grarup, N.; Veiga, P.; Oozeer, R.; Dervyn, R.; Layec, S.; Bruls, T.; Winogradski, Y.; Zoetendal, E.G.; Renault, D.; Sicheritz-Ponten,; Bork, P.; Wang, J.; Brunak, S.; Ehrlich, S.D.

    2014-01-01

    Most current approaches for analyzing metagenomic data rely on comparisons to reference genomes, but the microbial diversity of many environments extends far beyond what is covered by reference databases. De novo segregation of complex metagenomic data into specific biological entities, such as part

  9. Metagenomic covariation along densely sampled environmental gradients in the Red Sea

    KAUST Repository

    Thompson, Luke R

    2016-07-15

    Oceanic microbial diversity covaries with physicochemical parameters. Temperature, for example, explains approximately half of global variation in surface taxonomic abundance. It is unknown, however, whether covariation patterns hold over narrower parameter gradients and spatial scales, and extending to mesopelagic depths. We collected and sequenced 45 epipelagic and mesopelagic microbial metagenomes on a meridional transect through the eastern Red Sea. We asked which environmental parameters explain the most variation in relative abundances of taxonomic groups, gene ortholog groups, and pathways—at a spatial scale of <2000 km, along narrow but well-defined latitudinal and depth-dependent gradients. We also asked how microbes are adapted to gradients and extremes in irradiance, temperature, salinity, and nutrients, examining the responses of individual gene ortholog groups to these parameters. Functional and taxonomic metrics were equally well explained (75–79%) by environmental parameters. However, only functional and not taxonomic covariation patterns were conserved when comparing with an intruding water mass with different physicochemical properties. Temperature explained the most variation in each metric, followed by nitrate, chlorophyll, phosphate, and salinity. That nitrate explained more variation than phosphate suggested nitrogen limitation, consistent with low surface N:P ratios. Covariation of gene ortholog groups with environmental parameters revealed patterns of functional adaptation to the challenging Red Sea environment: high irradiance, temperature, salinity, and low nutrients. Nutrient-acquisition gene ortholog groups were anti-correlated with concentrations of their respective nutrient species, recapturing trends previously observed across much larger distances and environmental gradients. This dataset of metagenomic covariation along densely sampled environmental gradients includes online data exploration supplements, serving as a community

  10. Modular approach to customise sample preparation procedures for viral metagenomics: a reproducible protocol for virome analysis.

    Science.gov (United States)

    Conceição-Neto, Nádia; Zeller, Mark; Lefrère, Hanne; De Bruyn, Pieter; Beller, Leen; Deboutte, Ward; Yinda, Claude Kwe; Lavigne, Rob; Maes, Piet; Van Ranst, Marc; Heylen, Elisabeth; Matthijnssens, Jelle

    2015-11-12

    A major limitation for better understanding the role of the human gut virome in health and disease is the lack of validated methods that allow high throughput virome analysis. To overcome this, we evaluated the quantitative effect of homogenisation, centrifugation, filtration, chloroform treatment and random amplification on a mock-virome (containing nine highly diverse viruses) and a bacterial mock-community (containing four faecal bacterial species) using quantitative PCR and next-generation sequencing. This resulted in an optimised protocol that was able to recover all viruses present in the mock-virome and strongly alters the ratio of viral versus bacterial and 16S rRNA genetic material in favour of viruses (from 43.2% to 96.7% viral reads and from 47.6% to 0.19% bacterial reads). Furthermore, our study indicated that most of the currently used virome protocols, using small filter pores and/or stringent centrifugation conditions may have largely overlooked large viruses present in viromes. We propose NetoVIR (Novel enrichment technique of VIRomes), which allows for a fast, reproducible and high throughput sample preparation for viral metagenomics studies, introducing minimal bias. This procedure is optimised mainly for faecal samples, but with appropriate concentration steps can also be used for other sample types with lower initial viral loads.

  11. Metagenomics and future perspectives in virus discovery.

    Science.gov (United States)

    Mokili, John L; Rohwer, Forest; Dutilh, Bas E

    2012-02-01

    Monitoring the emergence and re-emergence of viral diseases with the goal of containing the spread of viral agents requires both adequate preparedness and quick response. Identifying the causative agent of a new epidemic is one of the most important steps for effective response to disease outbreaks. Traditionally, virus discovery required propagation of the virus in cell culture, a proven technique responsible for the identification of the vast majority of viruses known to date. However, many viruses cannot be easily propagated in cell culture, thus limiting our knowledge of viruses. Viral metagenomic analyses of environmental samples suggest that the field of virology has explored less than 1% of the extant viral diversity. In the last decade, the culture-independent and sequence-independent metagenomic approach has permitted the discovery of many viruses in a wide range of samples. Phylogenetically, some of these viruses are distantly related to previously discovered viruses. In addition, 60-99% of the sequences generated in different viral metagenomic studies are not homologous to known viruses. In this review, we discuss the advances in the area of viral metagenomics during the last decade and their relevance to virus discovery, clinical microbiology and public health. We discuss the potential of metagenomics for characterization of the normal viral population in a healthy community and identification of viruses that could pose a threat to humans through zoonosis. In addition, we propose a new model of the Koch's postulates named the 'Metagenomic Koch's Postulates'. Unlike the original Koch's postulates and the Molecular Koch's postulates as formulated by Falkow, the metagenomic Koch's postulates focus on the identification of metagenomic traits in disease cases. The metagenomic traits that can be traced after healthy individuals have been exposed to the source of the suspected pathogen.

  12. Growth dynamics of gut microbiota in health and disease inferred from single metagenomic samples

    Science.gov (United States)

    Avnit-Sagi, Tali; Pompan-Lotan, Maya; Matot, Elad; Jona, Ghil; Harmelin, Alon; Cohen, Nadav; Sirota-Madi, Alexandra; Thaiss, Christoph A.; Pevsner-Fischer, Meirav; Sorek, Rotem; Xavier, Ramnik; Elinav, Eran; Segal, Eran

    2016-01-01

    Metagenomic sequencing increased our understanding of the role of the microbiome in health and disease, yet it only provides a snapshot of a highly dynamic ecosystem. Here, we show that the pattern of metagenomic sequencing read coverage for different microbial genomes contains a single trough and a single peak, the latter coinciding with the bacterial origin of replication. Furthermore, the ratio of sequencing coverage between the peak and trough provides a quantitative measure of a species' growth rate. We demonstrate this in vitro and in vivo, under different growth conditions, and in complex bacterial communities. For several bacterial species, peak to trough coverage ratios, but not relative abundances, correlated with the manifestation of inflammatory bowel disease and type II diabetes. PMID:26229116

  13. Diagnostic metagenomics: potential applications to bacterial, viral and parasitic infections

    OpenAIRE

    Pallen, M. J.

    2014-01-01

    SUMMARY The term ‘shotgun metagenomics’ is applied to the direct sequencing of DNA extracted from a sample without culture or target-specific amplification or capture. In diagnostic metagenomics, this approach is applied to clinical samples in the hope of detecting and characterizing pathogens. Here, I provide a conceptual overview, before reviewing several recent promising proof-of-principle applications of metagenomics in virus discovery, analysis of outbreaks and detection of pathogens in ...

  14. Livermore Metagenomics Analysis Toolkit

    Energy Technology Data Exchange (ETDEWEB)

    2012-10-01

    LMAT is designed to take as input a collection of raw metagenomic sequencer reads, and search each read against a reference genome database and assign a taxonomic label and confidence value to each read and report a summary of the predicted taxonomic contents of the metagenomic sample.

  15. Identification of a novel human papillomavirus by metagenomic analysis of samples from patients with febrile respiratory illness.

    Directory of Open Access Journals (Sweden)

    John L Mokili

    Full Text Available As part of a virus discovery investigation using a metagenomic approach, a highly divergent novel Human papillomavirus type was identified in pooled convenience nasal/oropharyngeal swab samples collected from patients with febrile respiratory illness. Phylogenetic analysis of the whole genome and the L1 gene reveals that the new HPV identified in this study clusters with previously described gamma papillomaviruses, sharing only 61.1% (whole genome and 63.1% (L1 sequence identity with its closest relative in the Papillomavirus episteme (PAVE database. This new virus was named HPV_SD2 pending official classification. The complete genome of HPV-SD2 is 7,299 bp long (36.3% G/C and contains 7 open reading frames (L2, L1, E6, E7, E1, E2 and E4 and a non-coding long control region (LCR between L1 and E6. The metagenomic procedures, coupled with the bioinformatic methods described herein are well suited to detect small circular genomes such as those of human papillomaviruses.

  16. Virome profiling of bats from Myanmar by metagenomic analysis of tissue samples reveals more novel Mammalian viruses.

    Directory of Open Access Journals (Sweden)

    Biao He

    Full Text Available Bats are reservoir animals harboring many important pathogenic viruses and with the capability of transmitting these to humans and other animals. To establish an effective surveillance to monitor transboundary spread of bat viruses between Myanmar and China, complete organs from the thorax and abdomen from 853 bats of six species from two Myanmar counties close to Yunnan province, China, were collected and tested for their virome through metagenomics by Solexa sequencing and bioinformatic analysis. In total, 3,742,314 reads of 114 bases were generated, and over 86% were assembled into 1,649,512 contigs with an average length of 114 bp, of which 26,698 (2% contigs were recognizable viral sequences belonging to 24 viral families. Of the viral contigs 45% (12,086/26,698 were related to vertebrate viruses, 28% (7,443/26,698 to insect viruses, 27% (7,074/26,698 to phages and 95 contigs to plant viruses. The metagenomic results were confirmed by PCR of selected viruses in all bat samples followed by phylogenetic analysis, which has led to the discovery of some novel bat viruses of the genera Mamastrovirus, Bocavirus, Circovirus, Iflavirus and Orthohepadnavirus and to their prevalence rates in two bat species. In conclusion, the present study aims to present the bat virome in Myanmar, and the results obtained further expand the spectrum of viruses harbored by bats.

  17. Culture-independent detection and characterisation of Mycobacterium tuberculosis and M. africanum in sputum samples using shotgun metagenomics on a benchtop sequencer

    Directory of Open Access Journals (Sweden)

    Emma L. Doughty

    2014-09-01

    Full Text Available Tuberculosis remains a major global health problem. Laboratory diagnostic methods that allow effective, early detection of cases are central to management of tuberculosis in the individual patient and in the community. Since the 1880s, laboratory diagnosis of tuberculosis has relied primarily on microscopy and culture. However, microscopy fails to provide species- or lineage-level identification and culture-based workflows for diagnosis of tuberculosis remain complex, expensive, slow, technically demanding and poorly able to handle mixed infections. We therefore explored the potential of shotgun metagenomics, sequencing of DNA from samples without culture or target-specific amplification or capture, to detect and characterise strains from the Mycobacterium tuberculosis complex in smear-positive sputum samples obtained from The Gambia in West Africa. Eight smear- and culture-positive sputum samples were investigated using a differential-lysis protocol followed by a kit-based DNA extraction method, with sequencing performed on a benchtop sequencing instrument, the Illumina MiSeq. The number of sequence reads in each sputum-derived metagenome ranged from 989,442 to 2,818,238. The proportion of reads in each metagenome mapping against the human genome ranged from 20% to 99%. We were able to detect sequences from the M. tuberculosis complex in all eight samples, with coverage of the H37Rv reference genome ranging from 0.002X to 0.7X. By analysing the distribution of large sequence polymorphisms (deletions and the locations of the insertion element IS6110 and single nucleotide polymorphisms (SNPs, we were able to assign seven of eight metagenome-derived genomes to a species and lineage within the M. tuberculosis complex. Two metagenome-derived mycobacterial genomes were assigned to M. africanum, a species largely confined to West Africa; the others that could be assigned belonged to lineages T, H or LAM within the clade of “modern” M. tuberculosis

  18. Ocean microbial metagenomics

    Science.gov (United States)

    Kerkhof, Lee J.; Goodman, Robert M.

    2009-09-01

    Technology for accessing the genomic DNA of microorganisms, directly from environmental samples without prior cultivation, has opened new vistas to understanding microbial diversity and functions. Especially as applied to soils and the oceans, environments on Earth where microbial diversity is vast, metagenomics and its emergent approaches have the power to transform rapidly our understanding of environmental microbiology. Here we explore select recent applications of the metagenomic suite to ocean microbiology.

  19. Analysis,Comparison and Classification of Metagenomic Samples%宏基因组样本数据的分析比较与分类

    Institute of Scientific and Technical Information of China (English)

    程福东; 丁啸; 李晟; 孙啸

    2016-01-01

    宏基因组学研究试图通过测序并分析微生物群落的DNA序列,以理解环境微生物的组成及其与环境的交互作用。宏基因组学革命性地改变了微生物学,使得以免培养的方式研究复杂生物系统中的微生物群落成为可能。第二代测序技术的不断进步和生物信息学的高速发展促进了高通量宏基因组研究的发展,大批高质量的宏基因组数据不断产生并对科学界开放,宏基因组学的重要作用被科学界广泛认可。与此同时,对应个体不同健康状态和人体不同部位的大量宏基因组样本数据不断产生,使得比较和分类宏基因组样本在微生物学研究上变得更加重要,比较宏基因组学成为宏基因组学的重要分支。主要介绍了宏基因组数据的分析比较,以及样本分类的相关研究和算法。%Metagenomics attempts to understand the diversity of the environmental microbial community and the interaction between microorganisms and environment by analyzing the sequence data of metagenomic samples. Microbiology has been revolutionized by metagenomics,which makes it feasible to research the microbial communities in complex biological systems without cultivating the microbes. The high-throughput metagenomic study is promoted by the rapid development of next-generation sequencing technology and bioinformatics. As a mass of high-quality metagenomic sequencing data are produced,also are accessible to the scientific community,the role of metagenomics has been recognized by various scientific areas. On the other sides,huge metagenomic data for individuals with different health status,or for different habitats of the human body makes the comparison and classification of metagenomic samples more important,leading the comparative metagenomics to become an important branch of metagenomics. This review mainly introduces the related researches and algorithms in the analysis,comparison and classification

  20. Integration of Metagenomic and Biogeochemical Data from Soils Sampled from a Long-Term Reciprocal Transplant

    Science.gov (United States)

    Bailey, V. L.; Hess, N. J.; McCue, L. A.

    2014-12-01

    The long-term impacts of climate conditions on soil ecosystems are difficult to discern with sufficient resolution to underpin a predictive understanding of ecosystem response to global climate change. The structure and function of the microbial community is intimately linked to soil organic carbon (SOC) by both the deposition of new carbon, and metabolism and respiration of existing SOC. We are studying the resilience of the microbial community, and the vulnerability of the soil carbon reservoirs, to changing climate conditions using a reciprocal soil transplant experiment initiated in 1994 in eastern Washington. Soil cores were reciprocally transplanted between two elevations (310 m and 844 m); the lower site is warmer and drier with 0.8% soil carbon, and the upper site is cooler and wetter with 1.8% soil carbon. We resampled these cores in 2012-13 to analyze the structure of the microbial community, biochemical activities of carbohydrate-active enzymes, and the soil carbon and nitrogen content. We hypothesized that microbial and biochemical dynamics developed under cool, moist conditions would destabilize under hot, dry conditions, such that carbon and nitrogen losses would be faster in warmer climate soils than the accruals in cooler climate soils. Metagenomics data analyses show that the microbial communities below 5 cm depth in the transplanted soils are most similar to those in the native and control soils from their original (pre-1994) location, whereas the surface microbial community has been influenced by their new (post-1994) location. Enzyme activities are highest in soils from the cooler, moister location, and the activities of the reciprocally transplanted soils are shifting toward the activities typical of their new location. Integration of these results with high-resolution mass spectrometry data of the soil carbon moieties will contribute to our fundamental understanding of climate change effects on the terrestrial ecosystem carbon cycle.

  1. Metagenomics for pathogen detection in public health

    OpenAIRE

    Miller, Ruth R.; Montoya, Vincent; Gardy, Jennifer L; Patrick, David M.; Tang, Patrick

    2013-01-01

    Traditional pathogen detection methods in public health infectious disease surveillance rely upon the identification of agents that are already known to be associated with a particular clinical syndrome. The emerging field of metagenomics has the potential to revolutionize pathogen detection in public health laboratories by allowing the simultaneous detection of all microorganisms in a clinical sample, without a priori knowledge of their identities, through the use of next-generation DNA sequ...

  2. Computational tools for viral metagenomics and their application in clinical research

    OpenAIRE

    Fancello, L.; Raoult, D.; Desnues, C.

    2012-01-01

    There are 100 times more virus particles than eukaryotic cells in a healthy human body. The characterization of human-associated viral communities in a non-pathological state and the detection of viral pathogens in cases of infection are thus essential for medical care and epidemic surveillance. Viral metagenomics, the sequenced-based analysis of the complete collection of viral genomes directly isolated from an organism or an ecosystem, bypasses the “single-organism-level” point of view of c...

  3. Applying meta-pathway analyses through metagenomics to identify the functional properties of the major bacterial communities of a single spontaneous cocoa bean fermentation process sample.

    Science.gov (United States)

    Illeghems, Koen; Weckx, Stefan; De Vuyst, Luc

    2015-09-01

    A high-resolution functional metagenomic analysis of a representative single sample of a Brazilian spontaneous cocoa bean fermentation process was carried out to gain insight into its bacterial community functioning. By reconstruction of microbial meta-pathways based on metagenomic data, the current knowledge about the metabolic capabilities of bacterial members involved in the cocoa bean fermentation ecosystem was extended. Functional meta-pathway analysis revealed the distribution of the metabolic pathways between the bacterial members involved. The metabolic capabilities of the lactic acid bacteria present were most associated with the heterolactic fermentation and citrate assimilation pathways. The role of Enterobacteriaceae in the conversion of substrates was shown through the use of the mixed-acid fermentation and methylglyoxal detoxification pathways. Furthermore, several other potential functional roles for Enterobacteriaceae were indicated, such as pectinolysis and citrate assimilation. Concerning acetic acid bacteria, metabolic pathways were partially reconstructed, in particular those related to responses toward stress, explaining their metabolic activities during cocoa bean fermentation processes. Further, the in-depth metagenomic analysis unveiled functionalities involved in bacterial competitiveness, such as the occurrence of CRISPRs and potential bacteriocin production. Finally, comparative analysis of the metagenomic data with bacterial genomes of cocoa bean fermentation isolates revealed the applicability of the selected strains as functional starter cultures. PMID:25998815

  4. Applying meta-pathway analyses through metagenomics to identify the functional properties of the major bacterial communities of a single spontaneous cocoa bean fermentation process sample.

    Science.gov (United States)

    Illeghems, Koen; Weckx, Stefan; De Vuyst, Luc

    2015-09-01

    A high-resolution functional metagenomic analysis of a representative single sample of a Brazilian spontaneous cocoa bean fermentation process was carried out to gain insight into its bacterial community functioning. By reconstruction of microbial meta-pathways based on metagenomic data, the current knowledge about the metabolic capabilities of bacterial members involved in the cocoa bean fermentation ecosystem was extended. Functional meta-pathway analysis revealed the distribution of the metabolic pathways between the bacterial members involved. The metabolic capabilities of the lactic acid bacteria present were most associated with the heterolactic fermentation and citrate assimilation pathways. The role of Enterobacteriaceae in the conversion of substrates was shown through the use of the mixed-acid fermentation and methylglyoxal detoxification pathways. Furthermore, several other potential functional roles for Enterobacteriaceae were indicated, such as pectinolysis and citrate assimilation. Concerning acetic acid bacteria, metabolic pathways were partially reconstructed, in particular those related to responses toward stress, explaining their metabolic activities during cocoa bean fermentation processes. Further, the in-depth metagenomic analysis unveiled functionalities involved in bacterial competitiveness, such as the occurrence of CRISPRs and potential bacteriocin production. Finally, comparative analysis of the metagenomic data with bacterial genomes of cocoa bean fermentation isolates revealed the applicability of the selected strains as functional starter cultures.

  5. A primer on metagenomics.

    Science.gov (United States)

    Wooley, John C; Godzik, Adam; Friedberg, Iddo

    2010-02-26

    Metagenomics is a discipline that enables the genomic study of uncultured microorganisms. Faster, cheaper sequencing technologies and the ability to sequence uncultured microbes sampled directly from their habitats are expanding and transforming our view of the microbial world. Distilling meaningful information from the millions of new genomic sequences presents a serious challenge to bioinformaticians. In cultured microbes, the genomic data come from a single clone, making sequence assembly and annotation tractable. In metagenomics, the data come from heterogeneous microbial communities, sometimes containing more than 10,000 species, with the sequence data being noisy and partial. From sampling, to assembly, to gene calling and function prediction, bioinformatics faces new demands in interpreting voluminous, noisy, and often partial sequence data. Although metagenomics is a relative newcomer to science, the past few years have seen an explosion in computational methods applied to metagenomic-based research. It is therefore not within the scope of this article to provide an exhaustive review. Rather, we provide here a concise yet comprehensive introduction to the current computational requirements presented by metagenomics, and review the recent progress made. We also note whether there is software that implements any of the methods presented here, and briefly review its utility. Nevertheless, it would be useful if readers of this article would avail themselves of the comment section provided by this journal, and relate their own experiences. Finally, the last section of this article provides a few representative studies illustrating different facets of recent scientific discoveries made using metagenomics.

  6. Partial least squares regression can aid in detecting differential abundance of multiple features in sets of metagenomic samples

    Directory of Open Access Journals (Sweden)

    Ondrej eLibiger

    2015-12-01

    Full Text Available It is now feasible to examine the composition and diversity of microbial communities (i.e., `microbiomes‘ that populate different human organs and orifices using DNA sequencing and related technologies. To explore the potential links between changes in microbial communities and various diseases in the human body, it is essential to test associations involving different species within and across microbiomes, environmental settings and disease states. Although a number of statistical techniques exist for carrying out relevant analyses, it is unclear which of these techniques exhibit the greatest statistical power to detect associations given the complexity of most microbiome datasets. We compared the statistical power of principal component regression, partial least squares regression, regularized regression, distance-based regression, Hill's diversity measures, and a modified test implemented in the popular and widely used microbiome analysis methodology 'Metastats‘ across a wide range of simulated scenarios involving changes in feature abundance between two sets of metagenomic samples. For this purpose, simulation studies were used to change the abundance of microbial species in a real dataset from a published study examining human hands. Each technique was applied to the same data, and its ability to detect the simulated change in abundance was assessed. We hypothesized that a small subset of methods would outperform the rest in terms of the statistical power. Indeed, we found that the Metastats technique modified to accommodate multivariate analysis and partial least squares regression yielded high power under the models and data sets we studied. The statistical power of diversity measure-based tests, distance-based regression and regularized regression was significantly lower. Our results provide insight into powerful analysis strategies that utilize information on species counts from large microbiome data sets exhibiting skewed frequency

  7. Towards quantitative metagenomics of wild viruses and other ultra-low concentration DNA samples: a rigorous assessment and optimization of the linker amplification method.

    Science.gov (United States)

    Duhaime, Melissa B; Deng, Li; Poulos, Bonnie T; Sullivan, Matthew B

    2012-09-01

    Metagenomics generates and tests hypotheses about dynamics and mechanistic drivers in wild populations, yet commonly suffers from insufficient (metagenomics analyses include linear amplification for deep sequencing (LADS), which requires more DNA than is normally available, linker-amplified shotgun libraries (LASLs), which is prohibitively low throughput, and whole-genome amplification, which is significantly biased and thus non-quantitative. Here, we adapt the LASL approach to next generation sequencing by offering an alternate polymerase for challenging samples, developing a more efficient sizing step, integrating a 'reconditioning PCR' step to increase yield and minimize late-cycle PCR artefacts, and empirically documenting the quantitative capability of the optimized method with both laboratory isolate and wild community viral DNA. Our optimized linker amplification method requires as little as 1 pg of DNA and is the most precise and accurate available, with G + C content amplification biases less than 1.5-fold, even for complex samples as diverse as a wild virus community. While optimized here for 454 sequencing, this linker amplification method can be used to prepare metagenomics libraries for sequencing with next-generation platforms, including Illumina and Ion Torrent, the first of which we tested and present data for here.

  8. Metagenomic analyses of novel viruses and plasmids from a cultured environmental sample of hyperthermophilic neutrophiles

    DEFF Research Database (Denmark)

    Garrett, Roger Antony; Prangishvili, David; Shah, Shiraz Ali;

    2010-01-01

    Two novel viral genomes and four plasmids were assembled from an environmental sample collected from a hot spring at Yellowstone National Park, USA, and maintained anaerobically in a bioreactor at 85°C and pH 6. The double-stranded DNA viral genomes are linear (22.7 kb) and circular (17.7 kb), an...... respectively. Strategies are considered for assembling genomes of smaller genetic elements from complex environmental samples, and for establishing possible host identities on the basis of sequence similarity to host CRISPR immune systems....

  9. Databases of the marine metagenomics

    KAUST Repository

    Mineta, Katsuhiko

    2015-10-28

    The metagenomic data obtained from marine environments is significantly useful for understanding marine microbial communities. In comparison with the conventional amplicon-based approach of metagenomics, the recent shotgun sequencing-based approach has become a powerful tool that provides an efficient way of grasping a diversity of the entire microbial community at a sampling point in the sea. However, this approach accelerates accumulation of the metagenome data as well as increase of data complexity. Moreover, when metagenomic approach is used for monitoring a time change of marine environments at multiple locations of the seawater, accumulation of metagenomics data will become tremendous with an enormous speed. Because this kind of situation has started becoming of reality at many marine research institutions and stations all over the world, it looks obvious that the data management and analysis will be confronted by the so-called Big Data issues such as how the database can be constructed in an efficient way and how useful knowledge should be extracted from a vast amount of the data. In this review, we summarize the outline of all the major databases of marine metagenome that are currently publically available, noting that database exclusively on marine metagenome is none but the number of metagenome databases including marine metagenome data are six, unexpectedly still small. We also extend our explanation to the databases, as reference database we call, that will be useful for constructing a marine metagenome database as well as complementing important information with the database. Then, we would point out a number of challenges to be conquered in constructing the marine metagenome database.

  10. Databases of the marine metagenomics.

    Science.gov (United States)

    Mineta, Katsuhiko; Gojobori, Takashi

    2016-02-01

    The metagenomic data obtained from marine environments is significantly useful for understanding marine microbial communities. In comparison with the conventional amplicon-based approach of metagenomics, the recent shotgun sequencing-based approach has become a powerful tool that provides an efficient way of grasping a diversity of the entire microbial community at a sampling point in the sea. However, this approach accelerates accumulation of the metagenome data as well as increase of data complexity. Moreover, when metagenomic approach is used for monitoring a time change of marine environments at multiple locations of the seawater, accumulation of metagenomics data will become tremendous with an enormous speed. Because this kind of situation has started becoming of reality at many marine research institutions and stations all over the world, it looks obvious that the data management and analysis will be confronted by the so-called Big Data issues such as how the database can be constructed in an efficient way and how useful knowledge should be extracted from a vast amount of the data. In this review, we summarize the outline of all the major databases of marine metagenome that are currently publically available, noting that database exclusively on marine metagenome is none but the number of metagenome databases including marine metagenome data are six, unexpectedly still small. We also extend our explanation to the databases, as reference database we call, that will be useful for constructing a marine metagenome database as well as complementing important information with the database. Then, we would point out a number of challenges to be conquered in constructing the marine metagenome database.

  11. An Improved Method for High Quality Metagenomics DNA Extraction from Human and Environmental Samples

    Science.gov (United States)

    Bag, Satyabrata; Saha, Bipasa; Mehta, Ojasvi; Anbumani, D.; Kumar, Naveen; Dayal, Mayanka; Pant, Archana; Kumar, Pawan; Saxena, Shruti; Allin, Kristine H.; Hansen, Torben; Arumugam, Manimozhiyan; Vestergaard, Henrik; Pedersen, Oluf; Pereira, Verima; Abraham, Philip; Tripathi, Reva; Wadhwa, Nitya; Bhatnagar, Shinjini; Prakash, Visvanathan Gnana; Radha, Venkatesan; Anjana, R. M.; Mohan, V.; Takeda, Kiyoshi; Kurakawa, Takashi; Nair, G. Balakrish; Das, Bhabatosh

    2016-01-01

    To explore the natural microbial community of any ecosystems by high-resolution molecular approaches including next generation sequencing, it is extremely important to develop a sensitive and reproducible DNA extraction method that facilitate isolation of microbial DNA of sufficient purity and quantity from culturable and uncultured microbial species living in that environment. Proper lysis of heterogeneous community microbial cells without damaging their genomes is a major challenge. In this study, we have developed an improved method for extraction of community DNA from different environmental and human origin samples. We introduced a combination of physical, chemical and mechanical lysis methods for proper lysis of microbial inhabitants. The community microbial DNA was precipitated by using salt and organic solvent. Both the quality and quantity of isolated DNA was compared with the existing methodologies and the supremacy of our method was confirmed. Maximum recovery of genomic DNA in the absence of substantial amount of impurities made the method convenient for nucleic acid extraction. The nucleic acids obtained using this method are suitable for different downstream applications. This improved method has been named as the THSTI method to depict the Institute where the method was developed. PMID:27240745

  12. Microbial metagenomics: beyond the genome.

    Science.gov (United States)

    Gilbert, Jack A; Dupont, Christopher L

    2011-01-01

    Metagenomics literally means "beyond the genome." Marine microbial metagenomic databases presently comprise approximately 400 billion base pairs of DNA, only approximately 3% of that found in 1 ml of seawater. Very soon a trillion-base-pair sequence run will be feasible, so it is time to reflect on what we have learned from metagenomics. We review the impact of metagenomics on our understanding of marine microbial communities. We consider the studies facilitated by data generated through the Global Ocean Sampling expedition, as well as the revolution wrought at the individual laboratory level through next generation sequencing technologies. We review recent studies and discoveries since 2008, provide a discussion of bioinformatic analyses, including conceptual pipelines and sequence annotation and predict the future of metagenomics, with suggestions of collaborative community studies tailored toward answering some of the fundamental questions in marine microbial ecology.

  13. Microbial Metagenomics: Beyond the Genome

    Science.gov (United States)

    Gilbert, Jack A.; Dupont, Christopher L.

    2011-01-01

    Metagenomics literally means “beyond the genome.” Marine microbial metagenomic databases presently comprise ˜400 billion base pairs of DNA, only ˜3% of that found in 1 ml of seawater. Very soon a trillion-base-pair sequence run will be feasible, so it is time to reflect on what we have learned from metagenomics. We review the impact of metagenomics on our understanding of marine microbial communities. We consider the studies facilitated by data generated through the Global Ocean Sampling expedition, as well as the revolution wrought at the individual laboratory level through next generation sequencing technologies. We review recent studies and discoveries since 2008, provide a discussion of bioinformatic analyses, including conceptual pipelines and sequence annotation and predict the future of metagenomics, with suggestions of collaborative community studies tailored toward answering some of the fundamental questions in marine microbial ecology.

  14. SRIdent: A novel pipeline for real-time identification of species from high-throughput sequencing reads in Metagenomics and clinical diagnostic assays.

    Science.gov (United States)

    Karimi, Ramin; Hajdu, Andras

    2015-01-01

    New advances in rapid sequencing of large amounts of DNA have brought a great potential for the study of complex communities of microorganisms. One of the challenging problems is rapid identification of species from sequenced reads. Delays in the identification of pathogens are a barrier to the early diagnosis and proper treatment of infectious diseases. In this paper we proposed SRIdent (Short Read Identifier), an effective pipeline for real-time identification of species from high-throughput sequencing reads in Metagenomics and clinical diagnostic assays. This pipeline is based on generating k-mers from the short reads and searching the existence of DNA signatures in the Reads k-mers, by using Apache Hive data-warehousing. RkmerG (Read k-mers Generator) is a software program presented in this paper, for producing k-mers of the short reads, in order to use in the pipeline. The purpose of this study is to identify the species in a sample, directly from the reads without assembling and alignment.

  15. MetaCluster 5.0: a two-round binning approach for metagenomic data for low-abundance species in a noisy sample

    Science.gov (United States)

    Wang, Yi; Leung, Henry C.M.; Yiu, S.M.; Chin, Francis Y.L.

    2012-01-01

    Motivation: Metagenomic binning remains an important topic in metagenomic analysis. Existing unsupervised binning methods for next-generation sequencing (NGS) reads do not perform well on (i) samples with low-abundance species or (ii) samples (even with high abundance) when there are many extremely low-abundance species. These two problems are common for real metagenomic datasets. Binning methods that can solve these problems are desirable. Results: We proposed a two-round binning method (MetaCluster 5.0) that aims at identifying both low-abundance and high-abundance species in the presence of a large amount of noise due to many extremely low-abundance species. In summary, MetaCluster 5.0 uses a filtering strategy to remove noise from the extremely low-abundance species. It separate reads of high-abundance species from those of low-abundance species in two different rounds. To overcome the issue of low coverage for low-abundance species, multiple w values are used to group reads with overlapping w-mers, whereas reads from high-abundance species are grouped with high confidence based on a large w and then binning expands to low-abundance species using a relaxed (shorter) w. Compared to the recent tools, TOSS and MetaCluster 4.0, MetaCluster 5.0 can find more species (especially those with low abundance of say 6× to 10×) and can achieve better sensitivity and specificity using less memory and running time. Availability: http://i.cs.hku.hk/~alse/MetaCluster/ Contact: chin@cs.hku.hk PMID:22962452

  16. Metagenomics: An Application Based Perspective

    OpenAIRE

    Yasir Bashir; Salam Pradeep Singh; Bolin Kumar Konwar

    2014-01-01

    Metagenomics deals with the isolation of genetic material directly recovered from environmental samples. Metagenomics as an approach has emerged over the past two decades to elucidate a host of microbial communities inhabiting a specific niche with the goal of understanding their genetic diversity, population structure, and ecological role played by them. A number of new and novel molecules with significant functionalities and applications have been identified through this approach. In fact, ...

  17. Legionella detection from clinical and environmental samples

    Directory of Open Access Journals (Sweden)

    Maria Luisa Ricci

    2004-12-01

    Full Text Available

    Several methods are used for diagnosis of legionellosis: culture, urinary antigen detection, serologic tests, PCR etc., characterised by different level of specificity, sensitivity and rapidity. Microbiological diagnosis is an essential tool to for the prompt adoption of an adequate antimicrobial therapy and to understand the real diffusion of legionellosis.

    Likewise, the environmental samples analysis allows us to know the distribution of the Legionella in the environment and to detect the origin of the infection during an outbreak by comparing clinical and environmental strains.

  18. Beyond biodiversity: fish metagenomes.

    Directory of Open Access Journals (Sweden)

    Alba Ardura

    Full Text Available Biodiversity and intra-specific genetic diversity are interrelated and determine the potential of a community to survive and evolve. Both are considered together in Prokaryote communities treated as metagenomes or ensembles of functional variants beyond species limits.Many factors alter biodiversity in higher Eukaryote communities, and human exploitation can be one of the most important for some groups of plants and animals. For example, fisheries can modify both biodiversity and genetic diversity (intra specific. Intra-specific diversity can be drastically altered by overfishing. Intense fishing pressure on one stock may imply extinction of some genetic variants and subsequent loss of intra-specific diversity. The objective of this study was to apply a metagenome approach to fish communities and explore its value for rapid evaluation of biodiversity and genetic diversity at community level. Here we have applied the metagenome approach employing the barcoding target gene coi as a model sequence in catch from four very different fish assemblages exploited by fisheries: freshwater communities from the Amazon River and northern Spanish rivers, and marine communities from the Cantabric and Mediterranean seas.Treating all sequences obtained from each regional catch as a biological unit (exploited community we found that metagenomic diversity indices of the Amazonian catch sample here examined were lower than expected. Reduced diversity could be explained, at least partially, by overexploitation of the fish community that had been independently estimated by other methods.We propose using a metagenome approach for estimating diversity in Eukaryote communities and early evaluating genetic variation losses at multi-species level.

  19. Marine metagenomics as a source for bioprospecting

    KAUST Repository

    Kodzius, Rimantas

    2015-08-12

    This review summarizes usage of genome-editing technologies for metagenomic studies; these studies are used to retrieve and modify valuable microorganisms for production, particularly in marine metagenomics. Organisms may be cultivable or uncultivable. Metagenomics is providing especially valuable information for uncultivable samples. The novel genes, pathways and genomes can be deducted. Therefore, metagenomics, particularly genome engineering and system biology, allows for the enhancement of biological and chemical producers and the creation of novel bioresources. With natural resources rapidly depleting, genomics may be an effective way to efficiently produce quantities of known and novel foods, livestock feed, fuels, pharmaceuticals and fine or bulk chemicals.

  20. Marine metagenomics as a source for bioprospecting.

    Science.gov (United States)

    Kodzius, Rimantas; Gojobori, Takashi

    2015-12-01

    This review summarizes usage of genome-editing technologies for metagenomic studies; these studies are used to retrieve and modify valuable microorganisms for production, particularly in marine metagenomics. Organisms may be cultivable or uncultivable. Metagenomics is providing especially valuable information for uncultivable samples. The novel genes, pathways and genomes can be deducted. Therefore, metagenomics, particularly genome engineering and system biology, allows for the enhancement of biological and chemical producers and the creation of novel bioresources. With natural resources rapidly depleting, genomics may be an effective way to efficiently produce quantities of known and novel foods, livestock feed, fuels, pharmaceuticals and fine or bulk chemicals. PMID:26204808

  1. Marine metagenomics as a source for bioprospecting.

    Science.gov (United States)

    Kodzius, Rimantas; Gojobori, Takashi

    2015-12-01

    This review summarizes usage of genome-editing technologies for metagenomic studies; these studies are used to retrieve and modify valuable microorganisms for production, particularly in marine metagenomics. Organisms may be cultivable or uncultivable. Metagenomics is providing especially valuable information for uncultivable samples. The novel genes, pathways and genomes can be deducted. Therefore, metagenomics, particularly genome engineering and system biology, allows for the enhancement of biological and chemical producers and the creation of novel bioresources. With natural resources rapidly depleting, genomics may be an effective way to efficiently produce quantities of known and novel foods, livestock feed, fuels, pharmaceuticals and fine or bulk chemicals.

  2. A primer on metagenomics.

    OpenAIRE

    Wooley, John C.; Adam Godzik; Iddo Friedberg

    2010-01-01

    Metagenomics is a discipline that enables the genomic study of uncultured microorganisms. Faster, cheaper sequencing technologies and the ability to sequence uncultured microbes sampled directly from their habitats are expanding and transforming our view of the microbial world. Distilling meaningful information from the millions of new genomic sequences presents a serious challenge to bioinformaticians. In cultured microbes, the genomic data come from a single clone, making sequence assembly ...

  3. Metagenomic survey of methanesulfonic acid (MSA) catabolic genes in an Atlantic Ocean surface water sample and in a partial enrichment

    Science.gov (United States)

    Henriques, Ana C.; Azevedo, Rui M.S.

    2016-01-01

    Methanesulfonic acid (MSA) is a relevant intermediate of the biogeochemical cycle of sulfur and environmental microorganisms assume an important role in the mineralization of this compound. Several methylotrophic bacterial strains able to grow on MSA have been isolated from soil or marine water and two conserved operons, msmABCD coding for MSA monooxygenase and msmEFGH coding for a transport system, have been repeatedly encountered in most of these strains. Homologous sequences have also been amplified directly from the environment or observed in marine metagenomic data, but these showed a base composition (G + C content) very different from their counterparts from cultivated bacteria. The aim of this study was to understand which microorganisms within the coastal surface oceanic microflora responded to MSA as a nutrient and how the community evolved in the early phases of an enrichment by means of metagenome and gene-targeted amplicon sequencing. From the phylogenetic point of view, the community shifted significantly with the disappearance of all signals related to the Archaea, the Pelagibacteraceae and phylum SAR406, and the increase in methylotroph-harboring taxa, accompanied by other groups so far not known to comprise methylotrophs such as the Hyphomonadaceae. At the functional level, the abundance of several genes related to sulfur metabolism and methylotrophy increased during the enrichment and the allelic distribution of gene msmA diagnostic for MSA monooxygenase altered considerably. Even more dramatic was the disappearance of MSA import-related gene msmE, which suggests that alternative transporters must be present in the enriched community and illustrate the inadequacy of msmE as an ecofunctional marker for MSA degradation at sea. PMID:27761315

  4. Construction and screening of marine metagenomic libraries

    DEFF Research Database (Denmark)

    Weiland, Nancy; Löscher, Carolin; Metzger, Rebekka;

    2010-01-01

    microbial consortia on marine tissues of multicellular organisms are rich sources for isolating novel bioactive compounds and genes. Here we describe the sampling, construction of large-insert metagenomic libraries from marine habitats and exemplarily one function based screen of metagenomic clones....

  5. Metagenomics using next-generation sequencing.

    Science.gov (United States)

    Bragg, Lauren; Tyson, Gene W

    2014-01-01

    Traditionally, microbial genome sequencing has been restricted to the small number of species that can be grown in pure culture. The progressive development of culture-independent methods over the last 15 years now allows researchers to sequence microbial communities directly from environmental samples. This approach is commonly referred to as "metagenomics" or "community genomics". However, the term metagenomics is applied liberally in the literature to describe any culture-independent analysis of microbial communities. Here, we define metagenomics as shotgun ("random") sequencing of the genomic DNA of a sample taken directly from the environment. The metagenome can be thought of as a sampling of the collective genome of the microbial community. We outline the considerations and analyses that should be undertaken to ensure the success of a metagenomic sequencing project, including the choice of sequencing platform and methods for assembly, binning, annotation, and comparative analysis.

  6. A Delphi Technology Foresight Study: Mapping Social Construction of Scientific Evidence on Metagenomics Tests for Water Safety.

    Directory of Open Access Journals (Sweden)

    Stanislav Birko

    Full Text Available Access to clean water is a grand challenge in the 21st century. Water safety testing for pathogens currently depends on surrogate measures such as fecal indicator bacteria (e.g., E. coli. Metagenomics concerns high-throughput, culture-independent, unbiased shotgun sequencing of DNA from environmental samples that might transform water safety by detecting waterborne pathogens directly instead of their surrogates. Yet emerging innovations such as metagenomics are often fiercely contested. Innovations are subject to shaping/construction not only by technology but also social systems/values in which they are embedded, such as experts' attitudes towards new scientific evidence. We conducted a classic three-round Delphi survey, comprised of 107 questions. A multidisciplinary expert panel (n = 24 representing the continuum of discovery scientists and policymakers evaluated the emergence of metagenomics tests. To the best of our knowledge, we report here the first Delphi foresight study of experts' attitudes on (1 the top 10 priority evidentiary criteria for adoption of metagenomics tests for water safety, (2 the specific issues critical to governance of metagenomics innovation trajectory where there is consensus or dissensus among experts, (3 the anticipated time lapse from discovery to practice of metagenomics tests, and (4 the role and timing of public engagement in development of metagenomics tests. The ability of a test to distinguish between harmful and benign waterborne organisms, analytical/clinical sensitivity, and reproducibility were the top three evidentiary criteria for adoption of metagenomics. Experts agree that metagenomic testing will provide novel information but there is dissensus on whether metagenomics will replace the current water safety testing methods or impact the public health end points (e.g., reduction in boil water advisories. Interestingly, experts view the publics relevant in a "downstream capacity" for adoption of

  7. A Delphi Technology Foresight Study: Mapping Social Construction of Scientific Evidence on Metagenomics Tests for Water Safety.

    Science.gov (United States)

    Birko, Stanislav; Dove, Edward S; Özdemir, Vural

    2015-01-01

    Access to clean water is a grand challenge in the 21st century. Water safety testing for pathogens currently depends on surrogate measures such as fecal indicator bacteria (e.g., E. coli). Metagenomics concerns high-throughput, culture-independent, unbiased shotgun sequencing of DNA from environmental samples that might transform water safety by detecting waterborne pathogens directly instead of their surrogates. Yet emerging innovations such as metagenomics are often fiercely contested. Innovations are subject to shaping/construction not only by technology but also social systems/values in which they are embedded, such as experts' attitudes towards new scientific evidence. We conducted a classic three-round Delphi survey, comprised of 107 questions. A multidisciplinary expert panel (n = 24) representing the continuum of discovery scientists and policymakers evaluated the emergence of metagenomics tests. To the best of our knowledge, we report here the first Delphi foresight study of experts' attitudes on (1) the top 10 priority evidentiary criteria for adoption of metagenomics tests for water safety, (2) the specific issues critical to governance of metagenomics innovation trajectory where there is consensus or dissensus among experts, (3) the anticipated time lapse from discovery to practice of metagenomics tests, and (4) the role and timing of public engagement in development of metagenomics tests. The ability of a test to distinguish between harmful and benign waterborne organisms, analytical/clinical sensitivity, and reproducibility were the top three evidentiary criteria for adoption of metagenomics. Experts agree that metagenomic testing will provide novel information but there is dissensus on whether metagenomics will replace the current water safety testing methods or impact the public health end points (e.g., reduction in boil water advisories). Interestingly, experts view the publics relevant in a "downstream capacity" for adoption of metagenomics rather

  8. Exploring neighborhoods in the metagenome universe.

    Science.gov (United States)

    Aßhauer, Kathrin P; Klingenberg, Heiner; Lingner, Thomas; Meinicke, Peter

    2014-07-14

    The variety of metagenomes in current databases provides a rapidly growing source of information for comparative studies. However, the quantity and quality of supplementary metadata is still lagging behind. It is therefore important to be able to identify related metagenomes by means of the available sequence data alone. We have studied efficient sequence-based methods for large-scale identification of similar metagenomes within a database retrieval context. In a broad comparison of different profiling methods we found that vector-based distance measures are well-suitable for the detection of metagenomic neighbors. Our evaluation on more than 1700 publicly available metagenomes indicates that for a query metagenome from a particular habitat on average nine out of ten nearest neighbors represent the same habitat category independent of the utilized profiling method or distance measure. While for well-defined labels a neighborhood accuracy of 100% can be achieved, in general the neighbor detection is severely affected by a natural overlap of manually annotated categories. In addition, we present results of a novel visualization method that is able to reflect the similarity of metagenomes in a 2D scatter plot. The visualization method shows a similarly high accuracy in the reduced space as compared with the high-dimensional profile space. Our study suggests that for inspection of metagenome neighborhoods the profiling methods and distance measures can be chosen to provide a convenient interpretation of results in terms of the underlying features. Furthermore, supplementary metadata of metagenome samples in the future needs to comply with readily available ontologies for fine-grained and standardized annotation. To make profile-based k-nearest-neighbor search and the 2D-visualization of the metagenome universe available to the research community, we included the proposed methods in our CoMet-Universe server for comparative metagenome analysis.

  9. Exploring the viral world through metagenomics.

    Science.gov (United States)

    Rosario, Karyna; Breitbart, Mya

    2011-10-01

    Viral metagenomics, or shotgun sequencing of purified viral particles, has revolutionized the field of environmental virology by allowing the exploration of viral communities in a variety of sample types throughout the biosphere. The introduction of viral metagenomics has demonstrated that dominant viruses in environmental communities are not well-represented by the cultured viruses in existing sequence databases. Viral metagenomic studies have provided insights into viral ecology by elucidating the genetic potential, community structure, and biogeography of environmental viruses. In addition, viral metagenomics has expanded current knowledge of virus-host interactions by uncovering genes that may allow viruses to manipulate their hosts in unexpected ways. The intrinsic potential for virus discovery through viral metagenomics can help advance a wide array of disciplines including evolutionary biology, pathogen surveillance, and biotechnology.

  10. Metagenomics: An Application Based Perspective

    Directory of Open Access Journals (Sweden)

    Yasir Bashir

    2014-01-01

    Full Text Available Metagenomics deals with the isolation of genetic material directly recovered from environmental samples. Metagenomics as an approach has emerged over the past two decades to elucidate a host of microbial communities inhabiting a specific niche with the goal of understanding their genetic diversity, population structure, and ecological role played by them. A number of new and novel molecules with significant functionalities and applications have been identified through this approach. In fact, many investigators are engaged in this field to unlock the untapped genetic resources with funding from governments sector. The sustainable economic future of modern industrialized societies requires the development of novel molecules, enzymes, processes, products, and applications. Metagenomics can also be applied to solve practical challenges in the field of medicine, agriculture, sustainability, and ecology. Metagenomics promises to provide new molecules and novel enzymes with diverse functions and enhanced features compared to the enzymes from the culturable microorganisms. Besides the application of metagenomics for unlocking novel biocatalysts from nature, it also has found applications in fields as diverse as bioremediation, personalized medicine, xenobiotic metabolism, and so forth.

  11. Rapid 16S rRNA next-generation sequencing of polymicrobial clinical samples for diagnosis of complex bacterial infections.

    Directory of Open Access Journals (Sweden)

    Stephen J Salipante

    Full Text Available Classifying individual bacterial species comprising complex, polymicrobial patient specimens remains a challenge for culture-based and molecular microbiology techniques in common clinical use. We therefore adapted practices from metagenomics research to rapidly catalog the bacterial composition of clinical specimens directly from patients, without need for prior culture. We have combined a semiconductor deep sequencing protocol that produces reads spanning 16S ribosomal RNA gene variable regions 1 and 2 (∼360 bp with a de-noising pipeline that significantly improves the fraction of error-free sequences. The resulting sequences can be used to perform accurate genus- or species-level taxonomic assignment. We explore the microbial composition of challenging, heterogeneous clinical specimens by deep sequencing, culture-based strain typing, and Sanger sequencing of bulk PCR product. We report that deep sequencing can catalog bacterial species in mixed specimens from which usable data cannot be obtained by conventional clinical methods. Deep sequencing a collection of sputum samples from cystic fibrosis (CF patients reveals well-described CF pathogens in specimens where they were not detected by standard clinical culture methods, especially for low-prevalence or fastidious bacteria. We also found that sputa submitted for CF diagnostic workup can be divided into a limited number of groups based on the phylogenetic composition of the airway microbiota, suggesting that metagenomic profiling may prove useful as a clinical diagnostic strategy in the future. The described method is sufficiently rapid (theoretically compatible with same-day turnaround times and inexpensive for routine clinical use.

  12. Swine Fecal Metagenomics

    Science.gov (United States)

    Metagenomic approaches are providing rapid and more robust means to investigate the composition and functional genetic potential of complex microbial communities. In this study, we utilized a metagenomic approach to further understand the functional diversity of the swine gut. To...

  13. Bioprospecting metagenomes: glycosyl hydrolases for converting biomass.

    Science.gov (United States)

    Li, Luen-Luen; McCorkle, Sean R; Monchy, Sebastien; Taghavi, Safiyh; van der Lelie, Daniel

    2009-01-01

    Throughout immeasurable time, microorganisms evolved and accumulated remarkable physiological and functional heterogeneity, and now constitute the major reserve for genetic diversity on earth. Using metagenomics, namely genetic material recovered directly from environmental samples, this biogenetic diversification can be accessed without the need to cultivate cells. Accordingly, microbial communities and their metagenomes, isolated from biotopes with high turnover rates of recalcitrant biomass, such as lignocellulosic plant cell walls, have become a major resource for bioprospecting; furthermore, this material is a major asset in the search for new biocatalytics (enzymes) for various industrial processes, including the production of biofuels from plant feedstocks. However, despite the contributions from metagenomics technologies consequent upon the discovery of novel enzymes, this relatively new enterprise requires major improvements. In this review, we compare function-based metagenome screening and sequence-based metagenome data mining, discussing the advantages and limitations of both methods. We also describe the unusual enzymes discovered via metagenomics approaches, and discuss the future prospects for metagenome technologies. PMID:19450243

  14. Bioprospecting metagenomes: glycosyl hydrolases for converting biomass

    Directory of Open Access Journals (Sweden)

    Monchy Sebastien

    2009-05-01

    Full Text Available Abstract Throughout immeasurable time, microorganisms evolved and accumulated remarkable physiological and functional heterogeneity, and now constitute the major reserve for genetic diversity on earth. Using metagenomics, namely genetic material recovered directly from environmental samples, this biogenetic diversification can be accessed without the need to cultivate cells. Accordingly, microbial communities and their metagenomes, isolated from biotopes with high turnover rates of recalcitrant biomass, such as lignocellulosic plant cell walls, have become a major resource for bioprospecting; furthermore, this material is a major asset in the search for new biocatalytics (enzymes for various industrial processes, including the production of biofuels from plant feedstocks. However, despite the contributions from metagenomics technologies consequent upon the discovery of novel enzymes, this relatively new enterprise requires major improvements. In this review, we compare function-based metagenome screening and sequence-based metagenome data mining, discussing the advantages and limitations of both methods. We also describe the unusual enzymes discovered via metagenomics approaches, and discuss the future prospects for metagenome technologies.

  15. Sample size determination in clinical trials with multiple endpoints

    CERN Document Server

    Sozu, Takashi; Hamasaki, Toshimitsu; Evans, Scott R

    2015-01-01

    This book integrates recent methodological developments for calculating the sample size and power in trials with more than one endpoint considered as multiple primary or co-primary, offering an important reference work for statisticians working in this area. The determination of sample size and the evaluation of power are fundamental and critical elements in the design of clinical trials. If the sample size is too small, important effects may go unnoticed; if the sample size is too large, it represents a waste of resources and unethically puts more participants at risk than necessary. Recently many clinical trials have been designed with more than one endpoint considered as multiple primary or co-primary, creating a need for new approaches to the design and analysis of these clinical trials. The book focuses on the evaluation of power and sample size determination when comparing the effects of two interventions in superiority clinical trials with multiple endpoints. Methods for sample size calculation in clin...

  16. A catalog of the mouse gut metagenome

    DEFF Research Database (Denmark)

    Xiao, Liang; Feng, Qiang; Liang, Suisha;

    2015-01-01

    We established a catalog of the mouse gut metagenome comprising ∼2.6 million nonredundant genes by sequencing DNA from fecal samples of 184 mice. To secure high microbiome diversity, we used mouse strains of diverse genetic backgrounds, from different providers, kept in different housing laborato......We established a catalog of the mouse gut metagenome comprising ∼2.6 million nonredundant genes by sequencing DNA from fecal samples of 184 mice. To secure high microbiome diversity, we used mouse strains of diverse genetic backgrounds, from different providers, kept in different housing...... laboratories and fed either a low-fat or high-fat diet. Similar to the human gut microbiome, >99% of the cataloged genes are bacterial. We identified 541 metagenomic species and defined a core set of 26 metagenomic species found in 95% of the mice. The mouse gut microbiome is functionally similar to its human...

  17. [Pathology and viral metagenomics, a recent history].

    Science.gov (United States)

    Bernardo, Pauline; Albina, Emmanuel; Eloit, Marc; Roumagnac, Philippe

    2013-05-01

    Human, animal and plant viral diseases have greatly benefited from recent metagenomics developments. Viral metagenomics is a culture-independent approach used to investigate the complete viral genetic populations of a sample. During the last decade, metagenomics concepts and techniques that were first used by ecologists progressively spread into the scientific field of viral pathology. The sample, which was first for ecologists a fraction of ecosystem, became for pathologists an organism that hosts millions of microbes and viruses. This new approach, providing without a priori high resolution qualitative and quantitative data on the viral diversity, is now revolutionizing the way pathologists decipher viral diseases. This review describes the very last improvements of the high throughput next generation sequencing methods and discusses the applications of viral metagenomics in viral pathology, including discovery of novel viruses, viral surveillance and diagnostic, large-scale molecular epidemiology, and viral evolution.

  18. Metagenomics: Facts and Artifacts, and Computational Challenges*

    OpenAIRE

    Wooley, John C.; Ye, Yuzhen

    2009-01-01

    Metagenomics is the study of microbial communities sampled directly from their natural environment, without prior culturing. By enabling an analysis of populations including many (so-far) unculturable and often unknown microbes, metagenomics is revolutionizing the field of microbiology, and has excited researchers in many disciplines that could benefit from the study of environmental microbes, including those in ecology, environmental sciences, and biomedicine. Specific computational and stat...

  19. Toward Accurate and Quantitative Comparative Metagenomics

    Science.gov (United States)

    Nayfach, Stephen; Pollard, Katherine S.

    2016-01-01

    Shotgun metagenomics and computational analysis are used to compare the taxonomic and functional profiles of microbial communities. Leveraging this approach to understand roles of microbes in human biology and other environments requires quantitative data summaries whose values are comparable across samples and studies. Comparability is currently hampered by the use of abundance statistics that do not estimate a meaningful parameter of the microbial community and biases introduced by experimental protocols and data-cleaning approaches. Addressing these challenges, along with improving study design, data access, metadata standardization, and analysis tools, will enable accurate comparative metagenomics. We envision a future in which microbiome studies are replicable and new metagenomes are easily and rapidly integrated with existing data. Only then can the potential of metagenomics for predictive ecological modeling, well-powered association studies, and effective microbiome medicine be fully realized. PMID:27565341

  20. Toward Accurate and Quantitative Comparative Metagenomics.

    Science.gov (United States)

    Nayfach, Stephen; Pollard, Katherine S

    2016-08-25

    Shotgun metagenomics and computational analysis are used to compare the taxonomic and functional profiles of microbial communities. Leveraging this approach to understand roles of microbes in human biology and other environments requires quantitative data summaries whose values are comparable across samples and studies. Comparability is currently hampered by the use of abundance statistics that do not estimate a meaningful parameter of the microbial community and biases introduced by experimental protocols and data-cleaning approaches. Addressing these challenges, along with improving study design, data access, metadata standardization, and analysis tools, will enable accurate comparative metagenomics. We envision a future in which microbiome studies are replicable and new metagenomes are easily and rapidly integrated with existing data. Only then can the potential of metagenomics for predictive ecological modeling, well-powered association studies, and effective microbiome medicine be fully realized. PMID:27565341

  1. Comparative metagenomics of the Red Sea

    KAUST Repository

    Mineta, Katsuhiko

    2016-01-26

    Metagenome produces a tremendous amount of data that comes from the organisms living in the environments. This big data enables us to examine not only microbial genes but also the community structure, interaction and adaptation mechanisms at the specific location and condition. The Red Sea has several unique characteristics such as high salinity, high temperature and low nutrition. These features must contribute to form the unique microbial community during the evolutionary process. Since 2014, we started monthly samplings of the metagenomes in the Red Sea under KAUST-CCF project. In collaboration with Kitasato University, we also collected the metagenome data from the ocean in Japan, which shows contrasting features to the Red Sea. Therefore, the comparative metagenomics of those data provides a comprehensive view of the Red Sea microbes, leading to identify key microbes, genes and networks related to those environmental differences.

  2. Analysis and comparison of very large metagenomes with fast clustering and functional annotation

    Directory of Open Access Journals (Sweden)

    Li Weizhong

    2009-10-01

    Full Text Available Abstract Background The remarkable advance of metagenomics presents significant new challenges in data analysis. Metagenomic datasets (metagenomes are large collections of sequencing reads from anonymous species within particular environments. Computational analyses for very large metagenomes are extremely time-consuming, and there are often many novel sequences in these metagenomes that are not fully utilized. The number of available metagenomes is rapidly increasing, so fast and efficient metagenome comparison methods are in great demand. Results The new metagenomic data analysis method Rapid Analysis of Multiple Metagenomes with a Clustering and Annotation Pipeline (RAMMCAP was developed using an ultra-fast sequence clustering algorithm, fast protein family annotation tools, and a novel statistical metagenome comparison method that employs a unique graphic interface. RAMMCAP processes extremely large datasets with only moderate computational effort. It identifies raw read clusters and protein clusters that may include novel gene families, and compares metagenomes using clusters or functional annotations calculated by RAMMCAP. In this study, RAMMCAP was applied to the two largest available metagenomic collections, the "Global Ocean Sampling" and the "Metagenomic Profiling of Nine Biomes". Conclusion RAMMCAP is a very fast method that can cluster and annotate one million metagenomic reads in only hundreds of CPU hours. It is available from http://tools.camera.calit2.net/camera/rammcap/.

  3. A microRNA isolation method from clinical samples

    Directory of Open Access Journals (Sweden)

    Sepideh Zununi Vahed

    2016-03-01

    Conclusion: The current isolation method can be applied for most clinical samples including cells, formalin-fixed and paraffin-embedded (FFPE tissues and even body fluids with a wide applicability in molecular biology investigations.

  4. An Experimental Metagenome Data Management and AnalysisSystem

    Energy Technology Data Exchange (ETDEWEB)

    Markowitz, Victor M.; Korzeniewski, Frank; Palaniappan, Krishna; Szeto, Ernest; Ivanova, Natalia N.; Kyrpides, Nikos C.; Hugenholtz, Philip

    2006-03-01

    The application of shotgun sequencing to environmental samples has revealed a new universe of microbial community genomes (metagenomes) involving previously uncultured organisms. Metagenome analysis, which is expected to provide a comprehensive picture of the gene functions and metabolic capacity of microbial community, needs to be conducted in the context of a comprehensive data management and analysis system. We present in this paper IMG/M, an experimental metagenome data management and analysis system that is based on the Integrated Microbial Genomes (IMG) system. IMG/M provides tools and viewers for analyzing both metagenomes and isolate genomes individually or in a comparative context.

  5. Bacteriophages in clinical samples can interfere with microbiological diagnostic tools.

    Science.gov (United States)

    Brown-Jaque, Maryury; Muniesa, Maite; Navarro, Ferran

    2016-09-09

    Bacteriophages are viruses that infect bacteria, and they are found everywhere their bacterial hosts are present, including the human body. To explore the presence of phages in clinical samples, we assessed 65 clinical samples (blood, ascitic fluid, urine, cerebrospinal fluid, and serum). Infectious tailed phages were detected in >45% of ascitic fluid and urine samples. Three examples of phage interference with bacterial isolation were observed. Phages prevented the confluent bacterial growth required for an antibiogram assay when the inoculum was taken from an agar plate containing lysis plaques, but not when taken from a single colony in a phage-free area. In addition, bacteria were isolated directly from ascitic fluid, but not after liquid enrichment culture of the same samples, since phage propagation lysed the bacteria. Lastly, Gram-negative bacilli observed in a urine sample did not grow on agar plates due to the high densities of infectious phages in the sample.

  6. Bacteriophages in clinical samples can interfere with microbiological diagnostic tools

    Science.gov (United States)

    Brown-Jaque, Maryury; Muniesa, Maite; Navarro, Ferran

    2016-01-01

    Bacteriophages are viruses that infect bacteria, and they are found everywhere their bacterial hosts are present, including the human body. To explore the presence of phages in clinical samples, we assessed 65 clinical samples (blood, ascitic fluid, urine, cerebrospinal fluid, and serum). Infectious tailed phages were detected in >45% of ascitic fluid and urine samples. Three examples of phage interference with bacterial isolation were observed. Phages prevented the confluent bacterial growth required for an antibiogram assay when the inoculum was taken from an agar plate containing lysis plaques, but not when taken from a single colony in a phage-free area. In addition, bacteria were isolated directly from ascitic fluid, but not after liquid enrichment culture of the same samples, since phage propagation lysed the bacteria. Lastly, Gram-negative bacilli observed in a urine sample did not grow on agar plates due to the high densities of infectious phages in the sample. PMID:27609086

  7. Unbiased Detection of Respiratory Viruses by Use of RNA Sequencing-Based Metagenomics: a Systematic Comparison to a Commercial PCR Panel.

    Science.gov (United States)

    Graf, Erin H; Simmon, Keith E; Tardif, Keith D; Hymas, Weston; Flygare, Steven; Eilbeck, Karen; Yandell, Mark; Schlaberg, Robert

    2016-04-01

    Current infectious disease molecular tests are largely pathogen specific, requiring test selection based on the patient's symptoms. For many syndromes caused by a large number of viral, bacterial, or fungal pathogens, such as respiratory tract infections, this necessitates large panels of tests and has limited yield. In contrast, next-generation sequencing-based metagenomics can be used for unbiased detection of any expected or unexpected pathogen. However, barriers for its diagnostic implementation include incomplete understanding of analytical performance and complexity of sequence data analysis. We compared detection of known respiratory virus-positive (n= 42) and unselected (n= 67) pediatric nasopharyngeal swabs using an RNA sequencing (RNA-seq)-based metagenomics approach and Taxonomer, an ultrarapid, interactive, web-based metagenomics data analysis tool, with an FDA-cleared respiratory virus panel (RVP; GenMark eSensor). Untargeted metagenomics detected 86% of known respiratory virus infections, and additional PCR testing confirmed RVP results for only 2 (33%) of the discordant samples. In unselected samples, untargeted metagenomics had excellent agreement with the RVP (93%). In addition, untargeted metagenomics detected an additional 12 viruses that were either not targeted by the RVP or missed due to highly divergent genome sequences. Normalized viral read counts for untargeted metagenomics correlated with viral burden determined by quantitative PCR and showed high intrarun and interrun reproducibility. Partial or full-length viral genome sequences were generated in 86% of RNA-seq-positive samples, allowing assessment of antiviral resistance, strain-level typing, and phylogenetic relatedness. Overall, untargeted metagenomics had high agreement with a sensitive RVP, detected viruses not targeted by the RVP, and yielded epidemiologically and clinically valuable sequence information.

  8. Which Microbial Communities Are Present? Sequence-Based Metagenomics

    Science.gov (United States)

    Caffrey, Sean M.

    The use of metagenomic methods that directly sequence environmental samples has revealed the extraordinary microbial diversity missed by traditional culture-based methodologies. Therefore, to develop a complete and representative model of an environment's microbial community and activities, metagenomic analysis is an essential tool.

  9. Metagenomic analysis for detecting pathogens in culture-negative infective endocarditis.

    Science.gov (United States)

    Fukui, Yuto; Aoki, Kotaro; Okuma, Shinnosuke; Sato, Takahiro; Ishii, Yoshikazu; Tateda, Kazuhiro

    2015-12-01

    Pathogen identification is important for proper diagnosis and optimal treatment of infective endocarditis (IE). Blood and valve cultures are the gold standard for detecting pathogens responsible for IE. However, these tests only detect culturable pathogens, and have low sensitivity, especially in patients previously treated with antibiotics. Culture-negative IE is still a major clinical problem and a diagnostic challenge. Recently, metagenomic analysis using next generation sequencing has been used to detect pathogens directly from clinical samples. However, there are very few reports of the use of metagenomic analysis for pathogen identification in culture-negative IE cases and the usefulness of this new method is unknown. Here, we report a case of successful pathogen detection with metagenomic analysis in a patient of culture-negative IE. The patient underwent valve replacement surgery and received antibiotics for 5 weeks and survived. Using metagenomic analysis of resected vegetation, we detected Abiotrophia defectiva, which is often associated with culture-negative IE due to its fastidious growth. This method may be useful for pathogen identification in future cases of culture-negative IE.

  10. Yeasts isolated from clinical samples of AIDS patients

    Directory of Open Access Journals (Sweden)

    Neves Rejane Pereira

    2002-01-01

    Full Text Available In order to investigate yeasts in oropharyngeal secretion, urine, sputum and inguinal scales from AIDS patients, clinical samples were collected from one hundred patients interned in the Infectious and Parasitic Diseases Sector of the Hospital das Clínicas of the Universidade Federal de Pernambuco and in Hospital Universitário Osvaldo Cruz of the Universidade de Pernambuco. Yeasts were isolated from seventy-two out of one hundred and eight clinical samples. The isolated yeasts were: Candida albicans (sixty-two isolates, Candida tropicalis (four isolates, Candida glabrata (two isolates, Candida parapsilosis (two isolates, Candida krusei (one isolate and Trichosporon pullulans (one isolate.

  11. On an Approach to Bayesian Sample Sizing in Clinical Trials

    CERN Document Server

    Muirhead, Robb J

    2012-01-01

    This paper explores an approach to Bayesian sample size determination in clinical trials. The approach falls into the category of what is often called "proper Bayesian", in that it does not mix frequentist concepts with Bayesian ones. A criterion for a "successful trial" is defined in terms of a posterior probability, its probability is assessed using the marginal distribution of the data, and this probability forms the basis for choosing sample sizes. We illustrate with a standard problem in clinical trials, that of establishing superiority of a new drug over a control.

  12. Enhancing metagenomics investigations of microbial interactions with biofilm technology.

    Science.gov (United States)

    McLean, Robert J C; Kakirde, Kavita S

    2013-11-11

    Investigations of microbial ecology and diversity have been greatly enhanced by the application of culture-independent techniques. One such approach, metagenomics, involves sample collections from soil, water, and other environments. Extracted nucleic acids from bulk environmental samples are sequenced and analyzed, which allows microbial interactions to be inferred on the basis of bioinformatics calculations. In most environments, microbial interactions occur predominately in surface-adherent, biofilm communities. In this review, we address metagenomics sampling and biofilm biology, and propose an experimental strategy whereby the resolving power of metagenomics can be enhanced by incorporating a biofilm-enrichment step during sample acquisition.

  13. Shotgun metagenomic data streams: surfing without fear

    Energy Technology Data Exchange (ETDEWEB)

    Berendzen, Joel R [Los Alamos National Laboratory

    2010-12-06

    Timely information about bio-threat prevalence, consequence, propagation, attribution, and mitigation is needed to support decision-making, both routinely and in a crisis. One DNA sequencer can stream 25 Gbp of information per day, but sampling strategies and analysis techniques are needed to turn raw sequencing power into actionable knowledge. Shotgun metagenomics can enable biosurveillance at the level of a single city, hospital, or airplane. Metagenomics characterizes viruses and bacteria from complex environments such as soil, air filters, or sewage. Unlike targeted-primer-based sequencing, shotgun methods are not blind to sequences that are truly novel, and they can measure absolute prevalence. Shotgun metagenomic sampling can be non-invasive, efficient, and inexpensive while being informative. We have developed analysis techniques for shotgun metagenomic sequencing that rely upon phylogenetic signature patterns. They work by indexing local sequence patterns in a manner similar to web search engines. Our methods are laptop-fast and favorable scaling properties ensure they will be sustainable as sequencing methods grow. We show examples of application to soil metagenomic samples.

  14. Genetic analysis of bacteriophages from clinical and environmental samples

    OpenAIRE

    Knapik, Kamila Z.

    2013-01-01

    Bacteriophages, viruses infecting bacteria, are uniformly present in any location where there are high numbers of bacteria, both in the external environment and the human body. Knowledge of their diversity is limited by the difficulty to culture the host species and by the lack of the universal marker gene present in all viruses. Metagenomics is a powerful tool that can be used to analyse viral communities in their natural environments. The aim of this study was to investigate diverse populat...

  15. Functional Metagenome Mining of Soil for a Novel Gentamicin Resistance Gene.

    Science.gov (United States)

    Im, Hyunjoo; Kim, Kyung Mo; Lee, Sang-Heon; Ryu, Choong-Min

    2016-03-01

    Extensive use of antibiotics over recent decades has led to bacterial resistance against antibiotics, including gentamicin, one of the most effective aminoglycosides. The emergence of resistance is problematic for hospitals, since gentamicin is an important broad-spectrum antibiotic for the control of bacterial pathogens in the clinic. Previous study to identify gentamicin resistance genes from environmental samples have been conducted using culture-dependent screening methods. To overcome these limitations, we employed a metagenome-based culture-independent protocol to identify gentamicin resistance genes. Through functional screening of metagenome libraries derived from soil samples, a fosmid clone was selected as it conferred strong gentamicin resistance. To identify a specific functioning gene conferring gentamicin resistance from a selected fosmid clone (35-40 kb), a shot-gun library was constructed and four shot-gun clones (2-3 kb) were selected. Further characterization of these clones revealed that they contained sequences similar to that of the RNA ligase, T4 rnlA that is known as a toxin gene. The overexpression of the rnlA-like gene in Escherichia coli increased gentamicin resistance, indicating that this toxin gene modulates this trait. The results of our metagenome library analysis suggest that the rnlA-like gene may represent a new class of gentamicin resistance genes in pathogenic bacteria. In addition, we demonstrate that the soil metagenome can provide an important resource for the identification of antibiotic resistance genes, which are valuable molecular targets in efforts to overcome antibiotic resistance. PMID:26699755

  16. High throughput whole rumen metagenome profiling using untargeted massively parallel sequencing

    Directory of Open Access Journals (Sweden)

    Ross Elizabeth M

    2012-07-01

    Full Text Available Abstract Background Variation of microorganism communities in the rumen of cattle (Bos taurus is of great interest because of possible links to economically or environmentally important traits, such as feed conversion efficiency or methane emission levels. The resolution of studies investigating this variation may be improved by utilizing untargeted massively parallel sequencing (MPS, that is, sequencing without targeted amplification of genes. The objective of this study was to develop a method which used MPS to generate “rumen metagenome profiles”, and to investigate if these profiles were repeatable among samples taken from the same cow. Given faecal samples are much easier to obtain than rumen fluid samples; we also investigated whether rumen metagenome profiles were predictive of faecal metagenome profiles. Results Rather than focusing on individual organisms within the rumen, our method used MPS data to generate quantitative rumen micro-biome profiles, regardless of taxonomic classifications. The method requires a previously assembled reference metagenome. A number of such reference metagenomes were considered, including two rumen derived metagenomes, a human faecal microflora metagenome and a reference metagenome made up of publically available prokaryote sequences. Sequence reads from each test sample were aligned to these references. The “rumen metagenome profile” was generated from the number of the reads that aligned to each contig in the database. We used this method to test the hypothesis that rumen fluid microbial community profiles vary more between cows than within multiple samples from the same cow. Rumen fluid samples were taken from three cows, at three locations within the rumen. DNA from the samples was sequenced on the Illumina GAIIx. When the reads were aligned to a rumen metagenome reference, the rumen metagenome profiles were repeatable (P  Conclusions We have presented a simple and high throughput method of

  17. Viral metagenomics: are we missing the giants?

    Science.gov (United States)

    Halary, S; Temmam, S; Raoult, D; Desnues, C

    2016-06-01

    Amoeba-infecting giant viruses are recently discovered viruses that have been isolated from diverse environments all around the world. In parallel to isolation efforts, metagenomics confirmed their worldwide distribution from a broad range of environmental and host-associated samples, including humans, depicting them as a major component of eukaryotic viruses in nature and a possible resident of the human/animal virome whose role is still unclear. Nevertheless, metagenomics data about amoeba-infecting giant viruses still remain scarce, mainly because of methodological limitations. Efforts should be pursued both at the metagenomic sample preparation level and on in silico analyses to better understand their roles in the environment and in human/animal health and disease. PMID:26851442

  18. Combining electrochemical sensors with miniaturized sample preparation for rapid detection in clinical samples.

    Science.gov (United States)

    Bunyakul, Natinan; Baeumner, Antje J

    2015-01-01

    Clinical analyses benefit world-wide from rapid and reliable diagnostics tests. New tests are sought with greatest demand not only for new analytes, but also to reduce costs, complexity and lengthy analysis times of current techniques. Among the myriad of possibilities available today to develop new test systems, amperometric biosensors are prominent players-best represented by the ubiquitous amperometric-based glucose sensors. Electrochemical approaches in general require little and often enough only simple hardware components, are rugged and yet provide low limits of detection. They thus offer many of the desirable attributes for point-of-care/point-of-need tests. This review focuses on investigating the important integration of sample preparation with (primarily electrochemical) biosensors. Sample clean up requirements, miniaturized sample preparation strategies, and their potential integration with sensors will be discussed, focusing on clinical sample analyses. PMID:25558994

  19. Learning Adaptive Forecasting Models from Irregularly Sampled Multivariate Clinical Data

    Science.gov (United States)

    Liu, Zitao; Hauskrecht, Milos

    2016-01-01

    Building accurate predictive models of clinical multivariate time series is crucial for understanding of the patient condition, the dynamics of a disease, and clinical decision making. A challenging aspect of this process is that the model should be flexible and adaptive to reflect well patient-specific temporal behaviors and this also in the case when the available patient-specific data are sparse and short span. To address this problem we propose and develop an adaptive two-stage forecasting approach for modeling multivariate, irregularly sampled clinical time series of varying lengths. The proposed model (1) learns the population trend from a collection of time series for past patients; (2) captures individual-specific short-term multivariate variability; and (3) adapts by automatically adjusting its predictions based on new observations. The proposed forecasting model is evaluated on a real-world clinical time series dataset. The results demonstrate the benefits of our approach on the prediction tasks for multivariate, irregularly sampled clinical time series, and show that it can outperform both the population based and patient-specific time series prediction models in terms of prediction accuracy.

  20. Metagenomics and antibiotics.

    Science.gov (United States)

    Garmendia, L; Hernandez, A; Sanchez, M B; Martinez, J L

    2012-07-01

    Most of the bacterial species that form part of the biosphere have never been cultivated. In this situation, a comprehensive study of bacterial communities requires the utilization of non-culture-based methods, which have been named metagenomics. In this paper we review the use of different metagenomic techniques for understanding the effect of antibiotics on microbial communities, to synthesize new antimicrobial compounds and to analyse the distribution of antibiotic resistance genes in different ecosystems. These techniques include functional metagenomics, which serves to find new antibiotics or new antibiotic resistance genes, and descriptive metagenomics, which serves to analyse changes in the composition of the microbiota and to track the presence and abundance of already known antibiotic resistance genes in different ecosystems.

  1. Viral Metagenomics: MetaView Software

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, C; Smith, J

    2007-10-22

    The purpose of this report is to design and develop a tool for analysis of raw sequence read data from viral metagenomics experiments. The tool should compare read sequences of known viral nucleic acid sequence data and enable a user to attempt to determine, with some degree of confidence, what virus groups may be present in the sample. This project was conducted in two phases. In phase 1 we surveyed the literature and examined existing metagenomics tools to educate ourselves and to more precisely define the problem of analyzing raw read data from viral metagenomic experiments. In phase 2 we devised an approach and built a prototype code and database. This code takes viral metagenomic read data in fasta format as input and accesses all complete viral genomes from Kpath for sequence comparison. The system executes at the UNIX command line, producing output that is stored in an Oracle relational database. We provide here a description of the approach we came up with for handling un-assembled, short read data sets from viral metagenomics experiments. We include a discussion of the current MetaView code capabilities and additional functionality that we believe should be added, should additional funding be acquired to continue the work.

  2. Antibiotic Susceptibilities of Acinetobacter Baumanii Strains Isolated from Clinical Samples

    OpenAIRE

    Harun Aðca

    2013-01-01

         Aim :  In this study it was aimed to investigate the antibiotic susceptibilities of Acinetobacter baumanii strains isolated from various clinical samples sent to Tavsanli State Hospital Microbiology Laboratory retrospectively. Material and Method: All of the cultures were examined for the agent and antibiotic susceptibilities. For the identification of bacteria, various chemical tests and BBL Crystal E/NF (Beckton Dickinson, ABD) system was used. Antibiotic susce...

  3. Vancomycin Resistance Pattern of Staphylococcus Aureus among Clinical Samples

    OpenAIRE

    S Saadat; K Solhjoo; A. Kazemi; Erfanian, S. (MSc); Ashrafian, F. (MSc)

    2014-01-01

    Background and Objective: Vancomycin is used for treatment of methicillin-resistant S. Aureus (MRSA) infections; therefore, resistance to this antibiotic is increasing. We aimed to determine the antibiotic resistance pattern and frequency of vancomycin resistant S. Areas (VRSA) strains isolated from clinical samples. Material and Methods: In this cross-sectional study, 100 S. Aureus isolates collected from hospitals in Shiraz during six months, 2012, were identified by biochemical, microbiolo...

  4. PCR Detection of Helicobacter pylori in Clinical Samples

    OpenAIRE

    Rimbara, Emiko; Sasatsu, Masanori; David Y Graham

    2013-01-01

    Helicobacter pylori is an important pathogen whose primary niche is the human stomach. H. pylori is etio-logically associated with gastric inflammation (gastritis), peptic ulcer disease, and gastric cancer. Both noninvasive (e.g., urea breath and stool antigen tests) and invasive (gastric biopsy for histology, culture, or PCR) tests are used for diagnosis. PCR detection of H. pylori has been reported using a variety of clinical samples including gastric biopsy, gastric juice, saliva, dental p...

  5. Metagenomic analysis of viruses associated with field-grown and retail lettuce identifies human and animal viruses.

    Science.gov (United States)

    Aw, Tiong Gim; Wengert, Samantha; Rose, Joan B

    2016-04-16

    The emergence of culture- and sequence-independent metagenomic methods has not only provided great insight into the microbial community structure in a wide range of clinical and environmental samples but has also proven to be powerful tools for pathogen detection. Recent studies of the food microbiome have revealed the vast genetic diversity of bacteria associated with fresh produce. However, no work has been done to apply metagenomic methods to tackle viruses associated with fresh produce for addressing food safety. Thus, there is a little knowledge about the presence and diversity of viruses associated with fresh produce from farm-to-fork. To address this knowledge gap, we assessed viruses on commercial romaine and iceberg lettuces in fields and a produce distribution center using a shotgun metagenomic sequencing targeting both RNA and DNA viruses. Commercial lettuce harbors an immense assemblage of viruses that infect a wide range of hosts. As expected, plant pathogenic viruses dominated these communities. Sequences of rotaviruses and picobirnaviruses were also identified in both field-harvest and retail lettuce samples, suggesting an emerging foodborne transmission threat that has yet to be fully recognized. The identification of human and animal viruses in lettuce samples in the field emphasizes the importance of preventing viral contamination on leafy greens starting at the field. Although there are still some inherent experimental and bioinformatics challenges in applying viral metagenomic approaches for food safety testing, this work will facilitate further application of this unprecedented deep sequencing method to food samples. PMID:26894328

  6. Metagenomic analysis of viruses associated with field-grown and retail lettuce identifies human and animal viruses.

    Science.gov (United States)

    Aw, Tiong Gim; Wengert, Samantha; Rose, Joan B

    2016-04-16

    The emergence of culture- and sequence-independent metagenomic methods has not only provided great insight into the microbial community structure in a wide range of clinical and environmental samples but has also proven to be powerful tools for pathogen detection. Recent studies of the food microbiome have revealed the vast genetic diversity of bacteria associated with fresh produce. However, no work has been done to apply metagenomic methods to tackle viruses associated with fresh produce for addressing food safety. Thus, there is a little knowledge about the presence and diversity of viruses associated with fresh produce from farm-to-fork. To address this knowledge gap, we assessed viruses on commercial romaine and iceberg lettuces in fields and a produce distribution center using a shotgun metagenomic sequencing targeting both RNA and DNA viruses. Commercial lettuce harbors an immense assemblage of viruses that infect a wide range of hosts. As expected, plant pathogenic viruses dominated these communities. Sequences of rotaviruses and picobirnaviruses were also identified in both field-harvest and retail lettuce samples, suggesting an emerging foodborne transmission threat that has yet to be fully recognized. The identification of human and animal viruses in lettuce samples in the field emphasizes the importance of preventing viral contamination on leafy greens starting at the field. Although there are still some inherent experimental and bioinformatics challenges in applying viral metagenomic approaches for food safety testing, this work will facilitate further application of this unprecedented deep sequencing method to food samples.

  7. PCR detection of Helicobacter pylori in clinical samples.

    Science.gov (United States)

    Rimbara, Emiko; Sasatsu, Masanori; Graham, David Y

    2013-01-01

    Helicobacter pylori is an important pathogen whose primary niche is the human stomach. H. pylori is etiologically associated with gastric inflammation (gastritis), peptic ulcer disease, and gastric cancer. Both noninvasive (e.g., urea breath and stool antigen tests) and invasive (gastric biopsy for histology, culture, or PCR) tests are used for diagnosis. PCR detection of H. pylori has been reported using a variety of clinical samples including gastric biopsy, gastric juice, saliva, dental plaque, and stools as well as environmental samples. Whenever possibly, noninvasive tests are preferred over invasive tests. H. pylori are excreted in the stool. Culture from stool is variable whereas stool antigen testing is widely used. Stool consists of a complicated mixture of commensal bacteria and chemicals and often includes inhibitors of PCR. Nevertheless, simple extraction methods are available to efficiently extract DNA from human stools and nested-PCR targeting the 23S rRNA gene have proven to be highly sensitive for the detection of H. pylori. Detection of clarithromycin susceptibility/resistance is important clinically and the mutation of the 23S rRNA gene responsible for resistance can also be detected using stool. This described method can be modified for other clinical samples such as gastric juice or biopsy material. PMID:23104297

  8. FANTOM: Functional and taxonomic analysis of metagenomes

    Directory of Open Access Journals (Sweden)

    Sanli Kemal

    2013-02-01

    Full Text Available Abstract Background Interpretation of quantitative metagenomics data is important for our understanding of ecosystem functioning and assessing differences between various environmental samples. There is a need for an easy to use tool to explore the often complex metagenomics data in taxonomic and functional context. Results Here we introduce FANTOM, a tool that allows for exploratory and comparative analysis of metagenomics abundance data integrated with metadata information and biological databases. Importantly, FANTOM can make use of any hierarchical database and it comes supplied with NCBI taxonomic hierarchies as well as KEGG Orthology, COG, PFAM and TIGRFAM databases. Conclusions The software is implemented in Python, is platform independent, and is available at http://www.sysbio.se/Fantom.

  9. The metagenomic approach and causality in virology

    Directory of Open Access Journals (Sweden)

    Silvana Beres Castrignano

    2015-01-01

    Full Text Available Nowadays, the metagenomic approach has been a very important tool in the discovery of new viruses in environmental and biological samples. Here we discuss how these discoveries may help to elucidate the etiology of diseases and the criteria necessary to establish a causal association between a virus and a disease.

  10. Building on basic metagenomics with complementary technologies

    OpenAIRE

    Warnecke, Falk; Hugenholtz, Philip

    2007-01-01

    Metagenomics, the application of random shotgun sequencing to environmental samples, is a powerful approach for characterizing microbial communities. However, this method only represents the cornerstone of what can be achieved using a range of complementary technologies such as transcriptomics, proteomics, cell sorting and microfluidics. Together, these approaches hold great promise for the study of microbial ecology and evolution.

  11. [Research in metagenomics and its applications in translational medicine].

    Science.gov (United States)

    Jiahuan, Chen; Zheng, Sun; Xiaojun, Wang; Xiaoquan, Su; Kang, Ning

    2015-07-01

    Humans are born with microbiota, which have accompanied us through our life-span. There is an important symbiotic relationship between us and the microbial communities, thus microbial communities are of great importance to our health. All genomic information within this microbiota is referered to as "metagenomics" (also referred to as "human's second genome"). The analysis of high throughput metagenomic data generated from biomedical experiments would provide new approaches for translational research, and it have several applications in clinics. With the help of next generation sequencing technology and the emerging metagenomic approach (analysis of all genomic information in microbiota as a whole), we can overcome the pitfalls of tedious traditional method of isolation and cultivation of single microbial species. The metagenomic approach can also help us to analyze the whole microbial community efficiently and offer deep insights in human-microbe relationships as well as new ideas on many biomedical problems. In this review, we summarize frontiers in metagenomic research, including new concepts and methods. Then, we focus on the applications of metagenomic research in medical researches and clinical applications in recent years, which would clearly show the importance of metagenomic research in the field of translational medicine.

  12. Metagenomic mining for microbiologists.

    Science.gov (United States)

    Delmont, Tom O; Malandain, Cedric; Prestat, Emmanuel; Larose, Catherine; Monier, Jean-Michel; Simonet, Pascal; Vogel, Timothy M

    2011-12-01

    Microbial ecologists can now start digging into the accumulating mountains of metagenomic data to uncover the occurrence of functional genes and their correlations to microbial community members. Limitations and biases in DNA extraction and sequencing technologies impact sequence distributions, and therefore, have to be considered. However, when comparing metagenomes from widely differing environments, these fluctuations have a relatively minor role in microbial community discrimination. As a consequence, any functional gene or species distribution pattern can be compared among metagenomes originating from various environments and projects. In particular, global comparisons would help to define ecosystem specificities, such as involvement and response to climate change (for example, carbon and nitrogen cycle), human health risks (eg, presence of pathogen species, toxin genes and viruses) and biodegradation capacities. Although not all scientists have easy access to high-throughput sequencing technologies, they do have access to the sequences that have been deposited in databases, and therefore, can begin to intensively mine these metagenomic data to generate hypotheses that can be validated experimentally. Information about metabolic functions and microbial species compositions can already be compared among metagenomes from different ecosystems. These comparisons add to our understanding about microbial adaptation and the role of specific microbes in different ecosystems. Concurrent with the rapid growth of sequencing technologies, we have entered a new age of microbial ecology, which will enable researchers to experimentally confirm putative relationships between microbial functions and community structures. PMID:21593798

  13. Cladosporium Species Recovered from Clinical Samples in the United States.

    Science.gov (United States)

    Sandoval-Denis, Marcelo; Sutton, Deanna A; Martin-Vicente, Adela; Cano-Lira, José F; Wiederhold, Nathan; Guarro, Josep; Gené, Josepa

    2015-09-01

    Cladosporium species are ubiquitous, saprobic, dematiaceous fungi, only infrequently associated with human and animal opportunistic infections. We have studied a large set of Cladosporium isolates recovered from clinical samples in the United States to ascertain the predominant species there in light of recent taxonomic changes in this genus and to determine whether some could possibly be rare potential pathogens. A total of 92 isolates were identified using phenotypic and molecular methods, which included sequence analysis of the internal transcribed spacer (ITS) region and a fragment of the large subunit (LSU) of the nuclear ribosomal DNA (rDNA), as well as fragments of the translation elongation factor 1 alpha (EF-1α) and actin (Act) genes. The most frequent species was Cladosporium halotolerans (14.8%), followed by C. tenuissimum (10.2%), C. subuliforme (5.7%), and C. pseudocladosporioides (4.5%). However, 39.8% of the isolates did not correspond to any known species and were deemed to comprise at least 17 new lineages for Cladosporium. The most frequent anatomic site of isolation was the respiratory tract (54.5%), followed by superficial (28.4%) and deep tissues and fluids (14.7%). Species of the two recently described cladosporiumlike genera Toxicocladosporium and Penidiella are reported for the first time from clinical samples. In vitro susceptibility testing of 92 isolates against nine antifungal drugs showed a variety of results but high activity overall for the azoles, echinocandins, and terbinafine.

  14. Metagenomics of extreme environments.

    Science.gov (United States)

    Cowan, D A; Ramond, J-B; Makhalanyane, T P; De Maayer, P

    2015-06-01

    Whether they are exposed to extremes of heat or cold, or buried deep beneath the Earth's surface, microorganisms have an uncanny ability to survive under these conditions. This ability to survive has fascinated scientists for nearly a century, but the recent development of metagenomics and 'omics' tools has allowed us to make huge leaps in understanding the remarkable complexity and versatility of extremophile communities. Here, in the context of the recently developed metagenomic tools, we discuss recent research on the community composition, adaptive strategies and biological functions of extremophiles.

  15. Metagenomic Sequencing of an In Vitro-Simulated Microbial Community

    Energy Technology Data Exchange (ETDEWEB)

    Morgan, Jenna L.; Darling, Aaron E.; Eisen, Jonathan A.

    2009-12-01

    Background: Microbial life dominates the earth, but many species are difficult or even impossible to study under laboratory conditions. Sequencing DNA directly from the environment, a technique commonly referred to as metagenomics, is an important tool for cataloging microbial life. This culture-independent approach involves collecting samples that include microbes in them, extracting DNA from the samples, and sequencing the DNA. A sample may contain many different microorganisms, macroorganisms, and even free-floating environmental DNA. A fundamental challenge in metagenomics has been estimating the abundance of organisms in a sample based on the frequency with which the organism's DNA was observed in reads generated via DNA sequencing. Methodology/Principal Findings: We created mixtures of ten microbial species for which genome sequences are known. Each mixture contained an equal number of cells of each species. We then extracted DNA from the mixtures, sequenced the DNA, and measured the frequency with which genomic regions from each organism was observed in the sequenced DNA. We found that the observed frequency of reads mapping to each organism did not reflect the equal numbers of cells that were known to be included in each mixture. The relative organism abundances varied significantly depending on the DNA extraction and sequencing protocol utilized. Conclusions/Significance: We describe a new data resource for measuring the accuracy of metagenomic binning methods, created by in vitro-simulation of a metagenomic community. Our in vitro simulation can be used to complement previous in silico benchmark studies. In constructing a synthetic community and sequencing its metagenome, we encountered several sources of observation bias that likely affect most metagenomic experiments to date and present challenges for comparative metagenomic studies. DNA preparation methods have a particularly profound effect in our study, implying that samples prepared with

  16. Metagenomic sequencing of an in vitro-simulated microbial community.

    Directory of Open Access Journals (Sweden)

    Jenna L Morgan

    Full Text Available BACKGROUND: Microbial life dominates the earth, but many species are difficult or even impossible to study under laboratory conditions. Sequencing DNA directly from the environment, a technique commonly referred to as metagenomics, is an important tool for cataloging microbial life. This culture-independent approach involves collecting samples that include microbes in them, extracting DNA from the samples, and sequencing the DNA. A sample may contain many different microorganisms, macroorganisms, and even free-floating environmental DNA. A fundamental challenge in metagenomics has been estimating the abundance of organisms in a sample based on the frequency with which the organism's DNA was observed in reads generated via DNA sequencing. METHODOLOGY/PRINCIPAL FINDINGS: We created mixtures of ten microbial species for which genome sequences are known. Each mixture contained an equal number of cells of each species. We then extracted DNA from the mixtures, sequenced the DNA, and measured the frequency with which genomic regions from each organism was observed in the sequenced DNA. We found that the observed frequency of reads mapping to each organism did not reflect the equal numbers of cells that were known to be included in each mixture. The relative organism abundances varied significantly depending on the DNA extraction and sequencing protocol utilized. CONCLUSIONS/SIGNIFICANCE: We describe a new data resource for measuring the accuracy of metagenomic binning methods, created by in vitro-simulation of a metagenomic community. Our in vitro simulation can be used to complement previous in silico benchmark studies. In constructing a synthetic community and sequencing its metagenome, we encountered several sources of observation bias that likely affect most metagenomic experiments to date and present challenges for comparative metagenomic studies. DNA preparation methods have a particularly profound effect in our study, implying that samples

  17. Functional Metagenomics of the Bronchial Microbiome in COPD.

    Directory of Open Access Journals (Sweden)

    Laura Millares

    Full Text Available The course of chronic obstructive pulmonary disease (COPD is frequently aggravated by exacerbations, and changes in the composition and activity of the microbiome may be implicated in their appearance. The aim of this study was to analyse the composition and the gene content of the microbial community in bronchial secretions of COPD patients in both stability and exacerbation. Taxonomic data were obtained by 16S rRNA gene amplification and pyrosequencing, and metabolic information through shotgun metagenomics, using the Metagenomics RAST server (MG-RAST, and the PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States programme, which predict metagenomes from 16S data. Eight severe COPD patients provided good quality sputum samples, and no significant differences in the relative abundance of any phyla and genera were found between stability and exacerbation. Bacterial biodiversity (Chao1 and Shannon indexes did not show statistical differences and beta-diversity analysis (Bray-Curtis dissimilarity index showed a similar microbial composition in the two clinical situations. Four functional categories showed statistically significant differences with MG-RAST at KEGG level 2: in exacerbation, Cell growth and Death and Transport and Catabolism decreased in abundance [1.6 (0.2-2.3 vs 3.6 (3.3-6.9, p = 0.012; and 1.8 (0-3.3 vs 3.6 (1.8-5.1, p = 0.025 respectively], while Cancer and Carbohydrate Metabolism increased [0.8 (0-1.5 vs 0 (0-0.5, p = 0.043; and 7 (6.4-9 vs 5.9 (6.3-6.1, p = 0.012 respectively]. In conclusion, the bronchial microbiome as a whole is not significantly modified when exacerbation symptoms appear in severe COPD patients, but its functional metabolic capabilities show significant changes in several pathways.

  18. Functional Metagenomics of the Bronchial Microbiome in COPD.

    Science.gov (United States)

    Millares, Laura; Pérez-Brocal, Vicente; Ferrari, Rafaela; Gallego, Miguel; Pomares, Xavier; García-Núñez, Marian; Montón, Concepción; Capilla, Silvia; Monsó, Eduard; Moya, Andrés

    2015-01-01

    The course of chronic obstructive pulmonary disease (COPD) is frequently aggravated by exacerbations, and changes in the composition and activity of the microbiome may be implicated in their appearance. The aim of this study was to analyse the composition and the gene content of the microbial community in bronchial secretions of COPD patients in both stability and exacerbation. Taxonomic data were obtained by 16S rRNA gene amplification and pyrosequencing, and metabolic information through shotgun metagenomics, using the Metagenomics RAST server (MG-RAST), and the PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) programme, which predict metagenomes from 16S data. Eight severe COPD patients provided good quality sputum samples, and no significant differences in the relative abundance of any phyla and genera were found between stability and exacerbation. Bacterial biodiversity (Chao1 and Shannon indexes) did not show statistical differences and beta-diversity analysis (Bray-Curtis dissimilarity index) showed a similar microbial composition in the two clinical situations. Four functional categories showed statistically significant differences with MG-RAST at KEGG level 2: in exacerbation, Cell growth and Death and Transport and Catabolism decreased in abundance [1.6 (0.2-2.3) vs 3.6 (3.3-6.9), p = 0.012; and 1.8 (0-3.3) vs 3.6 (1.8-5.1), p = 0.025 respectively], while Cancer and Carbohydrate Metabolism increased [0.8 (0-1.5) vs 0 (0-0.5), p = 0.043; and 7 (6.4-9) vs 5.9 (6.3-6.1), p = 0.012 respectively]. In conclusion, the bronchial microbiome as a whole is not significantly modified when exacerbation symptoms appear in severe COPD patients, but its functional metabolic capabilities show significant changes in several pathways.

  19. MOCAT: A Metagenomics Assembly and Gene Prediction Toolkit

    OpenAIRE

    Kultima, Jens Roat; Sunagawa, Shinichi; Li, Junhua; Chen, Weineng; Chen, Hua; Mende, Daniel R; Arumugam, Manimozhiyan; Pan, Qi; Liu, Binghang; Qin, Junjie; Wang, Jun; Bork, Peer

    2012-01-01

    MOCAT is a highly configurable, modular pipeline for fast, standardized processing of single or paired-end sequencing data generated by the Illumina platform. The pipeline uses state-of-the-art programs to quality control, map, and assemble reads from metagenomic samples sequenced at a depth of several billion base pairs, and predict protein-coding genes on assembled metagenomes. Mapping against reference databases allows for read extraction or removal, as well as abundance calculations. Rele...

  20. Metagenomics for studying unculturable microorganisms: cutting the Gordian knot

    OpenAIRE

    Patrick D Schloss; Handelsman, Jo

    2005-01-01

    More than 99% of prokaryotes in the environment cannot be cultured in the laboratory, a phenomenon that limits our understanding of microbial physiology, genetics, and community ecology. One way around this problem is metagenomics, the culture-independent cloning and analysis of microbial DNA extracted directly from an environmental sample. Recent advances in shotgun sequencing and computational methods for genome assembly have advanced the field of metagenomics to provide glimpses into the l...

  1. Exploration of Metagenome Assemblies with an Interactive Visualization Tool

    Energy Technology Data Exchange (ETDEWEB)

    Cantor, Michael; Nordberg, Henrik; Smirnova, Tatyana; Andersen, Evan; Tringe, Susannah; Hess, Matthias; Dubchak, Inna

    2014-07-09

    Metagenomics, one of the fastest growing areas of modern genomic science, is the genetic profiling of the entire community of microbial organisms present in an environmental sample. Elviz is a web-based tool for the interactive exploration of metagenome assemblies. Elviz can be used with publicly available data sets from the Joint Genome Institute or with custom user-loaded assemblies. Elviz is available at genome.jgi.doe.gov/viz

  2. Social Cognition in a Clinical Sample of Personality Disorder Patients

    Directory of Open Access Journals (Sweden)

    Amparo eRuiz-Tagle

    2015-05-01

    Full Text Available Social cognition was assessed in a clinical sample of Personality Disorder (PD stable patients receiving ambulatory treatment (N=17 and healthy matched controls (N=17 using tests of recognition of emotions in faces and eyes, in a test of social faux pas and in theory of mind stories. Results indicated that when compared with healthy controls, individuals with PD showed a clear tendency to obtain lower scoring in tasks assessing recognition of emotion in faces (T=-2,602, p=0,014, eyes (T=-3,593, p=0,001, TOM stories (T=-4,706, p=0,000 and Faux pas (T=-2,227, p=0,035. In the present pilot study, PD individuals with a normal cognitive efficiency showed an impaired performance at social cognition assessment including emotion recognition and theory of mind.

  3. EBI metagenomics in 2016--an expanding and evolving resource for the analysis and archiving of metagenomic data.

    Science.gov (United States)

    Mitchell, Alex; Bucchini, Francois; Cochrane, Guy; Denise, Hubert; ten Hoopen, Petra; Fraser, Matthew; Pesseat, Sebastien; Potter, Simon; Scheremetjew, Maxim; Sterk, Peter; Finn, Robert D

    2016-01-01

    EBI metagenomics (https://www.ebi.ac.uk/metagenomics/) is a freely available hub for the analysis and archiving of metagenomic and metatranscriptomic data. Over the last 2 years, the resource has undergone rapid growth, with an increase of over five-fold in the number of processed samples and consequently represents one of the largest resources of analysed shotgun metagenomes. Here, we report the status of the resource in 2016 and give an overview of new developments. In particular, we describe updates to data content, a complete overhaul of the analysis pipeline, streamlining of data presentation via the website and the development of a new web based tool to compare functional analyses of sequence runs within a study. We also highlight two of the higher profile projects that have been analysed using the resource in the last year: the oceanographic projects Ocean Sampling Day and Tara Oceans.

  4. Recent progresses in metagenomics

    Science.gov (United States)

    Metagenomics addresses the collective genetic structure and functional composition of a microbial community at its native habitat. This approach has emerged as a powerful tool to study the structure and function of the microbiota for the past few years and is revolutionizing studies of microbial ec...

  5. High throughtput comparisons and profiling of metagenomes for industrially relevant enzymes

    KAUST Repository

    Alam, Intikhab

    2016-01-26

    More and more genomes and metagenomes are being sequenced since the advent of Next Generation Sequencing Technologies (NGS). Many metagenomic samples are collected from a variety of environments, each exhibiting a different environmental profile, e.g. temperature, environmental chemistry, etc… These metagenomes can be profiled to unearth enzymes relevant to several industries based on specific enzyme properties such as ability to work on extreme conditions, such as extreme temperatures, salinity, anaerobically, etc.. In this work, we present the DMAP platform comprising of a high-throughput metagenomic annotation pipeline and a data-warehouse for comparisons and profiling across large number of metagenomes. We developed two reference databases for profiling of important genes, one containing enzymes related to different industries and the other containing genes with potential bioactivity roles. In this presentation we describe an example analysis of a large number of publicly available metagenomic sample from TARA oceans study (Science 2015) that covers significant part of world oceans.

  6. Comparison of microbial DNA enrichment tools for metagenomic whole genome sequencing.

    Science.gov (United States)

    Thoendel, Matthew; Jeraldo, Patricio R; Greenwood-Quaintance, Kerryl E; Yao, Janet Z; Chia, Nicholas; Hanssen, Arlen D; Abdel, Matthew P; Patel, Robin

    2016-08-01

    Metagenomic whole genome sequencing for detection of pathogens in clinical samples is an exciting new area for discovery and clinical testing. A major barrier to this approach is the overwhelming ratio of human to pathogen DNA in samples with low pathogen abundance, which is typical of most clinical specimens. Microbial DNA enrichment methods offer the potential to relieve this limitation by improving this ratio. Two commercially available enrichment kits, the NEBNext Microbiome DNA Enrichment Kit and the Molzym MolYsis Basic kit, were tested for their ability to enrich for microbial DNA from resected arthroplasty component sonicate fluids from prosthetic joint infections or uninfected sonicate fluids spiked with Staphylococcus aureus. Using spiked uninfected sonicate fluid there was a 6-fold enrichment of bacterial DNA with the NEBNext kit and 76-fold enrichment with the MolYsis kit. Metagenomic whole genome sequencing of sonicate fluid revealed 13- to 85-fold enrichment of bacterial DNA using the NEBNext enrichment kit. The MolYsis approach achieved 481- to 9580-fold enrichment, resulting in 7 to 59% of sequencing reads being from the pathogens known to be present in the samples. These results demonstrate the usefulness of these tools when testing clinical samples with low microbial burden using next generation sequencing. PMID:27237775

  7. Soil metagenomics and tropical soil productivity

    OpenAIRE

    Garrett, Karen A.

    2009-01-01

    This presentation summarizes research in the soil metagenomics cross cutting research activity. Soil metagenomics studies soil microbial communities as contributors to soil health.C CCRA-4 (Soil Metagenomics)

  8. Improved metagenome assemblies and taxonomic binning using long-read circular consensus sequence data.

    OpenAIRE

    Frank, J. A.; Pan, Y.; Tooming-Klunderud, A; Eijsink, V. G. H.; A. C. McHardy; Nederbragt, A. J.; Pope, P.B.

    2016-01-01

    DNA assembly is a core methodological step in metagenomic pipelines used to study the structure and function within microbial communities. Here we investigate the utility of Pacific Biosciences long and high accuracy circular consensus sequencing (CCS) reads for metagenomic projects. We compared the application and performance of both PacBio CCS and Illumina HiSeq data with assembly and taxonomic binning algorithms using metagenomic samples representing a complex microbial community. Eight SM...

  9. Improved metagenome assemblies and taxonomic binning using long-read circular consensus sequence data

    OpenAIRE

    Frank, J. A.; Pan, Y.; Tooming-Klunderud, A; V. G. H. Eijsink; McHardy, A. C.; Nederbragt, A. J.; Pope, P.B.

    2016-01-01

    DNA assembly is a core methodological step in metagenomic pipelines used to study the structure and function within microbial communities. Here we investigate the utility of Pacific Biosciences long and high accuracy circular consensus sequencing (CCS) reads for metagenomic projects. We compared the application and performance of both PacBio CCS and Illumina HiSeq data with assembly and taxonomic binning algorithms using metagenomic samples representing a complex microbial community. Eight SM...

  10. Detection of Novel Integrons in the Metagenome of Human Saliva

    Science.gov (United States)

    Antepowicz, Agata; Mullany, Peter; Roberts, Adam P.

    2016-01-01

    Integrons are genetic elements capable of capturing and expressing open reading frames (ORFs) embedded within gene cassettes. They are involved in the dissemination of antibiotic resistance genes (ARGs) in clinically important pathogens. Although the ARGs are common in the oral cavity the association of integrons and antibiotic resistance has not been reported there. In this work, a PCR-based approach was used to investigate the presence of integrons and associated gene cassettes in human oral metagenomic DNA obtained from both the UK and Bangladesh. We identified a diverse array of gene cassettes containing ORFs predicted to confer antimicrobial resistance and other adaptive traits. The predicted proteins include a putative streptogramin A O-acetyltransferase, a bleomycin binding protein, cof-like hydrolase, competence and motility related proteins. This is the first study detecting integron gene cassettes directly from oral metagenomic DNA samples. The predicted proteins are likely to carry out a multitude of functions; however, the function of the majority is yet unknown. PMID:27304457

  11. Comparing viral metagenomics methods using a highly multiplexed human viral pathogens reagent.

    Science.gov (United States)

    Li, Linlin; Deng, Xutao; Mee, Edward T; Collot-Teixeira, Sophie; Anderson, Rob; Schepelmann, Silke; Minor, Philip D; Delwart, Eric

    2015-03-01

    Unbiased metagenomic sequencing holds significant potential as a diagnostic tool for the simultaneous detection of any previously genetically described viral nucleic acids in clinical samples. Viral genome sequences can also inform on likely phenotypes including drug susceptibility or neutralization serotypes. In this study, different variables of the laboratory methods often used to generate viral metagenomics libraries were compared for their abilities to detect multiple viruses and generate full genome coverage. A biological reagent consisting of 25 different human RNA and DNA viral pathogens was used to estimate the effect of filtration and nuclease digestion, DNA/RNA extraction methods, pre-amplification and the use of different library preparation kits on the detection of viral nucleic acids. Filtration and nuclease treatment led to slight decreases in the percentage of viral sequence reads and number of viruses detected. For nucleic acid extractions silica spin columns improved viral sequence recovery relative to magnetic beads and Trizol extraction. Pre-amplification using random RT-PCR while generating more viral sequence reads resulted in detection of fewer viruses, more overlapping sequences, and lower genome coverage. The ScriptSeq library preparation method retrieved more viruses and a greater fraction of their genomes than the TruSeq and Nextera methods. Viral metagenomics sequencing was able to simultaneously detect up to 22 different viruses in the biological reagent analyzed including all those detected by qPCR. Further optimization will be required for the detection of viruses in biologically more complex samples such as tissues, blood, or feces.

  12. Non-invasive prenatal chromosomal aneuploidy testing--clinical experience: 100,000 clinical samples.

    Directory of Open Access Journals (Sweden)

    Ron M McCullough

    Full Text Available OBJECTIVE: As the first laboratory to offer massively parallel sequencing-based noninvasive prenatal testing (NIPT for fetal aneuploidies, Sequenom Laboratories has been able to collect the largest clinical population experience data to date, including >100,000 clinical samples from all 50 U.S. states and 13 other countries. The objective of this study is to give a robust clinical picture of the current laboratory performance of the MaterniT21 PLUS LDT. STUDY DESIGN: The study includes plasma samples collected from patients with high-risk pregnancies in our CLIA-licensed, CAP-accredited laboratory between August 2012 to June 2013. Samples were assessed for trisomies 13, 18, 21 and for the presence of chromosome Y-specific DNA. Sample data and ad hoc outcome information provided by the clinician was compiled and reviewed to determine the characteristics of this patient population, as well as estimate the assay performance in a clinical setting. RESULTS: NIPT patients most commonly undergo testing at an average of 15 weeks, 3 days gestation; and average 35.1 years of age. The average turnaround time is 4.54 business days and an overall 1.3% not reportable rate. The positivity rate for Trisomy 21 was 1.51%, followed by 0.45% and 0.21% rate for Trisomies 18 and 13, respectively. NIPT positivity rates are similar to previous large clinical studies of aneuploidy in women of maternal age ≥ 35 undergoing amniocentesis. In this population 3519 patients had multifetal gestations (3.5% with 2.61% yielding a positive NIPT result. CONCLUSION: NIPT has been commercially offered for just over 2 years and the clinical use by patients and clinicians has increased significantly. The risks associated with invasive testing have been substantially reduced by providing another assessment of aneuploidy status in high-risk patients. The accuracy and NIPT assay positivity rate are as predicted by clinical validations and the test demonstrates improvement in the

  13. Parallel-META 2.0: enhanced metagenomic data analysis with functional annotation, high performance computing and advanced visualization.

    Science.gov (United States)

    Su, Xiaoquan; Pan, Weihua; Song, Baoxing; Xu, Jian; Ning, Kang

    2014-01-01

    The metagenomic method directly sequences and analyses genome information from microbial communities. The main computational tasks for metagenomic analyses include taxonomical and functional structure analysis for all genomes in a microbial community (also referred to as a metagenomic sample). With the advancement of Next Generation Sequencing (NGS) techniques, the number of metagenomic samples and the data size for each sample are increasing rapidly. Current metagenomic analysis is both data- and computation- intensive, especially when there are many species in a metagenomic sample, and each has a large number of sequences. As such, metagenomic analyses require extensive computational power. The increasing analytical requirements further augment the challenges for computation analysis. In this work, we have proposed Parallel-META 2.0, a metagenomic analysis software package, to cope with such needs for efficient and fast analyses of taxonomical and functional structures for microbial communities. Parallel-META 2.0 is an extended and improved version of Parallel-META 1.0, which enhances the taxonomical analysis using multiple databases, improves computation efficiency by optimized parallel computing, and supports interactive visualization of results in multiple views. Furthermore, it enables functional analysis for metagenomic samples including short-reads assembly, gene prediction and functional annotation. Therefore, it could provide accurate taxonomical and functional analyses of the metagenomic samples in high-throughput manner and on large scale.

  14. Parallel-META 2.0: enhanced metagenomic data analysis with functional annotation, high performance computing and advanced visualization.

    Directory of Open Access Journals (Sweden)

    Xiaoquan Su

    Full Text Available The metagenomic method directly sequences and analyses genome information from microbial communities. The main computational tasks for metagenomic analyses include taxonomical and functional structure analysis for all genomes in a microbial community (also referred to as a metagenomic sample. With the advancement of Next Generation Sequencing (NGS techniques, the number of metagenomic samples and the data size for each sample are increasing rapidly. Current metagenomic analysis is both data- and computation- intensive, especially when there are many species in a metagenomic sample, and each has a large number of sequences. As such, metagenomic analyses require extensive computational power. The increasing analytical requirements further augment the challenges for computation analysis. In this work, we have proposed Parallel-META 2.0, a metagenomic analysis software package, to cope with such needs for efficient and fast analyses of taxonomical and functional structures for microbial communities. Parallel-META 2.0 is an extended and improved version of Parallel-META 1.0, which enhances the taxonomical analysis using multiple databases, improves computation efficiency by optimized parallel computing, and supports interactive visualization of results in multiple views. Furthermore, it enables functional analysis for metagenomic samples including short-reads assembly, gene prediction and functional annotation. Therefore, it could provide accurate taxonomical and functional analyses of the metagenomic samples in high-throughput manner and on large scale.

  15. A Bioinformatician's Guide to Metagenomics

    Energy Technology Data Exchange (ETDEWEB)

    Kunin, Victor; Copeland, Alex; Lapidus, Alla; Mavromatis, Konstantinos; Hugenholtz, Philip

    2008-08-01

    As random shotgun metagenomic projects proliferate and become the dominant source of publicly available sequence data, procedures for best practices in their execution and analysis become increasingly important. Based on our experience at the Joint Genome Institute, we describe step-by-step the chain of decisions accompanying a metagenomic project from the viewpoint of a bioinformatician. We guide the reader through a standard workflow for a metagenomic project beginning with pre-sequencing considerations such as community composition and sequence data type that will greatly influence downstream analyses. We proceed with recommendations for sampling and data generation including sample and metadata collection, community profiling, construction of shotgun libraries and sequencing strategies. We then discuss the application of generic sequence processing steps (read preprocessing, assembly, and gene prediction and annotation) to metagenomic datasets by contrast to genome projects. Different types of data analyses particular to metagenomes are then presented including binning, dominant population analysis and gene-centric analysis. Finally data management systems and issues are presented and discussed. We hope that this review will assist bioinformaticians and biologists in making better-informed decisions on their journey during a metagenomic project.

  16. A Bioinformatician's Guide to Metagenomics

    OpenAIRE

    Kunin, Victor; Copeland, Alex; Lapidus, Alla; Mavromatis, Konstantinos; Hugenholtz, Philip

    2008-01-01

    Summary: As random shotgun metagenomic projects proliferate and become the dominant source of publicly available sequence data, procedures for the best practices in their execution and analysis become increasingly important. Based on our experience at the Joint Genome Institute, we describe the chain of decisions accompanying a metagenomic project from the viewpoint of the bioinformatic analysis step by step. We guide the reader through a standard workflow for a metagenomic project beginning ...

  17. Metagenomic analysis of medicinal Cannabis samples; pathogenic bacteria, toxigenic fungi, and beneficial microbes grow in culture-based yeast and mold tests [version 1; referees: 2 approved, 1 approved with reservations

    Directory of Open Access Journals (Sweden)

    Kevin McKernan

    2016-10-01

    Full Text Available Background: The presence of bacteria and fungi in medicinal or recreational Cannabis poses a potential threat to consumers if those microbes include pathogenic or toxigenic species. This study evaluated two widely used culture-based platforms for total yeast and mold (TYM testing marketed by 3M Corporation and Biomérieux, in comparison with a quantitative PCR (qPCR approach marketed by Medicinal Genomics Corporation. Methods: A set of 15 medicinal Cannabis samples were analyzed using 3M and Biomérieux culture-based platforms and by qPCR to quantify microbial DNA. All samples were then subjected to next-generation sequencing and metagenomics analysis to enumerate the bacteria and fungi present before and after growth on culture-based media. Results: Several pathogenic or toxigenic bacterial and fungal species were identified in proportions of >5% of classified reads on the samples, including Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Ralstonia pickettii, Salmonella enterica, Stenotrophomonas maltophilia, Aspergillus ostianus, Aspergillus sydowii, Penicillium citrinum and Penicillium steckii. Samples subjected to culture showed substantial shifts in the number and diversity of species present, including the failure of Aspergillus species to grow well on either platform. Substantial growth of Clostridium botulinum and other bacteria were frequently observed on one or both of the culture-based TYM platforms. The presence of plant growth promoting (beneficial fungal species further influenced the differential growth of species in the microbiome of each sample. Conclusions: These findings have important implications for the Cannabis and food safety testing industries.

  18. METAGENassist: a comprehensive web server for comparative metagenomics.

    Science.gov (United States)

    Arndt, David; Xia, Jianguo; Liu, Yifeng; Zhou, You; Guo, An Chi; Cruz, Joseph A; Sinelnikov, Igor; Budwill, Karen; Nesbø, Camilla L; Wishart, David S

    2012-07-01

    With recent improvements in DNA sequencing and sample extraction techniques, the quantity and quality of metagenomic data are now growing exponentially. This abundance of richly annotated metagenomic data and bacterial census information has spawned a new branch of microbiology called comparative metagenomics. Comparative metagenomics involves the comparison of bacterial populations between different environmental samples, different culture conditions or different microbial hosts. However, in order to do comparative metagenomics, one typically requires a sophisticated knowledge of multivariate statistics and/or advanced software programming skills. To make comparative metagenomics more accessible to microbiologists, we have developed a freely accessible, easy-to-use web server for comparative metagenomic analysis called METAGENassist. Users can upload their bacterial census data from a wide variety of common formats, using either amplified 16S rRNA data or shotgun metagenomic data. Metadata concerning environmental, culture, or host conditions can also be uploaded. During the data upload process, METAGENassist also performs an automated taxonomic-to-phenotypic mapping. Phenotypic information covering nearly 20 functional categories such as GC content, genome size, oxygen requirements, energy sources and preferred temperature range is automatically generated from the taxonomic input data. Using this phenotypically enriched data, users can then perform a variety of multivariate and univariate data analyses including fold change analysis, t-tests, PCA, PLS-DA, clustering and classification. To facilitate data processing, users are guided through a step-by-step analysis workflow using a variety of menus, information hyperlinks and check boxes. METAGENassist also generates colorful, publication quality tables and graphs that can be downloaded and used directly in the preparation of scientific papers. METAGENassist is available at http://www.metagenassist.ca.

  19. Antibiotic Susceptibilities of Acinetobacter Baumanii Strains Isolated from Clinical Samples

    Directory of Open Access Journals (Sweden)

    Harun Aðca

    2013-01-01

    Full Text Available      Aim :  In this study it was aimed to investigate the antibiotic susceptibilities of Acinetobacter baumanii strains isolated from various clinical samples sent to Tavsanli State Hospital Microbiology Laboratory retrospectively. Material and Method: All of the cultures were examined for the agent and antibiotic susceptibilities. For the identification of bacteria, various chemical tests and BBL Crystal E/NF (Beckton Dickinson, ABD system was used. Antibiotic susceptibilities were investigated according to CLSI criteria on Mueller Hinton agar by disc diffusion method. Results: There were 74 strains isolated and identified as Acinetobacter baumanii. Most of the strains were isolated from  tracheal aspirate specimens (46 % Most of the strains were isolated from nosocomial infections. Antibiotic resistance was high among strains. The most susceptible antibiotic was gentamicin (30%. Discussion: To prevent the development of resistance, antibiotics should be used carefully in appropriate doses and time, empirical  antibiotherapy should be determined for each centre according to resistance rates of the centre and should be regulated according to the antibiogram results. Increasing resistance rates in Acinetobacter strains leads to the usage of new alternative antibiotics.  

  20. Clinical audit for occupational therapy intervention for children with autism spectrum disorder: sampling steps and sample size calculation

    OpenAIRE

    Weeks, Scott; Atlas, Alvin

    2015-01-01

    A priori sample size calculations are used to determine the adequate sample size to estimate the prevalence of the target population with good precision. However, published audits rarely report a priori calculations for their sample size. This article discusses a process in health services delivery mapping to generate a comprehensive sampling frame, which was used to calculate an a priori sample size for a targeted clinical record audit. We describe how we approached methodological and defini...

  1. Estimating the extent of horizontal gene transfer in metagenomic sequences

    Directory of Open Access Journals (Sweden)

    Moya Andrés

    2008-03-01

    Full Text Available Abstract Background Although the extent of horizontal gene transfer (HGT in complete genomes has been widely studied, its influence in the evolution of natural communities of prokaryotes remains unknown. The availability of metagenomic sequences allows us to address the study of global patterns of prokaryotic evolution in samples from natural communities. However, the methods that have been commonly used for the study of HGT are not suitable for metagenomic samples. Therefore it is important to develop new methods or to adapt existing ones to be used with metagenomic sequences. Results We have created two different methods that are suitable for the study of HGT in metagenomic samples. The methods are based on phylogenetic and DNA compositional approaches, and have allowed us to assess the extent of possible HGT events in metagenomes for the first time. The methods are shown to be compatible and quite precise, although they probably underestimate the number of possible events. Our results show that the phylogenetic method detects HGT in between 0.8% and 1.5% of the sequences, while DNA compositional methods identify putative HGT in between 2% and 8% of the sequences. These ranges are very similar to these found in complete genomes by related approaches. Both methods act with a different sensitivity since they probably target HGT events of different ages: the compositional method mostly identifies recent transfers, while the phylogenetic is more suitable for the detections of older events. Nevertheless, the study of the number of HGT events in metagenomic sequences from different communities shows a consistent trend for both methods: the lower amount is found for the sequences of the Sargasso Sea metagenome, while the higher quantity is found in the whale fall metagenome from the bottom of the ocean. The significance of these observations is discussed. Conclusion The computational approaches that are used to find possible HGT events in complete

  2. Metagenomic analysis of soil microbial communities

    OpenAIRE

    Đokić Lidija; Savić M.; Narančić Tanja; Vasiljević Branka

    2010-01-01

    Ramonda serbica and Ramonda nathaliae, rare resurrection plants growing in the Balkan Peninsula, produce a high amount of phenolic compounds as a response to stress. The composition and size of bacterial communities in two rhizosphere soil samples of these plants were analyzed using a metagenomic approach. Fluorescent in situ hybridization (FISH) experiments together with DAPI staining showed that the metabolically active bacteria represent only a small fraction, approximately 5%, of total so...

  3. The YNP metagenome project

    DEFF Research Database (Denmark)

    Inskeep, William P.; Jay, Zackary J.; Tringe, Susannah G.;

    2013-01-01

    The Yellowstone geothermal complex contains over 10,000 diverse geothermal features that host numerous phylogenetically deeply rooted and poorly understood archaea, bacteria, and viruses. Microbial communities in high-temperature environments are generally less diverse than soil, marine, sediment......, and environmental variables. Twenty geochemically distinct geothermal ecosystems representing a broad spectrum of Yellowstone hot-spring environments were used for metagenomic and geochemical analysis and included approximately equal numbers of: (1) phototrophic mats, (2) “filamentous streamer” communities, and (3...

  4. Methods for comparative metagenomics

    OpenAIRE

    Auch Alexander F; Mitra Suparna; Richter Daniel C; Huson Daniel H; Schuster Stephan C

    2009-01-01

    Abstract Background Metagenomics is a rapidly growing field of research that aims at studying uncultured organisms to understand the true diversity of microbes, their functions, cooperation and evolution, in environments such as soil, water, ancient remains of animals, or the digestive system of animals and humans. The recent development of ultra-high throughput sequencing technologies, which do not require cloning or PCR amplification, and can produce huge numbers of DNA reads at an affordab...

  5. MetLab: An In Silico Experimental Design, Simulation and Analysis Tool for Viral Metagenomics Studies

    Science.gov (United States)

    Gourlé, Hadrien; Bongcam-Rudloff, Erik; Hayer, Juliette

    2016-01-01

    Metagenomics, the sequence characterization of all genomes within a sample, is widely used as a virus discovery tool as well as a tool to study viral diversity of animals. Metagenomics can be considered to have three main steps; sample collection and preparation, sequencing and finally bioinformatics. Bioinformatic analysis of metagenomic datasets is in itself a complex process, involving few standardized methodologies, thereby hampering comparison of metagenomics studies between research groups. In this publication the new bioinformatics framework MetLab is presented, aimed at providing scientists with an integrated tool for experimental design and analysis of viral metagenomes. MetLab provides support in designing the metagenomics experiment by estimating the sequencing depth needed for the complete coverage of a species. This is achieved by applying a methodology to calculate the probability of coverage using an adaptation of Stevens’ theorem. It also provides scientists with several pipelines aimed at simplifying the analysis of viral metagenomes, including; quality control, assembly and taxonomic binning. We also implement a tool for simulating metagenomics datasets from several sequencing platforms. The overall aim is to provide virologists with an easy to use tool for designing, simulating and analyzing viral metagenomes. The results presented here include a benchmark towards other existing software, with emphasis on detection of viruses as well as speed of applications. This is packaged, as comprehensive software, readily available for Linux and OSX users at https://github.com/norling/metlab. PMID:27479078

  6. MetLab: An In Silico Experimental Design, Simulation and Analysis Tool for Viral Metagenomics Studies.

    Science.gov (United States)

    Norling, Martin; Karlsson-Lindsjö, Oskar E; Gourlé, Hadrien; Bongcam-Rudloff, Erik; Hayer, Juliette

    2016-01-01

    Metagenomics, the sequence characterization of all genomes within a sample, is widely used as a virus discovery tool as well as a tool to study viral diversity of animals. Metagenomics can be considered to have three main steps; sample collection and preparation, sequencing and finally bioinformatics. Bioinformatic analysis of metagenomic datasets is in itself a complex process, involving few standardized methodologies, thereby hampering comparison of metagenomics studies between research groups. In this publication the new bioinformatics framework MetLab is presented, aimed at providing scientists with an integrated tool for experimental design and analysis of viral metagenomes. MetLab provides support in designing the metagenomics experiment by estimating the sequencing depth needed for the complete coverage of a species. This is achieved by applying a methodology to calculate the probability of coverage using an adaptation of Stevens' theorem. It also provides scientists with several pipelines aimed at simplifying the analysis of viral metagenomes, including; quality control, assembly and taxonomic binning. We also implement a tool for simulating metagenomics datasets from several sequencing platforms. The overall aim is to provide virologists with an easy to use tool for designing, simulating and analyzing viral metagenomes. The results presented here include a benchmark towards other existing software, with emphasis on detection of viruses as well as speed of applications. This is packaged, as comprehensive software, readily available for Linux and OSX users at https://github.com/norling/metlab. PMID:27479078

  7. MetLab: An In Silico Experimental Design, Simulation and Analysis Tool for Viral Metagenomics Studies.

    Science.gov (United States)

    Norling, Martin; Karlsson-Lindsjö, Oskar E; Gourlé, Hadrien; Bongcam-Rudloff, Erik; Hayer, Juliette

    2016-01-01

    Metagenomics, the sequence characterization of all genomes within a sample, is widely used as a virus discovery tool as well as a tool to study viral diversity of animals. Metagenomics can be considered to have three main steps; sample collection and preparation, sequencing and finally bioinformatics. Bioinformatic analysis of metagenomic datasets is in itself a complex process, involving few standardized methodologies, thereby hampering comparison of metagenomics studies between research groups. In this publication the new bioinformatics framework MetLab is presented, aimed at providing scientists with an integrated tool for experimental design and analysis of viral metagenomes. MetLab provides support in designing the metagenomics experiment by estimating the sequencing depth needed for the complete coverage of a species. This is achieved by applying a methodology to calculate the probability of coverage using an adaptation of Stevens' theorem. It also provides scientists with several pipelines aimed at simplifying the analysis of viral metagenomes, including; quality control, assembly and taxonomic binning. We also implement a tool for simulating metagenomics datasets from several sequencing platforms. The overall aim is to provide virologists with an easy to use tool for designing, simulating and analyzing viral metagenomes. The results presented here include a benchmark towards other existing software, with emphasis on detection of viruses as well as speed of applications. This is packaged, as comprehensive software, readily available for Linux and OSX users at https://github.com/norling/metlab.

  8. Random whole metagenomic sequencing for forensic discrimination of soils.

    Science.gov (United States)

    Khodakova, Anastasia S; Smith, Renee J; Burgoyne, Leigh; Abarno, Damien; Linacre, Adrian

    2014-01-01

    Here we assess the ability of random whole metagenomic sequencing approaches to discriminate between similar soils from two geographically distinct urban sites for application in forensic science. Repeat samples from two parklands in residential areas separated by approximately 3 km were collected and the DNA was extracted. Shotgun, whole genome amplification (WGA) and single arbitrarily primed DNA amplification (AP-PCR) based sequencing techniques were then used to generate soil metagenomic profiles. Full and subsampled metagenomic datasets were then annotated against M5NR/M5RNA (taxonomic classification) and SEED Subsystems (metabolic classification) databases. Further comparative analyses were performed using a number of statistical tools including: hierarchical agglomerative clustering (CLUSTER); similarity profile analysis (SIMPROF); non-metric multidimensional scaling (NMDS); and canonical analysis of principal coordinates (CAP) at all major levels of taxonomic and metabolic classification. Our data showed that shotgun and WGA-based approaches generated highly similar metagenomic profiles for the soil samples such that the soil samples could not be distinguished accurately. An AP-PCR based approach was shown to be successful at obtaining reproducible site-specific metagenomic DNA profiles, which in turn were employed for successful discrimination of visually similar soil samples collected from two different locations. PMID:25111003

  9. Metagenomic Analysis of Bacterial Communities of Antarctic Surface Snow

    Directory of Open Access Journals (Sweden)

    Anna eLopatina

    2016-03-01

    Full Text Available The diversity of bacteria present in surface snow around four Russian stations in Eastern Antarctica was studied by high throughput sequencing of amplified 16S rRNA gene fragments and shotgun metagenomic sequencing. Considerable class- and genus-level variation between the samples was revealed indicating a presence of inter-site diversity of bacteria in Antarctic snow. Flavobacterium was a major genus in one sampling site and was also detected in other sites. The diversity of flavobacterial type II-C CRISPR spacers in the samples was investigated by metagenome sequencing. Thousands of unique spacers were revealed with less than 35% overlap between the sampling sites, indicating an enormous natural variety of flavobacterial CRISPR spacers and, by extension, high level of adaptive activity of the corresponding CRISPR-Cas system. None of the spacers matched known spacers of flavobacterial isolates from the Northern hemisphere. Moreover, the percentage of spacers with matches with Antarctic metagenomic sequences obtained in this work was significantly higher than with sequences from much larger publically available environmental metagenomic database. The results indicate that despite the overall very high level of diversity, Antarctic Flavobacteria comprise a separate pool that experiences pressures from mobile genetic elements different from those present in other parts of the world. The results also establish analysis of metagenomic CRISPR spacer content as a powerful tool to study bacterial populations diversity.

  10. A retrospective metagenomics approach to studying Blastocystis.

    Science.gov (United States)

    Andersen, Lee O'Brien; Bonde, Ida; Nielsen, Henrik Bjørn; Stensvold, Christen Rune

    2015-07-01

    Blastocystis is a common single-celled intestinal parasitic genus, comprising several subtypes. Here, we screened data obtained by metagenomic analysis of faecal DNA for Blastocystis by searching for subtype-specific genes in coabundance gene groups, which are groups of genes that covary across a selection of 316 human faecal samples, hence representing genes originating from a single subtype. The 316 faecal samples were from 236 healthy individuals, 13 patients with Crohn's disease (CD) and 67 patients with ulcerative colitis (UC). The prevalence of Blastocystis was 20.3% in the healthy individuals and 14.9% in patients with UC. Meanwhile, Blastocystis was absent in patients with CD. Individuals with intestinal microbiota dominated by Bacteroides were much less prone to having Blastocystis-positive stool (Matthew's correlation coefficient = -0.25, P metagenomics approach. The study serves as an example of how it is possible to retrospectively investigate microbial eukaryotic communities in the gut using metagenomic datasets targeting the bacterial component of the intestinal microbiome and the interplay between these microbial communities.

  11. Metagenomic analysis of soil microbial communities

    Directory of Open Access Journals (Sweden)

    Đokić Lidija

    2010-01-01

    Full Text Available Ramonda serbica and Ramonda nathaliae, rare resurrection plants growing in the Balkan Peninsula, produce a high amount of phenolic compounds as a response to stress. The composition and size of bacterial communities in two rhizosphere soil samples of these plants were analyzed using a metagenomic approach. Fluorescent in situ hybridization (FISH experiments together with DAPI staining showed that the metabolically active bacteria represent only a small fraction, approximately 5%, of total soil bacteria. Using universal bacteria - specific primers 16S rDNA genes were amplified directly from metagenomic DNAs and two libraries were constructed. The Restriction Fragment Length Polymorphism (RLFP method was used in library screening. Amongst 192 clones, 35 unique operational taxonomic units (OTUs were determined from the rhizosphere of R. nathaliae, and 13 OTUs out of 80 clones in total from the library of R. serbica. Representative clones from each OTU were sequenced. The majority of sequences from metagenomes showed very little similarity to any cultured bacteria. In conclusion, the bacterial communities in the studied soil samples showed quite poor diversity. .

  12. Accurate genome relative abundance estimation based on shotgun metagenomic reads.

    Directory of Open Access Journals (Sweden)

    Li C Xia

    Full Text Available Accurate estimation of microbial community composition based on metagenomic sequencing data is fundamental for subsequent metagenomics analysis. Prevalent estimation methods are mainly based on directly summarizing alignment results or its variants; often result in biased and/or unstable estimates. We have developed a unified probabilistic framework (named GRAMMy by explicitly modeling read assignment ambiguities, genome size biases and read distributions along the genomes. Maximum likelihood method is employed to compute Genome Relative Abundance of microbial communities using the Mixture Model theory (GRAMMy. GRAMMy has been demonstrated to give estimates that are accurate and robust across both simulated and real read benchmark datasets. We applied GRAMMy to a collection of 34 metagenomic read sets from four metagenomics projects and identified 99 frequent species (minimally 0.5% abundant in at least 50% of the data-sets in the human gut samples. Our results show substantial improvements over previous studies, such as adjusting the over-estimated abundance for Bacteroides species for human gut samples, by providing a new reference-based strategy for metagenomic sample comparisons. GRAMMy can be used flexibly with many read assignment tools (mapping, alignment or composition-based even with low-sensitivity mapping results from huge short-read datasets. It will be increasingly useful as an accurate and robust tool for abundance estimation with the growing size of read sets and the expanding database of reference genomes.

  13. Current and future resources for functional metagenomics

    OpenAIRE

    Kathy Nguyen Lam; Jiujun eCheng; Katja eEngel; Josh David Neufeld; Trevor Carlos Charles

    2015-01-01

    Functional metagenomics is a powerful experimental approach for studying gene function, starting from the extracted DNA of mixed microbial populations. A functional approach relies on the construction and screening of metagenomic libraries – physical libraries that contain DNA cloned from environmental metagenomes. The information obtained from functional metagenomics can help in future annotations of gene function and serve as a complement to sequence-based metagenomics. In this Perspective,...

  14. Current and future resources for functional metagenomics

    OpenAIRE

    Lam, Kathy N.; Cheng, Jiujun; Engel, Katja; Neufeld, Josh D.; Charles, Trevor C.

    2015-01-01

    Functional metagenomics is a powerful experimental approach for studying gene function, starting from the extracted DNA of mixed microbial populations. A functional approach relies on the construction and screening of metagenomic libraries—physical libraries that contain DNA cloned from environmental metagenomes. The information obtained from functional metagenomics can help in future annotations of gene function and serve as a complement to sequence-based metagenomics. In this Perspective, w...

  15. Profiling critical cancer gene mutations in clinical tumor samples.

    Directory of Open Access Journals (Sweden)

    Laura E MacConaill

    Full Text Available BACKGROUND: Detection of critical cancer gene mutations in clinical tumor specimens may predict patient outcomes and inform treatment options; however, high-throughput mutation profiling remains underdeveloped as a diagnostic approach. We report the implementation of a genotyping and validation algorithm that enables robust tumor mutation profiling in the clinical setting. METHODOLOGY: We developed and implemented an optimized mutation profiling platform ("OncoMap" to interrogate approximately 400 mutations in 33 known oncogenes and tumor suppressors, many of which are known to predict response or resistance to targeted therapies. The performance of OncoMap was analyzed using DNA derived from both frozen and FFPE clinical material in a diverse set of cancer types. A subsequent in-depth analysis was conducted on histologically and clinically annotated pediatric gliomas. The sensitivity and specificity of OncoMap were 93.8% and 100% in fresh frozen tissue; and 89.3% and 99.4% in FFPE-derived DNA. We detected known mutations at the expected frequencies in common cancers, as well as novel mutations in adult and pediatric cancers that are likely to predict heightened response or resistance to existing or developmental cancer therapies. OncoMap profiles also support a new molecular stratification of pediatric low-grade gliomas based on BRAF mutations that may have immediate clinical impact. CONCLUSIONS: Our results demonstrate the clinical feasibility of high-throughput mutation profiling to query a large panel of "actionable" cancer gene mutations. In the future, this type of approach may be incorporated into both cancer epidemiologic studies and clinical decision making to specify the use of many targeted anticancer agents.

  16. From clinical sites to biorepositories: effectiveness in blood sample management.

    Science.gov (United States)

    Lefebvre, Céline; Tremblay, Nancy; Iverson, Bonnie; Wong, David; McWeeny, Kerri; Saghbini, Michael; Martinez, Heather; Hogan, Michael; Gaudet, Daniel; Arsenault, Steve

    2010-12-01

    Today's biobanks must work to take full advantage of collected samples, while maximizing sample quality and minimizing costs to sustain operations for a long period of time. This is a tall order that will require collaboration and compromise for both end-users and collection sites. This article discusses the efforts of the Génome Québec-Centre Hospitalier Affilié Universitaire Régional de Chicoutimi Biobank to fractionate blood samples for the simultaneous preservation of plasma and DNA-containing layers while minimizing resources required for shipping and transport. This article also describes methods for successful reproducible application of the plasma-depleted blood sample to GenPlates (GenVault, Carlsbad, CA).

  17. IMG/M: A data management and analysis system for metagenomes

    Energy Technology Data Exchange (ETDEWEB)

    Markowitz, Victor M.; Ivanova, Natalia N.; Szeto, Ernest; Palaniappan, Krishna; Chu, Ken; Dalevi, Daniel; Chen, I-Min A.; Grechkin,Yuri; Dubchak,Inna; Anderson, Iain; Lykidis, Athanasios; Mavromatis,Konstantinos; Hug enholtz, Phil; Kyrpides, Nikos C.

    2007-08-01

    IMG/M is a data management and analysis system for microbial community genomes (metagenomes) hosted at the Joint Genome Institute (JGI). IMG/M consists of metagenome data integrated with isolate microbial genomes from the Integrated Microbial Genomes (IMG) system. IMG/M provides IMG's comparative data analysis tools extended to handle metagenome data, together with metagenome-specific analysis tools. IMG/M is available at http://img.jgi.doe.gov/m. Studies of the collective genomes (also known as metagenomes) of environmental microbial communities (also known as microbiomes) are expected to lead to advances in environmental cleanup, agriculture, industrial processes, alternative energy production, and human health (1). Metagenomes of specific microbiome samples are sequenced by organizations worldwide, such as the Department of Energy's (DOE) Joint Genome Institute (JGI), the Venter Institute and the Washington University in St. Louis using different sequencing strategies, technology platforms, and annotation procedures. According to the Genomes OnLine Database, about 28 metagenome studies have been published to date, with over 60 other projects ongoing and more in the process of being launched (2). The Department of Energy's (DOE) Joint Genome Institute (JGI) is one of the major contributors of metagenome sequence data, currently sequencing more than 50% of the reported metagenome projects worldwide. Due to the higher complexity, inherent incompleteness, and lower quality of metagenome sequence data, traditional assembly, gene prediction, and annotation methods do not perform on these datasets as well as they do on isolate microbial genome sequences (3, 4). In spite of these limitations, metagenome data are amenable to a variety of analyses, as illustrated by several recent studies (5-10). Metagenome data analysis is usually set up in the context of reference isolate genomes and considers the questions of composition and functional or metabolic

  18. Introduction to the analysis of environmental sequences: metagenomics with MEGAN.

    Science.gov (United States)

    Huson, Daniel H; Mitra, Suparna

    2012-01-01

    Metagenomics is the study of microbial organisms using sequencing applied directly to environmental samples. Similarly, in metatranscriptomics and metaproteomics, the RNA and protein sequences of such samples are studied. The analysis of these kinds of data often starts by asking the questions of "who is out there?", "what are they doing?", and "how do they compare?". In this chapter, we describe how these computational questions can be addressed using MEGAN, the MEtaGenome ANalyzer program. We first show how to analyze the taxonomic and functional content of a single dataset and then show how such analyses can be performed in a comparative fashion. We demonstrate how to compare different datasets using ecological indices and other distance measures. The discussion is conducted using a number of published marine datasets comprising metagenomic, metatranscriptomic, metaproteomic, and 16S rRNA data.

  19. Sample size considerations for clinical research studies in nuclear cardiology.

    Science.gov (United States)

    Chiuzan, Cody; West, Erin A; Duong, Jimmy; Cheung, Ken Y K; Einstein, Andrew J

    2015-12-01

    Sample size calculation is an important element of research design that investigators need to consider in the planning stage of the study. Funding agencies and research review panels request a power analysis, for example, to determine the minimum number of subjects needed for an experiment to be informative. Calculating the right sample size is crucial to gaining accurate information and ensures that research resources are used efficiently and ethically. The simple question "How many subjects do I need?" does not always have a simple answer. Before calculating the sample size requirements, a researcher must address several aspects, such as purpose of the research (descriptive or comparative), type of samples (one or more groups), and data being collected (continuous or categorical). In this article, we describe some of the most frequent methods for calculating the sample size with examples from nuclear cardiology research, including for t tests, analysis of variance (ANOVA), non-parametric tests, correlation, Chi-squared tests, and survival analysis. For the ease of implementation, several examples are also illustrated via user-friendly free statistical software.

  20. Environmental Metagenomics: The Data Assembly and Data Analysis Perspectives

    Science.gov (United States)

    Kumar, Vinay; Maitra, S. S.; Shukla, Rohit Nandan

    2015-01-01

    Novel gene finding is one of the emerging fields in the environmental research. In the past decades the research was focused mainly on the discovery of microorganisms which were capable of degrading a particular compound. A lot of methods are available in literature about the cultivation and screening of these novel microorganisms. All of these methods are efficient for screening of microbes which can be cultivated in the laboratory. Microorganisms which live in extreme conditions like hot springs, frozen glaciers, acid mine drainage, etc. cannot be cultivated in the laboratory, this is because of incomplete knowledge about their growth requirements like temperature, nutrients and their mutual dependence on each other. The microbes that can be cultivated correspond only to less than 1 % of the total microbes which are present in the earth. Rest of the 99 % of uncultivated majority remains inaccessible. Metagenomics transcends the culture requirements of microbes. In metagenomics DNA is directly extracted from the environmental samples such as soil, seawater, acid mine drainage etc., followed by construction and screening of metagenomic library. With the ongoing research, a huge amount of metagenomic data is accumulating. Understanding this data is an essential step to extract novel genes of industrial importance. Various bioinformatics tools have been designed to analyze and annotate the data produced from the metagenome. The Bio-informatic requirements of metagenomics data analysis are different in theory and practice. This paper reviews the tools that are available for metagenomic data analysis and the capability such tools—what they can do and their web availability.

  1. Metagenomic Insights into Transferable Antibiotic Resistance in Oral Bacteria.

    Science.gov (United States)

    Sukumar, S; Roberts, A P; Martin, F E; Adler, C J

    2016-08-01

    Antibiotic resistance is considered one of the greatest threats to global public health. Resistance is often conferred by the presence of antibiotic resistance genes (ARGs), which are readily found in the oral microbiome. In-depth genetic analyses of the oral microbiome through metagenomic techniques reveal a broad distribution of ARGs (including novel ARGs) in individuals not recently exposed to antibiotics, including humans in isolated indigenous populations. This has resulted in a paradigm shift from focusing on the carriage of antibiotic resistance in pathogenic bacteria to a broader concept of an oral resistome, which includes all resistance genes in the microbiome. Metagenomics is beginning to demonstrate the role of the oral resistome and horizontal gene transfer within and between commensals in the absence of selective pressure, such as an antibiotic. At the chairside, metagenomic data reinforce our need to adhere to current antibiotic guidelines to minimize the spread of resistance, as such data reveal the extent of ARGs without exposure to antimicrobials and the ecologic changes created in the oral microbiome by even a single dose of antibiotics. The aim of this review is to discuss the role of metagenomics in the investigation of the oral resistome, including the transmission of antibiotic resistance in the oral microbiome. Future perspectives, including clinical implications of the findings from metagenomic investigations of oral ARGs, are also considered. PMID:27183895

  2. Clinical audit for occupational therapy intervention for children with autism spectrum disorder: sampling steps and sample size calculation.

    Science.gov (United States)

    Weeks, Scott; Atlas, Alvin

    2015-01-01

    A priori sample size calculations are used to determine the adequate sample size to estimate the prevalence of the target population with good precision. However, published audits rarely report a priori calculations for their sample size. This article discusses a process in health services delivery mapping to generate a comprehensive sampling frame, which was used to calculate an a priori sample size for a targeted clinical record audit. We describe how we approached methodological and definitional issues in the following steps: (1) target population definition, (2) sampling frame construction, and (3) a priori sample size calculation. We recommend this process for clinicians, researchers, or policy makers when detailed information on a reference population is unavailable. PMID:26122044

  3. Analytical artefacts in the speciation of arsenic in clinical samples

    Energy Technology Data Exchange (ETDEWEB)

    Slejkovec, Zdenka [Jozef Stefan Institute, Jamova 39, 1000 Ljubljana (Slovenia)], E-mail: zdenka.slejkovec@ijs.si; Falnoga, Ingrid [Jozef Stefan Institute, Jamova 39, 1000 Ljubljana (Slovenia); Goessler, Walter [Institute of Chemistry - Analytical Chemistry, Karl-Franzens University Graz, Universitaetsplatz 1, Graz (Austria); Elteren, Johannes T. van [National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana (Slovenia); Raml, Reingard [Institute of Chemistry - Analytical Chemistry, Karl-Franzens University Graz, Universitaetsplatz 1, Graz (Austria); Podgornik, Helena; Cernelc, Peter [University Medical Centre Ljubljana, Zaloska 7, 1000 Ljubljana (Slovenia)

    2008-01-21

    Urine and blood samples of cancer patients, treated with high doses of arsenic trioxide were analysed for arsenic species using HPLC-HGAFS and, in some cases, HPLC-ICPMS. Total arsenic was determined with either flow injection-HGAFS in urine or radiochemical neutron activation analysis in blood fractions (in serum/plasma, blood cells). The total arsenic concentrations (during prolonged, daily/weekly arsenic trioxide therapy) were in the {mu}g mL{sup -1} range for urine and in the ng g{sup -1} range for blood fractions. The main arsenic species found in urine were As(III), MA and DMA and in blood As(V), MA and DMA. With proper sample preparation and storage of urine (no preservation agents/storage in liquid nitrogen) no analytical artefacts were observed and absence of significant amounts of alleged trivalent metabolites was proven. On the contrary, in blood samples a certain amount of arsenic can get lost in the speciation procedure what was especially noticeable for the blood cells although also plasma/serum gave rise to some disappearance of arsenic. The latter losses may be attributed to precipitation of As(III)-containing proteins/peptides during the methanol/water extraction procedure whereas the former losses were due to loss of specific As(III)-complexing proteins/peptides (e.g. cysteine, metallothionein, reduced GSH, ferritin) on the column (Hamilton PRP-X100) during the separation procedure. Contemporary analytical protocols are not able to completely avoid artefacts due to losses from the sampling to the detection stage so that it is recommended to be careful with the explanation of results, particularly regarding metabolic and pharmacokinetic interpretations, and always aim to compare the sum of species with the total arsenic concentration determined independently.

  4. Metagenomic small molecule discovery methods

    OpenAIRE

    Charlop-Powers, Zachary; Milshteyn, Aleksandr; Brady, Sean F

    2014-01-01

    Metagenomic approaches to natural product discovery provide the means of harvesting bioactive small molecules synthesized by environmental bacteria without the requirement of first culturing these organisms. Advances in sequencing technologies and general metagenomic methods are beginning to provide the tools necessary to unlock the unexplored biosynthetic potential encoded by the genomes of uncultured environmental bacteria. Here, we highlight recent advances in sequence- and functional- bas...

  5. Promiscuous enzymes from functional metagenomics

    OpenAIRE

    Colin, Pierre-Yves; Kintses, Balint; Gielen, Fabrice; Miton, Charlotte; Fischer, Gerhard; Mahomed, Mark; HYVONEN, Marko; Morgavi, Diego P.; Janssen, Dick B.; Hollfelder, Florian

    2015-01-01

    Unculturable bacterial communities provide a rich source of biocatalysts, but their experimental discovery by functional metagenomics is difficult, because the odds are stacked against the experimentor. Here we demonstrate functional screening of a million-membered metagenomic library in microfluidic picolitre droplet compartments. Using bait substrates, new hydrolases for sulfate monoesters and phosphotriesters were identified, mostly based on promiscuous activities presumed not to be under ...

  6. Soil-specific limitations for access and analysis of soil microbial communities by metagenomics.

    Science.gov (United States)

    Lombard, Nathalie; Prestat, Emmanuel; van Elsas, Jan Dirk; Simonet, Pascal

    2011-10-01

    Metagenomics approaches represent an important way to acquire information on the microbial communities present in complex environments like soil. However, to what extent do these approaches provide us with a true picture of soil microbial diversity? Soil is a challenging environment to work with. Its physicochemical properties affect microbial distributions inside the soil matrix, metagenome extraction and its subsequent analyses. To better understand the bias inherent to soil metagenome 'processing', we focus on soil physicochemical properties and their effects on the perceived bacterial distribution. In the light of this information, each step of soil metagenome processing is then discussed, with an emphasis on strategies for optimal soil sampling. Then, the interaction of cells and DNA with the soil matrix and the consequences for microbial DNA extraction are examined. Soil DNA extraction methods are compared and the veracity of the microbial profiles obtained is discussed. Finally, soil metagenomic sequence analysis and exploitation methods are reviewed.

  7. A confirmatory factor analysis of the WMS-III in a clinical sample with crossvalidation in the standardization sample.

    Science.gov (United States)

    Bradley Burton, D; Ryan, Joseph J; Axelrod, Bradley N; Schellenberger, Tony; Richards, Heather M

    2003-08-01

    A maximum likelihood confirmatory factor analysis (CFA) of the Wechsler Memory Scale-III (WMS-III) was performed by applying LISREL 8 to a general clinical sample (n=281). Analyses were designed to determine which of seven hypothesized oblique factor solutions could best explain memory as measured by the WMS-III. Competing latent variable models were identified in previous studies. Results in the clinical sample were crossvalidated by testing all models in the WMS-III standardization samples (combined n=1,250). Findings in both the clinical and standardization samples supported a four-factor model containing auditory memory, visual memory, working memory, and learning factors. Our analysis differed from that presented in the WMS-III manual and by other authors. We tested our models in a clinical sample and included selected word list subtests in order to test the viability of a learning dimension. Consistent with prior research, we were also unable to empirically support the viability of the immediate and delayed memory indices, despite allowing the error terms between the immediate and delayed memory subtests to correlate.

  8. MetaStorm: A Public Resource for Customizable Metagenomics Annotation.

    Science.gov (United States)

    Arango-Argoty, Gustavo; Singh, Gargi; Heath, Lenwood S; Pruden, Amy; Xiao, Weidong; Zhang, Liqing

    2016-01-01

    Metagenomics is a trending research area, calling for the need to analyze large quantities of data generated from next generation DNA sequencing technologies. The need to store, retrieve, analyze, share, and visualize such data challenges current online computational systems. Interpretation and annotation of specific information is especially a challenge for metagenomic data sets derived from environmental samples, because current annotation systems only offer broad classification of microbial diversity and function. Moreover, existing resources are not configured to readily address common questions relevant to environmental systems. Here we developed a new online user-friendly metagenomic analysis server called MetaStorm (http://bench.cs.vt.edu/MetaStorm/), which facilitates customization of computational analysis for metagenomic data sets. Users can upload their own reference databases to tailor the metagenomics annotation to focus on various taxonomic and functional gene markers of interest. MetaStorm offers two major analysis pipelines: an assembly-based annotation pipeline and the standard read annotation pipeline used by existing web servers. These pipelines can be selected individually or together. Overall, MetaStorm provides enhanced interactive visualization to allow researchers to explore and manipulate taxonomy and functional annotation at various levels of resolution. PMID:27632579

  9. MetaStorm: A Public Resource for Customizable Metagenomics Annotation

    Science.gov (United States)

    Arango-Argoty, Gustavo; Singh, Gargi; Heath, Lenwood S.; Pruden, Amy; Xiao, Weidong; Zhang, Liqing

    2016-01-01

    Metagenomics is a trending research area, calling for the need to analyze large quantities of data generated from next generation DNA sequencing technologies. The need to store, retrieve, analyze, share, and visualize such data challenges current online computational systems. Interpretation and annotation of specific information is especially a challenge for metagenomic data sets derived from environmental samples, because current annotation systems only offer broad classification of microbial diversity and function. Moreover, existing resources are not configured to readily address common questions relevant to environmental systems. Here we developed a new online user-friendly metagenomic analysis server called MetaStorm (http://bench.cs.vt.edu/MetaStorm/), which facilitates customization of computational analysis for metagenomic data sets. Users can upload their own reference databases to tailor the metagenomics annotation to focus on various taxonomic and functional gene markers of interest. MetaStorm offers two major analysis pipelines: an assembly-based annotation pipeline and the standard read annotation pipeline used by existing web servers. These pipelines can be selected individually or together. Overall, MetaStorm provides enhanced interactive visualization to allow researchers to explore and manipulate taxonomy and functional annotation at various levels of resolution. PMID:27632579

  10. Selection in coastal Synechococcus (cyanobacteria populations evaluated from environmental metagenomes.

    Directory of Open Access Journals (Sweden)

    Vera Tai

    Full Text Available Environmental metagenomics provides snippets of genomic sequences from all organisms in an environmental sample and are an unprecedented resource of information for investigating microbial population genetics. Current analytical methods, however, are poorly equipped to handle metagenomic data, particularly of short, unlinked sequences. A custom analytical pipeline was developed to calculate dN/dS ratios, a common metric to evaluate the role of selection in the evolution of a gene, from environmental metagenomes sequenced using 454 technology of flow-sorted populations of marine Synechococcus, the dominant cyanobacteria in coastal environments. The large majority of genes (98% have evolved under purifying selection (dN/dS1, 77 out of 83 (93% were hypothetical. Notable among annotated genes, ribosomal protein L35 appears to be under positive selection in one Synechococcus population. Other annotated genes, in particular a possible porin, a large-conductance mechanosensitive channel, an ATP binding component of an ABC transporter, and a homologue of a pilus retraction protein had regions of the gene with elevated dN/dS. With the increasing use of next-generation sequencing in metagenomic investigations of microbial diversity and ecology, analytical methods need to accommodate the peculiarities of these data streams. By developing a means to analyze population diversity data from these environmental metagenomes, we have provided the first insight into the role of selection in the evolution of Synechococcus, a globally significant primary producer.

  11. Microbial community profiling of human saliva using shotgun metagenomic sequencing.

    Directory of Open Access Journals (Sweden)

    Nur A Hasan

    Full Text Available Human saliva is clinically informative of both oral and general health. Since next generation shotgun sequencing (NGS is now widely used to identify and quantify bacteria, we investigated the bacterial flora of saliva microbiomes of two healthy volunteers and five datasets from the Human Microbiome Project, along with a control dataset containing short NGS reads from bacterial species representative of the bacterial flora of human saliva. GENIUS, a system designed to identify and quantify bacterial species using unassembled short NGS reads was used to identify the bacterial species comprising the microbiomes of the saliva samples and datasets. Results, achieved within minutes and at greater than 90% accuracy, showed more than 175 bacterial species comprised the bacterial flora of human saliva, including bacteria known to be commensal human flora but also Haemophilus influenzae, Neisseria meningitidis, Streptococcus pneumoniae, and Gamma proteobacteria. Basic Local Alignment Search Tool (BLASTn analysis in parallel, reported ca. five times more species than those actually comprising the in silico sample. Both GENIUS and BLAST analyses of saliva samples identified major genera comprising the bacterial flora of saliva, but GENIUS provided a more precise description of species composition, identifying to strain in most cases and delivered results at least 10,000 times faster. Therefore, GENIUS offers a facile and accurate system for identification and quantification of bacterial species and/or strains in metagenomic samples.

  12. A Short Version of the Revised ‘Experience of Close Relationships Questionnaire’: Investigating Non-Clinical and Clinical Samples

    OpenAIRE

    Wongpakaran, Tinakon; Wongpakaran, Nahathai

    2012-01-01

    Aim: This study seeks to investigate the psychometric properties of the short version of the revised ‘Experience of Close Relationships’ questionnaire, comparing non-clinical and clinical samples. Methods: In total 702 subjects participated in this study, of whom 531 were non-clinical participants and 171 were psychiatric patients. They completed the short version of the revised ‘Experience of Close Relationships’ questionnaire (ECR-R-18), the Perceived Stress Scale-10(PSS-10), the Rosenberg ...

  13. Recovering full-length viral genomes from metagenomes

    Directory of Open Access Journals (Sweden)

    Saskia eSmits

    2015-10-01

    Full Text Available Infectious disease metagenomics is driven by the question: what is causing the disease? in contrast to classical metagenome studies which are guided by what is out there?. In case of a novel virus, a first step to eventually establishing etiology can be to recover a full-length viral genome from a metagenomic sample. However retrieval of a full-length genome of a divergent virus is technically challenging and can be time-consuming and costly. Here we discuss different assembly and fragment linkage strategies such as iterative assembly, motif searches, k-mer frequency profiling, coverage profile binning and other strategies used to recover genomes of potential viral pathogens in a timely and cost-effective manner.

  14. Fast and sensitive taxonomic classification for metagenomics with Kaiju

    DEFF Research Database (Denmark)

    Menzel, Peter; Ng, Kim Lee; Krogh, Anders

    2016-01-01

    The constantly decreasing cost and increasing output of current sequencing technologies enable large scale metagenomic studies of microbial communities from diverse habitats. Therefore, fast and accurate methods for taxonomic classification are needed, which can operate on increasingly larger...... datasets and reference databases. Recently, several fast metagenomic classifiers have been developed, which are based on comparison of genomic k-mers. However, nucleotide comparison using a fixed k-mer length often lacks the sensitivity to overcome the evolutionary distance between sampled species...... and genomes in the reference database. Here, we present the novel metagenome classifier Kaiju for fast assignment of reads to taxa. Kaiju finds maximum exact matches on the protein-level using the Borrows-Wheeler transform, and can optionally allow amino acid substitutions in the search using a greedy...

  15. Genovo: De Novo Assembly for Metagenomes

    Science.gov (United States)

    Laserson, Jonathan; Jojic, Vladimir; Koller, Daphne

    Next-generation sequencing technologies produce a large number of noisy reads from the DNA in a sample. Metagenomics and population sequencing aim to recover the genomic sequences of the species in the sample, which could be of high diversity. Methods geared towards single sequence reconstruction are not sensitive enough when applied in this setting. We introduce a generative probabilistic model of read generation from environmental samples and present Genovo, a novel de novo sequence assembler that discovers likely sequence reconstructions under the model. A Chinese restaurant process prior accounts for the unknown number of genomes in the sample. Inference is made by applying a series of hill-climbing steps iteratively until convergence. We compare the performance of Genovo to three other short read assembly programs across one synthetic dataset and eight metagenomic datasets created using the 454 platform, the largest of which has 311k reads. Genovo's reconstructions cover more bases and recover more genes than the other methods, and yield a higher assembly score.

  16. Reliable single cell array CGH for clinical samples.

    Directory of Open Access Journals (Sweden)

    Zbigniew T Czyż

    Full Text Available BACKGROUND: Disseminated cancer cells (DCCs and circulating tumor cells (CTCs are extremely rare, but comprise the precursors cells of distant metastases or therapy resistant cells. The detailed molecular analysis of these cells may help to identify key events of cancer cell dissemination, metastatic colony formation and systemic therapy escape. METHODOLOGY/PRINCIPAL FINDINGS: Using the Ampli1™ whole genome amplification (WGA technology and high-resolution oligonucleotide aCGH microarrays we optimized conditions for the analysis of structural copy number changes. The protocol presented here enables reliable detection of numerical genomic alterations as small as 0.1 Mb in a single cell. Analysis of single cells from well-characterized cell lines and single normal cells confirmed the stringent quantitative nature of the amplification and hybridization protocol. Importantly, fixation and staining procedures used to detect DCCs showed no significant impact on the outcome of the analysis, proving the clinical usability of our method. In a proof-of-principle study we tracked the chromosomal changes of single DCCs over a full course of high-dose chemotherapy treatment by isolating and analyzing DCCs of an individual breast cancer patient at four different time points. CONCLUSIONS/SIGNIFICANCE: The protocol enables detailed genome analysis of DCCs and thereby assessment of the clonal evolution during the natural course of the disease and under selection pressures. The results from an exemplary patient provide evidence that DCCs surviving selective therapeutic conditions may be recruited from a pool of genomically less advanced cells, which display a stable subset of specific genomic alterations.

  17. Metagenomic species profiling using universal phylogenetic marker genes

    NARCIS (Netherlands)

    Sunagawa, S.; Mende, D.R.; Zeller, G.; Izquierdo-Carrasco, F.; Berger, S.A.; Kultima, J.R.; Coelho, L.P.; Arumugam, M.; Tap, J.; Nielsen, H.B.; Rasmussen, S.; Brunak, S.; Pedersen, O.; Guarner, F.; Vos, de W.M.; Wang, J.; Li, J.; Doré, J.; Ehrlich, S.D.; Stamatakis, A.; Bork, P.

    2013-01-01

    To quantify known and unknown microorganisms at species-level resolution using shotgun sequencing data, we developed a method that establishes metagenomic operational taxonomic units (mOTUs) based on single-copy phylogenetic marker genes. Applied to 252 human fecal samples, the method revealed that

  18. Metagenomics and other Methods for Measuring Antibiotic Resistance in Agroecosystems

    Science.gov (United States)

    Background: There is broad concern regarding antibiotic resistance on farms and in fields, however there is no standard method for defining or measuring antibiotic resistance in environmental samples. Methods: We used metagenomic, culture-based, and molecular methods to characterize the amount, t...

  19. Metagenomic species profiling using universal phylogenetic marker genes

    DEFF Research Database (Denmark)

    Sunagawa, Shinichi; Mende, Daniel R; Zeller, Georg;

    2013-01-01

    To quantify known and unknown microorganisms at species-level resolution using shotgun sequencing data, we developed a method that establishes metagenomic operational taxonomic units (mOTUs) based on single-copy phylogenetic marker genes. Applied to 252 human fecal samples, the method revealed...

  20. Prevalence of dermatophytes and other fungal agents isolated from clinical samples

    OpenAIRE

    Kannan P.; Janaki C; Selvi G

    2006-01-01

    The common cause of skin infections are dermatophytes and opportunistic fungi. Aim of this study was to isolate and identify the fungal agents from clinical samples from patients with different mycoses. Clinical samples from 165 patients were subjected to potassium hydroxide (KOH) examination and culture isolation; causative agents were identified macroscopically and microscopically. All the 165 specimens were KOH positive and 110/165 (66.7%) samples were culture positive. Of these, hi...

  1. The Reliability and Validity of the Clinical Perfectionism Questionnaire in Eating Disorder and Community Samples

    OpenAIRE

    Egan, Sarah J.; Shafran, Roz; Lee, Michelle; Fairburn, Christopher G.; Cooper, Zafra; Doll, Helen A.; Palmer, Robert L.; Watson, Hunna J.

    2015-01-01

    Background: Clinical perfectionism is a risk and maintaining factor for anxiety disorders, depression and eating disorders. Aims: The aim was to examine the psychometric properties of the 12-item Clinical Perfectionism Questionnaire (CPQ). Method: The research involved two samples. Study 1 comprised a nonclinical sample (n = 206) recruited via the internet. Study 2 comprised individuals in treatment for an eating disorder (n = 129) and a community sample (n = 80). Results: Study 1 factor anal...

  2. Machine learning for metagenomics: methods and tools

    OpenAIRE

    Soueidan, Hayssam; Nikolski, Macha

    2015-01-01

    Owing to the complexity and variability of metagenomic studies, modern machine learning approaches have seen increased usage to answer a variety of question encompassing the full range of metagenomic NGS data analysis. We review here the contribution of machine learning techniques for the field of metagenomics, by presenting known successful approaches in a unified framework. This review focuses on five important metagenomic problems: OTU-clustering, binning, taxonomic profling and assignment...

  3. [From molecular to genomic and metagenomic epidemiology].

    Science.gov (United States)

    Zhebrun, A B

    2014-01-01

    The notion "molecular epidemiology" was introduced into scientific literature by Kilburn E. et al. in 1973. The first period of development of infectious diseases molecular epidemiology may be called "genotypic" (1980-1990s). During this period methodology of molecular marking of pathogens for purposes of monitoring of their spread and outbreak detection (novel nomenclature of diphtheria corynebacteria based on ribotyping; international network PulseNet for monitoring food source infections; international database of tuberculosis mycobacteria spoligotypes) was created. The second--"genomic" period started in the 2000s. Molecular epidemiology rapidly went through single markers (genotypes or single genes) to deciphering the whole genome of pathoge "mobileome", "resistome", "virulome" etc. took an important place in the studies of emerging and pandemic infections. Knowledge on genetic mechanisms leading to emergence and global dissemination of novel pathogens give molecular epidemiology its own scientific content and transforms it from a methodical approach to an independent field of epidemiology. The third--"metagenomic" period starts nowadays based on meta-genomic approach that allows to determine the whole set ofgenomes in the studied sample without the cultivation procedure. In the short-term this would lead to a change of a century-long paradigm of diagnostics and control of infections: instead of search of separate (key) pathogens--characteristics of the full specter of microorganisms in the material from patients and environmental samples with its identification up to any taxonomic depth. In the systems of regional and global epidemiologic control a universal monitoring of all known and re-emerging pathogens with construction and maintenance of metagenomic passports of human habitats will be realized. PMID:25286516

  4. [From molecular to genomic and metagenomic epidemiology].

    Science.gov (United States)

    Zhebrun, A B

    2014-01-01

    The notion "molecular epidemiology" was introduced into scientific literature by Kilburn E. et al. in 1973. The first period of development of infectious diseases molecular epidemiology may be called "genotypic" (1980-1990s). During this period methodology of molecular marking of pathogens for purposes of monitoring of their spread and outbreak detection (novel nomenclature of diphtheria corynebacteria based on ribotyping; international network PulseNet for monitoring food source infections; international database of tuberculosis mycobacteria spoligotypes) was created. The second--"genomic" period started in the 2000s. Molecular epidemiology rapidly went through single markers (genotypes or single genes) to deciphering the whole genome of pathoge "mobileome", "resistome", "virulome" etc. took an important place in the studies of emerging and pandemic infections. Knowledge on genetic mechanisms leading to emergence and global dissemination of novel pathogens give molecular epidemiology its own scientific content and transforms it from a methodical approach to an independent field of epidemiology. The third--"metagenomic" period starts nowadays based on meta-genomic approach that allows to determine the whole set ofgenomes in the studied sample without the cultivation procedure. In the short-term this would lead to a change of a century-long paradigm of diagnostics and control of infections: instead of search of separate (key) pathogens--characteristics of the full specter of microorganisms in the material from patients and environmental samples with its identification up to any taxonomic depth. In the systems of regional and global epidemiologic control a universal monitoring of all known and re-emerging pathogens with construction and maintenance of metagenomic passports of human habitats will be realized.

  5. Application of 16S rRNA metagenomics to analyze bacterial communities at a respiratory care centre in Taiwan.

    Science.gov (United States)

    Tang, Chuan Yi; Yiu, Siu-Ming; Kuo, Han-Yueh; Tan, Te-Sheng; Liao, Ki-Hok; Liu, Chih-Chin; Hon, Wing-Kai; Liou, Ming-Li

    2015-03-01

    In this study, we applied a 16S ribosomal RNA (rRNA) metagenomics approach to survey inanimate hospital environments (IHEs) in a respiratory care center (RCC). A total of 16 samples, including 9 from medical devices and 7 from workstations, were analyzed. Besides, clinical isolates were retrospectively analyzed during the sampling period in the RCC. A high amount of microbial diversity was detected, with an average of 1,836 phylotypes per sample. In addition to Acinetobacter, more than 60 % of the bacterial communities present among the top 25 abundant genera were dominated by skin-associated bacteria. Differences in bacterial profiles were restricted to individual samples. Furthermore, compliance with hand hygiene guidelines may be unsatisfactory among hospital staff according to a principal coordinate analysis that indicated clustering of bacterial communities between devices and workstations for most of the sampling sites. Compared to the high incidence of clinical isolates in the RCC, only Staphylococcus and Acinetobacter were highly abundant in the IHEs. Despite Acinetobacter was the most abundant genus present in IHEs of the RCC, potential pathogens, e.g., Acinetobacter baumannii, might remain susceptible to carbapenem. This study is the first in Taiwan to demonstrate a high diversity of human-associated bacteria in the RCC via 16S rRNA metagenomics, which allows for new assessment of potential health risks in RCCs, aids in the evaluation of existing sanitation protocols, and furthers our understanding of the development of healthcare-associated infections.

  6. Exploring Metagenomics in the Laboratory of an Introductory Biology Course †

    OpenAIRE

    Brian B. Gibbens; Scott, Cheryl L.; Hoff, Courtney D.; Janet L. Schottel

    2015-01-01

    Four laboratory modules were designed for introductory biology students to explore the field of metagenomics. Students collected microbes from environmental samples, extracted the DNA, and amplified 16S rRNA gene sequences using polymerase chain reaction (PCR). Students designed functional metagenomics screens to determine and compare antibiotic resistance profiles among the samples. Bioinformatics tools were used to generate and interpret phylogenetic trees and identify homologous genes. A p...

  7. Web Resources for Metagenomics Studies

    Institute of Scientific and Technical Information of China (English)

    Pravin Dudhagara; Sunil Bhavsar; Chintan Bhagat; Anjana Ghelani; Shreyas Bhatt; Rajesh Patel

    2015-01-01

    The development of next-generation sequencing (NGS) platforms spawned an enormous volume of data. This explosion in data has unearthed new scalability challenges for existing bioinformatics tools. The analysis of metagenomic sequences using bioinformatics pipelines is complicated by the substantial complexity of these data. In this article, we review several commonly-used online tools for metagenomics data analysis with respect to their quality and detail of analysis using simulated metagenomics data. There are at least a dozen such software tools presently available in the public domain. Among them, MGRAST, IMG/M, and METAVIR are the most well-known tools according to the number of citations by peer-reviewed scientific media up to mid-2015. Here, we describe 12 online tools with respect to their web link, annotation pipelines, clustering methods, online user support, and availability of data storage. We have also done the rating for each tool to screen more potential and preferential tools and evaluated five best tools using synthetic metagenome. The article comprehensively deals with the contemporary problems and the prospects of metagenomics from a bioinformatics viewpoint.

  8. Web Resources for Metagenomics Studies.

    Science.gov (United States)

    Dudhagara, Pravin; Bhavsar, Sunil; Bhagat, Chintan; Ghelani, Anjana; Bhatt, Shreyas; Patel, Rajesh

    2015-10-01

    The development of next-generation sequencing (NGS) platforms spawned an enormous volume of data. This explosion in data has unearthed new scalability challenges for existing bioinformatics tools. The analysis of metagenomic sequences using bioinformatics pipelines is complicated by the substantial complexity of these data. In this article, we review several commonly-used online tools for metagenomics data analysis with respect to their quality and detail of analysis using simulated metagenomics data. There are at least a dozen such software tools presently available in the public domain. Among them, MGRAST, IMG/M, and METAVIR are the most well-known tools according to the number of citations by peer-reviewed scientific media up to mid-2015. Here, we describe 12 online tools with respect to their web link, annotation pipelines, clustering methods, online user support, and availability of data storage. We have also done the rating for each tool to screen more potential and preferential tools and evaluated five best tools using synthetic metagenome. The article comprehensively deals with the contemporary problems and the prospects of metagenomics from a bioinformatics viewpoint.

  9. More than a (negative feeling: Validity of the perceived stress scale in Serbian clinical and non-clinical samples

    Directory of Open Access Journals (Sweden)

    Jovanović Veljko

    2015-01-01

    Full Text Available The goal of the present study was to test the validity of a Serbian version of the Perceived Stress Scale. The PSS was administered to 157 psychiatric outpatients, 165 adults from the non-clinical population, and 283 university students. The results of the confirmatory factor analysis supported a bifactor model of the PSS with one general factor and two specific factors reflecting perceived distress and perceived self-efficacy. Internal consistencies of the scale and its two subscales were adequate across clinical and non-clinical samples. Results supported the ability of the scale to discriminate between clinical and non-clinical samples. The PSS evidenced good convergent validity, showing moderate to high positive correlations with measures of unpleasant emotional states and moderate negative correlations with positive affect and life satisfaction. All but one correlation remained significant after controlling for the measures of emotional distress. The results of the present research support validity of the PSS and its use in both clinical and non-clinical samples. [Projekat Ministarstva nauke Republike Srbije, br. 179006

  10. Toward molecular trait-based ecology through integration of biogeochemical, geographical and metagenomic data

    DEFF Research Database (Denmark)

    Raes, Jeroen; Letunic, Ivica; Yamada, Takuji;

    2011-01-01

    Using metagenomic 'parts lists' to infer global patterns on microbial ecology remains a significant challenge. To deduce important ecological indicators such as environmental adaptation, molecular trait dispersal, diversity variation and primary production from the gene pool of an ecosystem, we...... integrated 25 ocean metagenomes with geographical, meteorological and geophysicochemical data. We find that climatic factors (temperature, sunlight) are the major determinants of the biomolecular repertoire of each sample and the main limiting factor on functional trait dispersal (absence of biogeographic...... composition derived from metagenomes is an important quantitative readout for molecular trait-based biogeography and ecology....

  11. Quality Control and Pre-Qualification of NGS Libraries Made from Clinical Samples

    OpenAIRE

    Langmore, John

    2013-01-01

    We have compared sequencing metrics from different types of clinical samples and different methods of making NGS libraries, for purposes of quality control of the samples and sample preps. We have performed the metrics using the HiSeq and MiSeq, however the same QC metrics could be measured on other platforms. By choosing metrics that can be measured from small amounts of data (e.g., 300,000 reads), we can measure the quality of the clinical samples and NGS libraries in a highly multiplexed f...

  12. Clinical, radiological and molecular diagnosis correlation in serum samples from patients with osteoarticular tuberculosis

    Directory of Open Access Journals (Sweden)

    Guadalupe García-Elorriaga

    2014-07-01

    Conclusions: Nested PCR in serum samples is a rapid, highly sensitive and specific modality for OTB detection. PCR should be performed in addition to clinical evaluation, imaging studies, acid-fast bacilli staining, culture and histopathology diagnosis, if possible.

  13. Whole metagenome profiling reveals skin microbiome-dependent susceptibility to atopic dermatitis flare.

    Science.gov (United States)

    Chng, Kern Rei; Tay, Angeline Su Ling; Li, Chenhao; Ng, Amanda Hui Qi; Wang, Jingjing; Suri, Bani Kaur; Matta, Sri Anusha; McGovern, Naomi; Janela, Baptiste; Wong, Xuan Fei Colin C; Sio, Yang Yie; Au, Bijin Veonice; Wilm, Andreas; De Sessions, Paola Florez; Lim, Thiam Chye; Tang, Mark Boon Yang; Ginhoux, Florent; Connolly, John E; Lane, E Birgitte; Chew, Fook Tim; Common, John E A; Nagarajan, Niranjan

    2016-01-01

    Whole metagenome analysis has the potential to reveal functional triggers of skin diseases, but issues of cost, robustness and sampling efficacy have limited its application. Here, we have established an alternative, clinically practical and robust metagenomic analysis protocol and applied it to 80 skin microbiome samples epidemiologically stratified for atopic dermatitis (AD). We have identified distinct non-flare, baseline skin microbiome signatures enriched for Streptococcus and Gemella but depleted for Dermacoccus in AD-prone versus normal healthy skin. Bacterial challenge assays using keratinocytes and monocyte-derived dendritic cells established distinct IL-1-mediated, innate and Th1-mediated adaptive immune responses with Staphylococcus aureus and Staphylococcus epidermidis. Bacterial differences were complemented by perturbations in the eukaryotic community and functional shifts in the microbiome-wide gene repertoire, which could exacerbate a dry and alkaline phenotype primed for pathogen growth and inflammation in AD-susceptible skin. These findings provide insights into how the skin microbial community, skin surface microenvironment and immune system cross-modulate each other, escalating the destructive feedback cycle between them that leads to AD flare. PMID:27562258

  14. Psychotic experiences in a mental health clinic sample : implications for suicidality, multimorbidity and functioning

    NARCIS (Netherlands)

    Kelleher, I.; Devlin, N.; Wigman, J. T. W.; Kehoe, A.; Murtagh, A.; Fitzpatrick, C.; Cannon, M.

    2014-01-01

    Background Recent community-based research has suggested that psychotic experiences act as markers of severity of psychopathology. There has, however, been a lack of clinic-based research. We wished to investigate, in a clinical sample of adolescents referred to a state-funded mental health service,

  15. Validity of the Aberrant Behavior Checklist in a Clinical Sample of Toddlers

    Science.gov (United States)

    Karabekiroglu, Koray; Aman, Michael G.

    2009-01-01

    We investigated the congruent and criterion validity of the Aberrant Behavior Checklist (ABC) in a clinical sample of toddlers seen over 1 year in Turkey. All consecutive patients (N = 93), 14-43 months old (mean, 30.6 mos.), in a child psychiatry outpatient clinic were included. The ABC, Autism Behavior Checklist (AuBC), and Child Behavior…

  16. Metagenomic analysis of microbial communities and beyond

    DEFF Research Database (Denmark)

    Schreiber, Lars

    2014-01-01

    From small clone libraries to large next-generation sequencing datasets – the field of community genomics or metagenomics has developed tremendously within the last years. This chapter will summarize some of these developments and will also highlight pitfalls of current metagenomic analyses....... It will illustrate the general workflow of a metagenomic study and introduce the three different metagenomic approaches: (1) the random shotgun approach that focuses on the metagenome as a whole, (2) the targeted approach that focuses on metagenomic amplicon sequences, and (3) the function-driven approach that uses...... heterologous expression of metagenomic DNA fragments to discover novel metabolic functions. Lastly, the chapter will shortly discuss the meta-analysis of gene expression of microbial communities, more precisely metatranscriptomics and metaproteomics....

  17. The Dutch version of the Child Posttraumatic Cognitions Inventory: validation in a clinical sample and a school sample

    Directory of Open Access Journals (Sweden)

    Julia Diehle

    2015-02-01

    Full Text Available Background: With the inclusion of trauma-related cognitions in the DSM-5 criteria for posttraumatic stress disorder (PTSD, the assessment of these cognitions has become essential. Therefore, valid tools for the assessment of these cognitions are warranted. Objective: The current study aimed at validating the Dutch version of the Child Posttraumatic Cognitions Inventory (CPTCI. Method: We included children aged 8–19 years in our study and assessed the factor structure, reliability and validity of the CPTCI in a clinical sample (n=184 and a school sample (n=318. Results: Our results supported the two-factor structure of the CPTCI and showed good internal consistency for the total scale and the two subscales. We found significant positive correlations between the CPTCI and measures of PTSD, depression, and anxiety disorder. The CPTCI correlated negatively with a measure of quality of life. Furthermore, we found significantly higher scores in the clinical sample than in the school sample. For children who received treatment, we found that a decrease in CPTCI scores was accompanied by a decrease in posttraumatic stress symptoms and comorbid problems indicating that the CPTCI is able to detect treatment effects. Conclusion: Overall, our results suggest that the Dutch CPTCI is a reliable and valid instrument.

  18. The single-species metagenome: subtyping Staphylococcus aureus core genome sequences from shotgun metagenomic data

    Science.gov (United States)

    Li, Ben; Petit III, Robert A.; Qin, Zhaohui S.; Darrow, Lyndsey

    2016-01-01

    In this study we developed a genome-based method for detecting Staphylococcus aureus subtypes from metagenome shotgun sequence data. We used a binomial mixture model and the coverage counts at >100,000 known S. aureus SNP (single nucleotide polymorphism) sites derived from prior comparative genomic analysis to estimate the proportion of 40 subtypes in metagenome samples. We were able to obtain >87% sensitivity and >94% specificity at 0.025X coverage for S. aureus. We found that 321 and 149 metagenome samples from the Human Microbiome Project and metaSUB analysis of the New York City subway, respectively, contained S. aureus at genome coverage >0.025. In both projects, CC8 and CC30 were the most common S. aureus clonal complexes encountered. We found evidence that the subtype composition at different body sites of the same individual were more similar than random sampling and more limited evidence that certain body sites were enriched for particular subtypes. One surprising finding was the apparent high frequency of CC398, a lineage often associated with livestock, in samples from the tongue dorsum. Epidemiologic analysis of the HMP subject population suggested that high BMI (body mass index) and health insurance are possibly associated with S. aureus carriage but there was limited power to identify factors linked to carriage of even the most common subtype. In the NYC subway data, we found a small signal of geographic distance affecting subtype clustering but other unknown factors influence taxonomic distribution of the species around the city. PMID:27781166

  19. Use of whole genome shotgun metagenomics: a practical guide for the microbiome-minded physician scientist.

    Science.gov (United States)

    Ma, Jun; Prince, Amanda; Aagaard, Kjersti M

    2014-01-01

    Whole genome shotgun sequencing (WGS) has been increasingly recognized as the most comprehensive and robust approach for metagenomics research. When compared with 16S-based metagenomics, it offers the advantage of identification of species level taxonomy and the estimation of metabolic pathway activities from human and environmental samples. Several large-scale metagenomic projects have been recently conducted or are currently underway utilizing WGS. With the generation of vast amounts of data, the bioinformatics and computational analysis of WGS results become vital for the success of a metagenomics study. However, each step in the WGS data analysis, including metagenome assembly, gene prediction, taxonomy identification, function annotation, and pathway analysis, is complicated by the shear amount of data. Algorithms and tools have been developed specifically to handle WGS-generated metagenomics data with the hope of reducing the requirement on computational time and storage space. Here, we present an overview of the current state of metagenomics through WGS sequencing, challenges frequently encountered, and up-to-date solutions. Several applications that are uniquely applicable to microbiome studies in reproductive and perinatal medicine are also discussed.

  20. Comparative metagenomics demonstrating different degradative capacity of activated biomass treating hydrocarbon contaminated wastewater.

    Science.gov (United States)

    Yadav, Trilok Chandra; Pal, Rajesh Ramavadh; Shastri, Sunita; Jadeja, Niti B; Kapley, Atya

    2015-01-01

    This study demonstrates the diverse degradative capacity of activated biomass, when exposed to different levels of total dissolved solids (TDS) using a comparative metagenomics approach. The biomass was collected at two time points to examine seasonal variations. Four metagenomes were sequenced on Illumina Miseq platform and analysed using MG-RAST. STAMP tool was used to analyse statistically significant differences amongst different attributes of metagenomes. Metabolic pathways related to degradation of aromatics via the central and peripheral pathways were found to be dominant in low TDS metagenome, while pathways corresponding to central carbohydrate metabolism, nitrogen, organic acids were predominant in high TDS sample. Seasonal variation was seen to affect catabolic gene abundance as well as diversity of the microbial community. Degradation of model compounds using activated sludge demonstrated efficient utilisation of single aromatic ring compounds in both samples but cyclic compounds were not efficiently utilised by biomass exposed to high TDS.

  1. The Stice model of overeating: Tests in clinical and non-clinical samples

    NARCIS (Netherlands)

    Strien, T. van; Engels, R.C.M.E.; Leeuwe, J.F.J. van; Snoek, H.M.

    2005-01-01

    The present study tested the dual pathway model of Stice [Stice, E (1994). A review of the evidence for a sociocultural model of bulimia nervosa and an exploration of the mechanisms of action. Clinical Psychology Review, 14, 633-661 and Stice, E. (2001). A prospective test of the dual-pathway model

  2. Detection of classical swine fever virus (CSFV) in clinical samples by RT-PCR assay in clinical samples by RT-PCR assay using different pairs of primers

    International Nuclear Information System (INIS)

    The aim was to compare the efficiency of RT-PCT assays using four pairs of primers selected from different regions of the CSFV genome for the detection of CSFV in clinical samples of swine and wild boars. The four RT-PCR assays were able to detect CSFV in all 20 clinical samples which had been collected from dead swine and wild boars during the outbreaks of CSF in Slovakia in 1993 and 1994. The quality of the selected RT-PCR primers was determined as follows: gp55L/gp55U (E2), 324/326 (5'-NC), S1/S2 (NS5B) and gp54L/gp54U (NS2 genomic region). We conclude that gp55L/gp55U primers are the most suitable for direct detection of CSFV by RT-PCR in tissue homogenates of diseased animals

  3. Unreliability of Results of PCR Detection of Helicobacter pylori in Clinical or Environmental Samples ▿ †

    OpenAIRE

    Sugimoto, Mitsushige; Wu, Jeng-Yih; Abudayyeh, Suhaib; Hoffman, Jill; Brahem, Hajer; Al-Khatib, Khaldun; Yamaoka, Yoshio; David Y Graham

    2009-01-01

    The aim of this study was to compare published Helicobacter pylori primer pairs for their ability to reliably detect H. pylori in gastric biopsy specimens and salivary samples. Detection limits of the 26 PCR primer pairs previously described for detection of H. pylori DNA in clinical samples were determined. Sensitivity and specificity were determined using primers with detection limits of

  4. A retrospective metagenomics approach to studying Blastocystis

    DEFF Research Database (Denmark)

    Andersen, Lee O'Brien; Bonde, Ida; Nielsen, Henrik Bjørn;

    2015-01-01

    a selection of 316 human faecal samples, hence representing genes originating from a single subtype. The 316 faecal samples were from 236 healthy individuals, 13 patients with Crohn's disease (CD) and 67 patients with ulcerative colitis (UC). The prevalence of Blastocystis was 20.3% in the healthy individuals...... metagenomic datasets targeting the bacterial component of the intestinal microbiome and the interplay between these microbial communities....

  5. Enhancing Metagenomics Investigations of Microbial Interactions with Biofilm Technology

    OpenAIRE

    Kakirde, Kavita S.; McLean, Robert J. C.

    2013-01-01

    Investigations of microbial ecology and diversity have been greatly enhanced by the application of culture-independent techniques. One such approach, metagenomics, involves sample collections from soil, water, and other environments. Extracted nucleic acids from bulk environmental samples are sequenced and analyzed, which allows microbial interactions to be inferred on the basis of bioinformatics calculations. In most environments, microbial interactions occur predominately in surface-adheren...

  6. The Microbiome of Brazilian Mangrove Sediments as Revealed by Metagenomics

    OpenAIRE

    Fernando Dini Andreote; Diego Javier Jiménez; Diego Chaves; Armando Cavalcante Franco Dias; Danice Mazzer Luvizotto; Francisco Dini-Andreote; Cristiane Cipola Fasanella; Maryeimy Varon Lopez; Sandra Baena; Rodrigo Gouvêa Taketani; Itamar Soares de Melo

    2012-01-01

    Here we embark in a deep metagenomic survey that revealed the taxonomic and potential metabolic pathways aspects of mangrove sediment microbiology. The extraction of DNA from sediment samples and the direct application of pyrosequencing resulted in approximately 215 Mb of data from four distinct mangrove areas (BrMgv01 to 04) in Brazil. The taxonomic approaches applied revealed the dominance of Deltaproteobacteria and Gammaproteobacteria in the samples. Paired statistical analysis showed high...

  7. Rapid whole genome sequencing for the detection and characterization of microorganisms directly from clinical samples

    DEFF Research Database (Denmark)

    Hasman, Henrik; Saputra, Dhany; Sicheritz-Pontén, Thomas;

    2014-01-01

    Whole genome sequencing (WGS) is becoming available as a routine tool for clinical microbiology. If applied directly on clinical samples this could further reduce diagnostic time and thereby improve control and treatment. A major bottle-neck is the availability of fast and reliable bioinformatics...... information and drastically reduce diagnostic time. This may prove very useful, but the need for data analysis is still a hurdle to clinical implementation. To overcome this problem a publicly available bioinformatics tool was developed in this study....... tools. This study was conducted to evaluate the applicability of WGS directly on clinical samples and to develop easy-to-use bioinformatics tools for analysis of the sequencing data. Thirty-five random urine samples from patients with suspected urinary tract infections were examined using conventional...

  8. A statistical toolbox for metagenomics: assessing functional diversity in microbial communities

    OpenAIRE

    Handelsman Jo; Schloss Patrick D

    2008-01-01

    Abstract Background The 99% of bacteria in the environment that are recalcitrant to culturing have spurred the development of metagenomics, a culture-independent approach to sample and characterize microbial genomes. Massive datasets of metagenomic sequences have been accumulated, but analysis of these sequences has focused primarily on the descriptive comparison of the relative abundance of proteins that belong to specific functional categories. More robust statistical methods are needed to ...

  9. Metagenomic data of fungal internal transcribed spacer from serofluid dish, a traditional Chinese fermented food

    OpenAIRE

    Peng Chen; Yang Zhao; Zhengrong Wu; Ronghui Liu; Ruixiang Xu; Lei Yan; Hongyu Li

    2015-01-01

    Serofluid dish (or Jiangshui, in Chinese), a traditional food in the Chinese culture for thousands of years, is made from vegetables by fermentation. In this work, microorganism community of the fermented serofluid dish was investigated by the culture-independent method. The metagenomic data in this article contains the sequences of fungal internal transcribed spacer (ITS) regions of rRNA genes from 12 different serofluid dish samples. The metagenome comprised of 50,865 average raw reads with...

  10. Metagenome Fragment Classification Using -Mer Frequency Profiles

    Directory of Open Access Journals (Sweden)

    Gail Rosen

    2008-01-01

    Full Text Available A vast amount of microbial sequencing data is being generated through large-scale projects in ecology, agriculture, and human health. Efficient high-throughput methods are needed to analyze the mass amounts of metagenomic data, all DNA present in an environmental sample. A major obstacle in metagenomics is the inability to obtain accuracy using technology that yields short reads. We construct the unique -mer frequency profiles of 635 microbial genomes publicly available as of February 2008. These profiles are used to train a naive Bayes classifier (NBC that can be used to identify the genome of any fragment. We show that our method is comparable to BLAST for small 25 bp fragments but does not have the ambiguity of BLAST's tied top scores. We demonstrate that this approach is scalable to identify any fragment from hundreds of genomes. It also performs quite well at the strain, species, and genera levels and achieves strain resolution despite classifying ubiquitous genomic fragments (gene and nongene regions. Cross-validation analysis demonstrates that species-accuracy achieves 90% for highly-represented species containing an average of 8 strains. We demonstrate that such a tool can be used on the Sargasso Sea dataset, and our analysis shows that NBC can be further enhanced.

  11. Sample size and power for comparing two or more treatment groups in clinical trials.

    OpenAIRE

    Day, S. J.; Graham, D F

    1989-01-01

    Methods for determining sample size and power when comparing two groups in clinical trials are widely available. Studies comparing three or more treatments are not uncommon but are more difficult to analyse. A linear nomogram was devised to help calculate the sample size required when comparing up to five parallel groups. It may also be used retrospectively to determine the power of a study of given sample size. In two worked examples the nomogram was efficient. Although the nomogram offers o...

  12. Multivariate analysis of functional metagenomes

    Directory of Open Access Journals (Sweden)

    Elizabeth A. Dinsdale

    2013-04-01

    Full Text Available Metagenomics is a primary tool for the description of microbial and viral communities. The sheer magnitude of the data generated in each metagenome makes identifying key differences in the function and taxonomy between communities difficult to elucidate. Here we discuss the application of seven different data mining and statistical analyses by comparing and contrasting the metabolic functions of 212 microbial metagenomes within and between 10 environments. Not all approaches are appropriate for all questions, and researchers should decide which approach addresses their questions. This work demonstrated the use of each approach: for example, random forests provided a robust and enlightening description of both the clustering of metagenomes and the metabolic processes that were important in separating microbial communities from different environments. All analyses identified that the presence of phage genes within the microbial community was a predictor of whether the microbial community was host associated or free living. Several analyses identified the subtle differences that occur with environments, such as those seen in different regions of the marine environment.

  13. Estimating richness from phage metagenomes

    Science.gov (United States)

    Bacteriophages are important drivers of ecosystem functions, yet little is known about the vast majority of phages. Phage metagenomics, or the study of the collective genome of an assemblage of phages, enables the investigation of broad ecological questions in phage communities. One ecological cha...

  14. Functional Intestinal Metagenomics (Chapter 18)

    NARCIS (Netherlands)

    Bogert, van den B.; Leimena, M.M.; Vos, de W.M.; Zoetendal, E.G.; Kleerebezem, M.

    2011-01-01

    The premiere two-volume reference on revelations from studying complex microbial communities in many distinct habitats Metagenomics is an emerging field that has changed the way microbiologists study microorganisms. It involves the genomic analysis of microorganisms by extraction and cloning of DNA

  15. Gene prediction in metagenomic fragments: A large scale machine learning approach

    Directory of Open Access Journals (Sweden)

    Morgenstern Burkhard

    2008-04-01

    Full Text Available Abstract Background Metagenomics is an approach to the characterization of microbial genomes via the direct isolation of genomic sequences from the environment without prior cultivation. The amount of metagenomic sequence data is growing fast while computational methods for metagenome analysis are still in their infancy. In contrast to genomic sequences of single species, which can usually be assembled and analyzed by many available methods, a large proportion of metagenome data remains as unassembled anonymous sequencing reads. One of the aims of all metagenomic sequencing projects is the identification of novel genes. Short length, for example, Sanger sequencing yields on average 700 bp fragments, and unknown phylogenetic origin of most fragments require approaches to gene prediction that are different from the currently available methods for genomes of single species. In particular, the large size of metagenomic samples requires fast and accurate methods with small numbers of false positive predictions. Results We introduce a novel gene prediction algorithm for metagenomic fragments based on a two-stage machine learning approach. In the first stage, we use linear discriminants for monocodon usage, dicodon usage and translation initiation sites to extract features from DNA sequences. In the second stage, an artificial neural network combines these features with open reading frame length and fragment GC-content to compute the probability that this open reading frame encodes a protein. This probability is used for the classification and scoring of gene candidates. With large scale training, our method provides fast single fragment predictions with good sensitivity and specificity on artificially fragmented genomic DNA. Additionally, this method is able to predict translation initiation sites accurately and distinguishes complete from incomplete genes with high reliability. Conclusion Large scale machine learning methods are well-suited for gene

  16. Marine Metagenome as A Resource for Novel Enzymes

    KAUST Repository

    Alma’abadi, Amani D.

    2015-11-10

    More than 99% of identified prokaryotes, including many from the marine environment, cannot be cultured in the laboratory. This lack of capability restricts our knowledge of microbial genetics and community ecology. Metagenomics, the culture-independent cloning of environmental DNAs that are isolated directly from an environmental sample, has already provided a wealth of information about the uncultured microbial world. It has also facilitated the discovery of novel biocatalysts by allowing researchers to probe directly into a huge diversity of enzymes within natural microbial communities. Recent advances in these studies have led to great interest in recruiting microbial enzymes for the development of environmentally-friendly industry. Although the metagenomics approach has many limitations, it is expected to provide not only scientific insights but also economic benefits, especially in industry. This review highlights the importance of metagenomics in mining microbial lipases, as an example, by using high-throughput techniques. In addition, we discuss challenges in the metagenomics as an important part of bioinformatics analysis in big data.

  17. Marine Metagenome as A Resource for Novel Enzymes

    Institute of Scientific and Technical Information of China (English)

    Amani D. Alma’abadi; Takashi Gojobori; Katsuhiko Mineta

    2015-01-01

    More than 99%of identified prokaryotes, including many from the marine environment, cannot be cultured in the laboratory. This lack of capability restricts our knowledge of microbial genetics and community ecology. Metagenomics, the culture-independent cloning of environmental DNAs that are isolated directly from an environmental sample, has already provided a wealth of information about the uncultured microbial world. It has also facilitated the discovery of novel bio-catalysts by allowing researchers to probe directly into a huge diversity of enzymes within natural microbial communities. Recent advances in these studies have led to a great interest in recruiting microbial enzymes for the development of environmentally-friendly industry. Although the metage-nomics approach has many limitations, it is expected to provide not only scientific insights but also economic benefits, especially in industry. This review highlights the importance of metagenomics in mining microbial lipases, as an example, by using high-throughput techniques. In addition, we dis-cuss challenges in the metagenomics as an important part of bioinformatics analysis in big data.

  18. Marine Metagenome as A Resource for Novel Enzymes

    Directory of Open Access Journals (Sweden)

    Amani D. Alma’abadi

    2015-10-01

    Full Text Available More than 99% of identified prokaryotes, including many from the marine environment, cannot be cultured in the laboratory. This lack of capability restricts our knowledge of microbial genetics and community ecology. Metagenomics, the culture-independent cloning of environmental DNAs that are isolated directly from an environmental sample, has already provided a wealth of information about the uncultured microbial world. It has also facilitated the discovery of novel biocatalysts by allowing researchers to probe directly into a huge diversity of enzymes within natural microbial communities. Recent advances in these studies have led to a great interest in recruiting microbial enzymes for the development of environmentally-friendly industry. Although the metagenomics approach has many limitations, it is expected to provide not only scientific insights but also economic benefits, especially in industry. This review highlights the importance of metagenomics in mining microbial lipases, as an example, by using high-throughput techniques. In addition, we discuss challenges in the metagenomics as an important part of bioinformatics analysis in big data.

  19. The Frequency of the Accidental Contamination with Laboratory Samples in Yazd Clinical Laboratories’ personnel in 2011

    Directory of Open Access Journals (Sweden)

    Jafari, AA. (PhD

    2014-05-01

    Full Text Available Background and Objective: laboratory personnel have always accidental exposure to clinical samples, which can cause the transmission of infection. This threat can be prevented and controlled by education for the use of safety instruments. The purpose was to determine the frequency of accidental exposure to laboratory samples among Yazd laboratory personnel in 2011. Material and Methods: This descriptive cross-sectional study was conducted on 100 of Yazd clinical laboratory personnel. The data was collected, using a valid and reliable questioner, via interview and analyzed by means of SPSS software. Results: Eighty-six percent of the subjects reported an experience of accidental exposure to clinical samples, such as blood, serum and urine. The causes were carelessness (41% and work overload (29%. Needle- stick was the most prevalent injury (52% particularly in sampler workers (51% and in their hands (69%. There wasn’t significant relationship between accidental exposure to laboratory samples and the variables such as private and governmental laboratories (p=0.517, kind of employment (p=0.411, record of services (p=0.439 and academic degree (p=0.454. The subjects aged 20-29 (p=0.034 and worked in sampling unit had the highest accidental exposure. Conclusion: based on the results, inexperience of the personnel especially in sampling room, overload at work and ignorance of applying safety instruments are known as the most important reasons for accidental exposure to clinical samples. Keywords: Contamination; accidental Exposure; Infectious agents; laboratory; personnel

  20. Diversity Indices as Measures of Functional Annotation Methods in Metagenomics Studies

    KAUST Repository

    Jankovic, Boris

    2016-01-26

    Applications of high-throughput techniques in metagenomics studies produce massive amounts of data. Fragments of genomic, transcriptomic and proteomic molecules are all found in metagenomics samples. Laborious and meticulous effort in sequencing and functional annotation are then required to, amongst other objectives, reconstruct a taxonomic map of the environment that metagenomics samples were taken from. In addition to computational challenges faced by metagenomics studies, the analysis is further complicated by the presence of contaminants in the samples, potentially resulting in skewed taxonomic analysis. The functional annotation in metagenomics can utilize all available omics data and therefore different methods that are associated with a particular type of data. For example, protein-coding DNA, non-coding RNA or ribosomal RNA data can be used in such an analysis. These methods would have their advantages and disadvantages and the question of comparison among them naturally arises. There are several criteria that can be used when performing such a comparison. Loosely speaking, methods can be evaluated in terms of computational complexity or in terms of the expected biological accuracy. We propose that the concept of diversity that is used in the ecosystems and species diversity studies can be successfully used in evaluating certain aspects of the methods employed in metagenomics studies. We show that when applying the concept of Hill’s diversity, the analysis of variations in the diversity order provides valuable clues into the robustness of methods used in the taxonomical analysis.

  1. Genetic diversity of multidrug resistant Staphylococcus aureus isolated from clinical and non clinical samples in Egypt.

    Science.gov (United States)

    Bendary, M M; Solyman, S M; Azab, M M; Mahmoud, N F; Hanora, A M

    2016-01-01

    In recent years, the increasing incidence of diseases caused by Staphylococcus aureus (S. aureus) has been noted in the university hospitals of El-Sharkia and Assuit governorates - Egypt. Therefore, we studied the genetic relatedness of multidrug resistant S. aureus isolates from different sources in the above mentioned governorates. One hundred and fifty six S. aureus isolates were divided into 5 different groups, 1 non clinical isolates from different food products and 4 different clinical isolates of human and animal sources in the 2 different governorates. Epidemiological characteristics of 156 S. aureus isolates were determined by phenotypic methods including quantitative antibiogram typing and biofilm production. Genetic typing of 35 multidrug resistant (MDR) isolates (7 from each group) based on 16S rRNA gene sequence, virulence and antimicrobial resistance gene profiles was done. The genetic relatedness of the highest virulent strain from each group was detected based on different single locus sequence typing and multi-locus sequence typing (MLST). S. aureus strains isolated from different sources and geographical areas showed high diversity. The genetic typing revealed different sequence types and different sequences of coa and spa genes. S. aureus isolates were found highly diverse in Egypt. PMID:27609475

  2. Functional metagenomics to decipher food-microbe-host crosstalk.

    Science.gov (United States)

    Larraufie, Pierre; de Wouters, Tomas; Potocki-Veronese, Gabrielle; Blottière, Hervé M; Doré, Joël

    2015-02-01

    The recent developments of metagenomics permit an extremely high-resolution molecular scan of the intestinal microbiota giving new insights and opening perspectives for clinical applications. Beyond the unprecedented vision of the intestinal microbiota given by large-scale quantitative metagenomics studies, such as the EU MetaHIT project, functional metagenomics tools allow the exploration of fine interactions between food constituents, microbiota and host, leading to the identification of signals and intimate mechanisms of crosstalk, especially between bacteria and human cells. Cloning of large genome fragments, either from complex intestinal communities or from selected bacteria, allows the screening of these biological resources for bioactivity towards complex plant polymers or functional food such as prebiotics. This permitted identification of novel carbohydrate-active enzyme families involved in dietary fibre and host glycan breakdown, and highlighted unsuspected bacterial players at the top of the intestinal microbial food chain. Similarly, exposure of fractions from genomic and metagenomic clones onto human cells engineered with reporter systems to track modulation of immune response, cell proliferation or cell metabolism has allowed the identification of bioactive clones modulating key cell signalling pathways or the induction of specific genes. This opens the possibility to decipher mechanisms by which commensal bacteria or candidate probiotics can modulate the activity of cells in the intestinal epithelium or even in distal organs such as the liver, adipose tissue or the brain. Hence, in spite of our inability to culture many of the dominant microbes of the human intestine, functional metagenomics open a new window for the exploration of food-microbe-host crosstalk. PMID:25417646

  3. Functional metagenomics to decipher food-microbe-host crosstalk.

    Science.gov (United States)

    Larraufie, Pierre; de Wouters, Tomas; Potocki-Veronese, Gabrielle; Blottière, Hervé M; Doré, Joël

    2015-02-01

    The recent developments of metagenomics permit an extremely high-resolution molecular scan of the intestinal microbiota giving new insights and opening perspectives for clinical applications. Beyond the unprecedented vision of the intestinal microbiota given by large-scale quantitative metagenomics studies, such as the EU MetaHIT project, functional metagenomics tools allow the exploration of fine interactions between food constituents, microbiota and host, leading to the identification of signals and intimate mechanisms of crosstalk, especially between bacteria and human cells. Cloning of large genome fragments, either from complex intestinal communities or from selected bacteria, allows the screening of these biological resources for bioactivity towards complex plant polymers or functional food such as prebiotics. This permitted identification of novel carbohydrate-active enzyme families involved in dietary fibre and host glycan breakdown, and highlighted unsuspected bacterial players at the top of the intestinal microbial food chain. Similarly, exposure of fractions from genomic and metagenomic clones onto human cells engineered with reporter systems to track modulation of immune response, cell proliferation or cell metabolism has allowed the identification of bioactive clones modulating key cell signalling pathways or the induction of specific genes. This opens the possibility to decipher mechanisms by which commensal bacteria or candidate probiotics can modulate the activity of cells in the intestinal epithelium or even in distal organs such as the liver, adipose tissue or the brain. Hence, in spite of our inability to culture many of the dominant microbes of the human intestine, functional metagenomics open a new window for the exploration of food-microbe-host crosstalk.

  4. Symptoms of Children with Autism Spectrum Disorder,a clinical sample

    Directory of Open Access Journals (Sweden)

    Ali Alavi Shooshtari

    2009-12-01

    Full Text Available "n Objective: "n "nThe aim of this report was to study the gender role on autismsymptoms distribution and severity in a clinical sample from Iran. Then, the results were compared with the published study from the same community population sample, Iran. "nMethod: The subjects of this retrospective study were a convenient clinical sample of the referrals of children with pervasive developmental disorders. The diagnosis was made according to DSM-IV diagnostic criteria. "nResults: "nMost of the subjects were boys. Boys were referred for evaluation more frequently than girls. The sample included 61 children and adolescents aged 2.1 to 15 years; of whom, 49 had autism. The mean age of children with autism was 7.2(SD=3.2 years. The mean of age, the diagnosis and severity of the symptoms were not related to gender . "n "n "nConclusion: Usually, those with severe cases of autism refer to clinics for treatment. Therefore, the clinical sample of children with autism is just the tip of the iceberg and they may not be the actual representative of community sample of children with autism. Preventive programs should be more focused on the screening and referring of inflected girls for service utilization .

  5. Subtractive assembly for comparative metagenomics, and its application to type 2 diabetes metagenomes

    OpenAIRE

    Wang, Mingjie; Doak, Thomas G.; Ye, Yuzhen

    2015-01-01

    Comparative metagenomics remains challenging due to the size and complexity of metagenomic datasets. Here we introduce subtractive assembly, a de novo assembly approach for comparative metagenomics that directly assembles only the differential reads that distinguish between two groups of metagenomes. Using simulated datasets, we show it improves both the efficiency of the assembly and the assembly quality of the differential genomes and genes. Further, its application to type 2 diabetes (T2D)...

  6. Experimental Design and Bioinformatics Analysis for the Application of Metagenomics in Environmental Sciences and Biotechnology.

    Science.gov (United States)

    Ju, Feng; Zhang, Tong

    2015-11-01

    Recent advances in DNA sequencing technologies have prompted the widespread application of metagenomics for the investigation of novel bioresources (e.g., industrial enzymes and bioactive molecules) and unknown biohazards (e.g., pathogens and antibiotic resistance genes) in natural and engineered microbial systems across multiple disciplines. This review discusses the rigorous experimental design and sample preparation in the context of applying metagenomics in environmental sciences and biotechnology. Moreover, this review summarizes the principles, methodologies, and state-of-the-art bioinformatics procedures, tools and database resources for metagenomics applications and discusses two popular strategies (analysis of unassembled reads versus assembled contigs/draft genomes) for quantitative or qualitative insights of microbial community structure and functions. Overall, this review aims to facilitate more extensive application of metagenomics in the investigation of uncultured microorganisms, novel enzymes, microbe-environment interactions, and biohazards in biotechnological applications where microbial communities are engineered for bioenergy production, wastewater treatment, and bioremediation.

  7. Use of simulated data sets to evaluate the fidelity of metagenomic processing methods

    Energy Technology Data Exchange (ETDEWEB)

    Mavromatis, K [U.S. Department of Energy, Joint Genome Institute; Ivanova, N [U.S. Department of Energy, Joint Genome Institute; Barry, Kerrie [U.S. Department of Energy, Joint Genome Institute; Shapiro, Harris [U.S. Department of Energy, Joint Genome Institute; Goltsman, Eugene [U.S. Department of Energy, Joint Genome Institute; McHardy, Alice C. [IBM T. J. Watson Research Center; Rigoutsos, Isidore [IBM T. J. Watson Research Center; Salamov, Asaf [U.S. Department of Energy, Joint Genome Institute; Korzeniewski, Frank [U.S. Department of Energy, Joint Genome Institute; Land, Miriam L [ORNL; Lapidus, Alla L. [U.S. Department of Energy, Joint Genome Institute; Grigoriev, Igor [U.S. Department of Energy, Joint Genome Institute; Hugenholtz, Philip [U.S. Department of Energy, Joint Genome Institute; Kyrpides, Nikos C [U.S. Department of Energy, Joint Genome Institute

    2007-01-01

    Metagenomics is a rapidly emerging field of research for studying microbial communities. To evaluate methods presently used to process metagenomic sequences, we constructed three simulated data sets of varying complexity by combining sequencing reads randomly selected from 113 isolate genomes. These data sets were designed to model real metagenomes in terms of complexity and phylogenetic composition. We assembled sampled reads using three commonly used genome assemblers (Phrap, Arachne and JAZZ), and predicted genes using two popular gene-finding pipelines (fgenesb and CRITICA/GLIMMER). The phylogenetic origins of the assembled contigs were predicted using one sequence similarity-based ( blast hit distribution) and two sequence composition-based (PhyloPythia, oligonucleotide frequencies) binning methods. We explored the effects of the simulated community structure and method combinations on the fidelity of each processing step by comparison to the corresponding isolate genomes. The simulated data sets are available online to facilitate standardized benchmarking of tools for metagenomic analysis.

  8. Metagenomic data of fungal internal transcribed spacer from serofluid dish, a traditional Chinese fermented food.

    Science.gov (United States)

    Chen, Peng; Zhao, Yang; Wu, Zhengrong; Liu, Ronghui; Xu, Ruixiang; Yan, Lei; Li, Hongyu

    2016-03-01

    Serofluid dish (or Jiangshui, in Chinese), a traditional food in the Chinese culture for thousands of years, is made from vegetables by fermentation. In this work, microorganism community of the fermented serofluid dish was investigated by the culture-independent method. The metagenomic data in this article contains the sequences of fungal internal transcribed spacer (ITS) regions of rRNA genes from 12 different serofluid dish samples. The metagenome comprised of 50,865 average raw reads with an average of 8,958,220 bp and G + C content is 45.62%. This is the first report on metagenomic data of fungal ITS from serofluid dish employing Illumina platform to profile the fungal communities of this little known fermented food from Gansu Province, China. The Metagenomic data of fungal internal transcribed spacer can be accessed at NCBI, SRA database accession no. SRP067411. PMID:26981389

  9. Metagenomic data of fungal internal transcribed spacer from serofluid dish, a traditional Chinese fermented food

    Directory of Open Access Journals (Sweden)

    Peng Chen

    2016-03-01

    Full Text Available Serofluid dish (or Jiangshui, in Chinese, a traditional food in the Chinese culture for thousands of years, is made from vegetables by fermentation. In this work, microorganism community of the fermented serofluid dish was investigated by the culture-independent method. The metagenomic data in this article contains the sequences of fungal internal transcribed spacer (ITS regions of rRNA genes from 12 different serofluid dish samples. The metagenome comprised of 50,865 average raw reads with an average of 8,958,220 bp and G + C content is 45.62%. This is the first report on metagenomic data of fungal ITS from serofluid dish employing Illumina platform to profile the fungal communities of this little known fermented food from Gansu Province, China. The Metagenomic data of fungal internal transcribed spacer can be accessed at NCBI, SRA database accession no. SRP067411.

  10. Coextraction of microbial metagenomic DNA and RNA from deep-sea sediment

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    A protocol to coextract the microbial metagenomic DNA and RNA from deep-sea sediment was developed for the microbiological study of environmental samples. The obtained pure metagenomic DNA with the size larger than 23 kb and stable RNA could be used directly for PCR and reverse transcription-PCR (RT-PCR) respectively. The direct lysis including the treatments of SDS, proteinase and lysozyme was applied to acquiring the metagenomic DNA and RNA furthest. Prior to the lysis treatment, the glass bead and denaturing solution were added to enhance the lysis efficiency and keep the integrity of RNA respectively. Denaturing gradient gel electrophoresis (DGGE) was applied in accessing the microbial 16S rRNA diversity by PCR and RT-PCR amplification from a single extraction. The pattern obtained by this analysis revealed some differences between them, indicating the efficiency of the protocol in extracting the metagenomic DNA and total RNA from deep-sea sediment.

  11. An application of statistics to comparative metagenomics

    Directory of Open Access Journals (Sweden)

    Rohwer Forest

    2006-03-01

    Full Text Available Abstract Background Metagenomics, sequence analyses of genomic DNA isolated directly from the environments, can be used to identify organisms and model community dynamics of a particular ecosystem. Metagenomics also has the potential to identify significantly different metabolic potential in different environments. Results Here we use a statistical method to compare curated subsystems, to predict the physiology, metabolism, and ecology from metagenomes. This approach can be used to identify those subsystems that are significantly different between metagenome sequences. Subsystems that were overrepresented in the Sargasso Sea and Acid Mine Drainage metagenome when compared to non-redundant databases were identified. Conclusion The methodology described herein applies statistics to the comparisons of metabolic potential in metagenomes. This analysis reveals those subsystems that are more, or less, represented in the different environments that are compared. These differences in metabolic potential lead to several testable hypotheses about physiology and metabolism of microbes from these ecosystems.

  12. Establishment of Detecting Tembusu Virus Method from Duck Source Samples Based on Metagenomics%基于宏基因组学的鸭源样本中坦布苏病毒检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    孙涛; 王超; 邓明俊; 郑小龙; 王群; 徐彪

    2016-01-01

    利用二代高通量测序技术建立检测鸭源样品中坦布苏病毒的方法。首先将样品过滤和经核酸酶处理,再利用等温链位移扩增(SPIA)技术快速制备样品总 cDNA,最后经磁珠纯化和文库构建后,通过Ion Torrent 进行高通量测序获得基因组信息。结果显示,通过 SPIA 扩增和核酸文库制备的优化,可从微量病原样本中获得327 pmol/μL 的核酸文库样本。Ion Torrent 测序共获得1725436条 reads,约6.2 M 数据,平均109 bp。数据拼接并 Blast 发现,病原样品的核酸序列可注释鸭坦布苏病毒全部基因组数据,与首次发生在我国的鸭坦布苏病毒 BYD-1株参考序列的同源性为100%。将所测病毒序列与其他5种黄病毒科成员进行同源性比对,发现与近3年发生在我国的鸭坦布苏病毒同源性最高,在98%以上。且 NS 基因进化树分析与鸭坦布苏病毒处于同一分支上,符合鸭坦布苏病毒特征。本研究建立的基于未知病原 SPIA 扩增结合高通量测序检测方法,可同步获知坦布苏病毒基因的核酸信息。%To explore the methods of high-throughput sequencing for Tembusu virus from duck source sam-ples based on metagenomics,first of all,interference was removed through filtering and nucleic acid enzyme treatment.Then,total cDNA was prepared by using isothermal strand-displacement amplification (SPIA) technology.The purification was made by using magnetic beads and the nucleotid library building,the cD-NA amplicons were sequenced by Ion Torrent,and the genome information was acquired.The results showed that 327 pmol/μL nucleic acid library was prepared from pathogenic samples through SPIA and op-timizing preparation of DNA library.1 725 436 reads,about 6.2 M data,were got by Ion Torrent sequen-cing,and average read was 109 bp.The nucleic acid sequence of pathogen samples can note entire genome data by Blast on NCBI with the pathogen analysis software

  13. Evaluation of a nonradiometric system (BACTEC 9000 MB) for detection of mycobacteria in human clinical samples.

    OpenAIRE

    ZANETTI, S.; Ardito, F; SECHI, L.; Sanguinetti, M.; Molicotti, P.; Delogu, G.; Pinna, M P; NACCI, A.; Fadda, G.

    1997-01-01

    This study was carried out to evaluate the rate of recovery and time required for detection of mycobacteria from pulmonary and extrapulmonary human clinical samples, by using a fluorescence-quenching-based oxygen sensor (BACTEC 9000 MB; Becton Dickinson Microbiology Systems, Sparks, Md.). The results were compared with those obtained by microscopy, conventional culture in Lowenstein-Jensen (LJ) medium, and a BACTEC radiometric system (BACTEC 460 TB; Becton Dickinson). Of the 779 clinical samp...

  14. A Novel Abundance-Based Algorithm for Binning Metagenomic Sequences Using l-Tuples

    Science.gov (United States)

    Wu, Yu-Wei; Ye, Yuzhen

    Metagenomics is the study of microbial communities sampled directly from their natural environment, without prior culturing. Among the computational tools recently developed for metagenomic sequence analysis, binning tools attempt to classify all (or most) of the sequences in a metagenomic dataset into different bins (i.e., species), based on various DNA composition patterns (e.g., the tetramer frequencies) of various genomes. Composition-based binning methods, however, cannot be used to classify very short fragments, because of the substantial variation of DNA composition patterns within a single genome. We developed a novel approach (AbundanceBin) for metagenomics binning by utilizing the different abundances of species living in the same environment. AbundanceBin is an application of the Lander-Waterman model to metagenomics, which is based on the l-tuple content of the reads. AbundanceBin achieved accurate, unsupervised, clustering of metagenomic sequences into different bins, such that the reads classified in a bin belong to species of identical or very similar abundances in the sample. In addition, AbundanceBin gave accurate estimations of species abundances, as well as their genome sizes - two important parameters for characterizing a microbial community. We also show that AbundanceBin performed well when the sequence lengths are very short (e.g. 75 bp) or have sequencing errors.

  15. An application of statistics to comparative metagenomics

    OpenAIRE

    Rohwer Forest; Rodriguez-Brito Beltran; Edwards Robert A

    2006-01-01

    Abstract Background Metagenomics, sequence analyses of genomic DNA isolated directly from the environments, can be used to identify organisms and model community dynamics of a particular ecosystem. Metagenomics also has the potential to identify significantly different metabolic potential in different environments. Results Here we use a statistical method to compare curated subsystems, to predict the physiology, metabolism, and ecology from metagenomes. This approach can be used to identify t...

  16. Indications, results, and clinical impact of endoscopic ultrasound (EUS)-guided sampling in gastroenterology

    DEFF Research Database (Denmark)

    Dumonceau, J-M; Polkowski, M; Larghi, A;

    2011-01-01

    This article is part of a combined publication that expresses the current view of the European Society of Gastrointestinal Endoscopy (ESGE) about endoscopic ultrasound (EUS)-guided sampling in gastroenterology, including EUS-guided fine needle aspiration (EUS-FNA) and EUS-guided trucut biopsy (EUS......-positive cytopathological results and needle tract seeding are also discussed. The present Clinical Guideline describes the results of EUS-guided sampling in the different clinical settings, considers the role of this technique in patient management, and makes recommendations on circumstances that warrant its use. A two...

  17. PhyloSift: phylogenetic analysis of genomes and metagenomes

    Directory of Open Access Journals (Sweden)

    Aaron E. Darling

    2014-01-01

    Full Text Available Like all organisms on the planet, environmental microbes are subject to the forces of molecular evolution. Metagenomic sequencing provides a means to access the DNA sequence of uncultured microbes. By combining DNA sequencing of microbial communities with evolutionary modeling and phylogenetic analysis we might obtain new insights into microbiology and also provide a basis for practical tools such as forensic pathogen detection. In this work we present an approach to leverage phylogenetic analysis of metagenomic sequence data to conduct several types of analysis. First, we present a method to conduct phylogeny-driven Bayesian hypothesis tests for the presence of an organism in a sample. Second, we present a means to compare community structure across a collection of many samples and develop direct associations between the abundance of certain organisms and sample metadata. Third, we apply new tools to analyze the phylogenetic diversity of microbial communities and again demonstrate how this can be associated to sample metadata. These analyses are implemented in an open source software pipeline called PhyloSift. As a pipeline, PhyloSift incorporates several other programs including LAST, HMMER, and pplacer to automate phylogenetic analysis of protein coding and RNA sequences in metagenomic datasets generated by modern sequencing platforms (e.g., Illumina, 454.

  18. Validation of the Novaco Anger Scale-Provocation Inventory (Danish) With Nonclinical, Clinical, and Offender Samples

    DEFF Research Database (Denmark)

    Moeller, Stine Bjerrum; Novaco, Raymond; Heinola-Nielsen, Vivian;

    2015-01-01

    Anger has high prevalence in clinical and forensic settings, and it is associated with aggressive behavior and ward atmosphere on psychiatric units. Dysregulated anger is a clinical problem in Danish mental health care systems, but no anger assessment instruments have been validated in Danish....... Because the Novaco Anger Scale and Provocation Inventory (NAS-PI) has been extensively validated with different clinical populations and lends itself to clinical case formulation, it was selected for translation and evaluation in the present multistudy project. Psychometric properties of the NAS-PI were...... investigated with samples of 477 nonclinical, 250 clinical, 167 male prisoner, and 64 male forensic participants. Anger prevalence and its relationship with other anger measures, anxiety/depression, and aggression were examined. NAS-PI was found to have high reliability, concurrent validity, and discriminant...

  19. A metagenomic analysis of pandemic influenza A (2009 H1N1 infection in patients from North America.

    Directory of Open Access Journals (Sweden)

    Alexander L Greninger

    Full Text Available Although metagenomics has been previously employed for pathogen discovery, its cost and complexity have prevented its use as a practical front-line diagnostic for unknown infectious diseases. Here we demonstrate the utility of two metagenomics-based strategies, a pan-viral microarray (Virochip and deep sequencing, for the identification and characterization of 2009 pandemic H1N1 influenza A virus. Using nasopharyngeal swabs collected during the earliest stages of the pandemic in Mexico, Canada, and the United States (n = 17, the Virochip was able to detect a novel virus most closely related to swine influenza viruses without a priori information. Deep sequencing yielded reads corresponding to 2009 H1N1 influenza in each sample (percentage of aligned sequences corresponding to 2009 H1N1 ranging from 0.0011% to 10.9%, with up to 97% coverage of the influenza genome in one sample. Detection of 2009 H1N1 by deep sequencing was possible even at titers near the limits of detection for specific RT-PCR, and the percentage of sequence reads was linearly correlated with virus titer. Deep sequencing also provided insights into the upper respiratory microbiota and host gene expression in response to 2009 H1N1 infection. An unbiased analysis combining sequence data from all 17 outbreak samples revealed that 90% of the 2009 H1N1 genome could be assembled de novo without the use of any reference sequence, including assembly of several near full-length genomic segments. These results indicate that a streamlined metagenomics detection strategy can potentially replace the multiple conventional diagnostic tests required to investigate an outbreak of a novel pathogen, and provide a blueprint for comprehensive diagnosis of unexplained acute illnesses or outbreaks in clinical and public health settings.

  20. Indications, results, and clinical impact of endoscopic ultrasound (EUS)-guided sampling in gastroenterology

    DEFF Research Database (Denmark)

    Dumonceau, J-M; Polkowski, M; Larghi, A;

    2011-01-01

    This article is part of a combined publication that expresses the current view of the European Society of Gastrointestinal Endoscopy (ESGE) about endoscopic ultrasound (EUS)-guided sampling in gastroenterology, including EUS-guided fine needle aspiration (EUS-FNA) and EUS-guided trucut biopsy (EUS......-positive cytopathological results and needle tract seeding are also discussed. The present Clinical Guideline describes the results of EUS-guided sampling in the different clinical settings, considers the role of this technique in patient management, and makes recommendations on circumstances that warrant its use. A two......-page executive summary of evidence statements and recommendations is provided. A separate Technical Guideline describes the general technique of EUS-guided sampling, particular techniques to maximize the diagnostic yield depending on the nature of the target lesion, and sample processing. The target readership...

  1. Exploration of noncoding sequences in metagenomes.

    Directory of Open Access Journals (Sweden)

    Fabián Tobar-Tosse

    Full Text Available Environment-dependent genomic features have been defined for different metagenomes, whose genes and their associated processes are related to specific environments. Identification of ORFs and their functional categories are the most common methods for association between functional and environmental features. However, this analysis based on finding ORFs misses noncoding sequences and, therefore, some metagenome regulatory or structural information could be discarded. In this work we analyzed 23 whole metagenomes, including coding and noncoding sequences using the following sequence patterns: (G+C content, Codon Usage (Cd, Trinucleotide Usage (Tn, and functional assignments for ORF prediction. Herein, we present evidence of a high proportion of noncoding sequences discarded in common similarity-based methods in metagenomics, and the kind of relevant information present in those. We found a high density of trinucleotide repeat sequences (TRS in noncoding sequences, with a regulatory and adaptive function for metagenome communities. We present associations between trinucleotide values and gene function, where metagenome clustering correlate with microorganism adaptations and kinds of metagenomes. We propose here that noncoding sequences have relevant information to describe metagenomes that could be considered in a whole metagenome analysis in order to improve their organization, classification protocols, and their relation with the environment.

  2. Metagenomic Assembly: Overview, Challenges and Applications

    Science.gov (United States)

    Ghurye, Jay S.; Cepeda-Espinoza, Victoria; Pop, Mihai

    2016-01-01

    Advances in sequencing technologies have led to the increased use of high throughput sequencing in characterizing the microbial communities associated with our bodies and our environment. Critical to the analysis of the resulting data are sequence assembly algorithms able to reconstruct genes and organisms from complex mixtures. Metagenomic assembly involves new computational challenges due to the specific characteristics of the metagenomic data. In this survey, we focus on major algorithmic approaches for genome and metagenome assembly, and discuss the new challenges and opportunities afforded by this new field. We also review several applications of metagenome assembly in addressing interesting biological problems. PMID:27698619

  3. Ruminations on rumination: anger and sadness rumination in a normative and clinical sample

    OpenAIRE

    Peled, Maya

    2006-01-01

    Anger rumination and sadness rumination were examined concurrently in a normative sample of adults (Study 1) and a clinical adolescent sample (Study 2). The purpose of this research was to assess if rumination on anger and sadness have distinct emotional and behavioural associations, and whether it is warranted to conceptualize them as separate constructs. In both studies, factor analysis indicated that items from analogous anger rumination and sadness rumination measures loaded onto two fact...

  4. Functional metagenomics of extreme environments.

    Science.gov (United States)

    Mirete, Salvador; Morgante, Verónica; González-Pastor, José Eduardo

    2016-04-01

    The bioprospecting of enzymes that operate under extreme conditions is of particular interest for many biotechnological and industrial processes. Nevertheless, there is a considerable limitation to retrieve novel enzymes as only a small fraction of microorganisms derived from extreme environments can be cultured under standard laboratory conditions. Functional metagenomics has the advantage of not requiring the cultivation of microorganisms or previous sequence information to known genes, thus representing a valuable approach for mining enzymes with new features. In this review, we summarize studies showing how functional metagenomics was employed to retrieve genes encoding for proteins involved not only in molecular adaptation and resistance to extreme environmental conditions but also in other enzymatic activities of biotechnological interest.

  5. Metagenomics, Metatranscriptomics, and Metabolomics Approaches for Microbiome Analysis

    OpenAIRE

    Vanessa Aguiar-Pulido; Wenrui Huang; Victoria Suarez-Ulloa; Trevor Cickovski; Kalai Mathee; Giri Narasimhan

    2016-01-01

    Microbiomes are ubiquitous and are found in the ocean, the soil, and in/on other living organisms. Changes in the microbiome can impact the health of the environmental niche in which they reside. In order to learn more about these communities, different approaches based on data from multiple omics have been pursued. Metagenomics produces a taxonomical profile of the sample, metatranscriptomics helps us to obtain a functional profile, and metabolomics completes the picture by determining which...

  6. Subtractive assembly for comparative metagenomics, and its application to type 2 diabetes metagenomes.

    Science.gov (United States)

    Wang, Mingjie; Doak, Thomas G; Ye, Yuzhen

    2015-11-02

    Comparative metagenomics remains challenging due to the size and complexity of metagenomic datasets. Here we introduce subtractive assembly, a de novo assembly approach for comparative metagenomics that directly assembles only the differential reads that distinguish between two groups of metagenomes. Using simulated datasets, we show it improves both the efficiency of the assembly and the assembly quality of the differential genomes and genes. Further, its application to type 2 diabetes (T2D) metagenomic datasets reveals clear signatures of the T2D gut microbiome, revealing new phylogenetic and functional features of the gut microbial communities associated with T2D.

  7. Expanding the marine virosphere using metagenomics.

    Directory of Open Access Journals (Sweden)

    Carolina Megumi Mizuno

    Full Text Available Viruses infecting prokaryotic cells (phages are the most abundant entities of the biosphere and contain a largely uncharted wealth of genomic diversity. They play a critical role in the biology of their hosts and in ecosystem functioning at large. The classical approaches studying phages require isolation from a pure culture of the host. Direct sequencing approaches have been hampered by the small amounts of phage DNA present in most natural habitats and the difficulty in applying meta-omic approaches, such as annotation of small reads and assembly. Serendipitously, it has been discovered that cellular metagenomes of highly productive ocean waters (the deep chlorophyll maximum contain significant amounts of viral DNA derived from cells undergoing the lytic cycle. We have taken advantage of this phenomenon to retrieve metagenomic fosmids containing viral DNA from a Mediterranean deep chlorophyll maximum sample. This method allowed description of complete genomes of 208 new marine phages. The diversity of these genomes was remarkable, contributing 21 genomic groups of tailed bacteriophages of which 10 are completely new. Sequence based methods have allowed host assignment to many of them. These predicted hosts represent a wide variety of important marine prokaryotic microbes like members of SAR11 and SAR116 clades, Cyanobacteria and also the newly described low GC Actinobacteria. A metavirome constructed from the same habitat showed that many of the new phage genomes were abundantly represented. Furthermore, other available metaviromes also indicated that some of the new phages are globally distributed in low to medium latitude ocean waters. The availability of many genomes from the same sample allows a direct approach to viral population genomics confirming the remarkable mosaicism of phage genomes.

  8. Automated Broad-Range Molecular Detection of Bacteria in Clinical Samples.

    Science.gov (United States)

    Budding, Andries E; Hoogewerf, Martine; Vandenbroucke-Grauls, Christina M J E; Savelkoul, Paul H M

    2016-04-01

    Molecular detection methods, such as quantitative PCR (qPCR), have found their way into clinical microbiology laboratories for the detection of an array of pathogens. Most routinely used methods, however, are directed at specific species. Thus, anything that is not explicitly searched for will be missed. This greatly limits the flexibility and universal application of these techniques. We investigated the application of a rapid universal bacterial molecular identification method, IS-pro, to routine patient samples received in a clinical microbiology laboratory. IS-pro is a eubacterial technique based on the detection and categorization of 16S-23S rRNA gene interspace regions with lengths that are specific for each microbial species. As this is an open technique, clinicians do not need to decide in advance what to look for. We compared routine culture to IS-pro using 66 samples sent in for routine bacterial diagnostic testing. The samples were obtained from patients with infections in normally sterile sites (without a resident microbiota). The results were identical in 20 (30%) samples, IS-pro detected more bacterial species than culture in 31 (47%) samples, and five of the 10 culture-negative samples were positive with IS-pro. The case histories of the five patients from whom these culture-negative/IS-pro-positive samples were obtained suggest that the IS-pro findings are highly clinically relevant. Our findings indicate that an open molecular approach, such as IS-pro, may have a high added value for clinical practice. PMID:26763956

  9. Maternal Drug Abuse History, Maltreatment, and Functioning in a Clinical Sample of Urban Children

    Science.gov (United States)

    Onigu-Otite, Edore C.; Belcher, Harolyn M. E.

    2012-01-01

    Objective: This study examined the association between maternal drug abuse history, maltreatment exposure, and functioning, in a clinical sample of young children seeking therapy for maltreatment. Methods: Data were collected on 91 children, mean age 5.3 years (SD 1.0). The Preschool and Early Childhood Functional Assessment Scales (PECFAS) was…

  10. A Mediation Model of Interparental Collaboration, Parenting Practices, and Child Externalizing Behavior in a Clinical Sample

    Science.gov (United States)

    Kjobli, John; Hagen, Kristine Amlund

    2009-01-01

    The present study examined maternal and paternal parenting practices as mediators of the link between interparental collaboration and children's externalizing behavior. Parent gender was tested as a moderator of the associations. A clinical sample consisting of 136 children with externalizing problems and their families participated in the study.…

  11. Protein Profile study of clinical samples using Laser Induced Fluorescence as the detection method

    DEFF Research Database (Denmark)

    Karemore, Gopal Raghunath; Raja, Sujatha N.; Rai, Lavanya;

    2009-01-01

    by using hard and Fuzzy clustering methods. The study was performed to test the utility of the HPLC-LIF protein profiling method for classification of tissue samples as well as to establish a complementary method for histopathology for clinical diagnosis of the tissue as normal or malignant.  ...

  12. Radioimmunoassay in situ used to detect products of cloning mycoplasma pneumoniae DNA and clinical micro samples

    International Nuclear Information System (INIS)

    From the recombinants, which constructed in the laboratory, using plasmids as vector, one strain which express antigen of Mycoplasma pneumoniae by detecting in situ radioimmunoassay was obtained. The authors detected 7 children samples which were throat washing liquids of children suspected of atypical pneumoniae, 4 of them positive by using above method. This data indicated that it was suitable for clinical diagnosis directly

  13. Intrusions, avoidance and overgeneral memory in a non-clinical sample

    NARCIS (Netherlands)

    Hauer, Beatrijs J. A.; Wessel, I.; Merckelbach, H.

    2006-01-01

    Previous studies have shown a positive relationship between intrusions, effortful avoidance and overgeneral memory in people suffering from (mild) depression or PTSD. The purpose of the present study was to investigate these relationships in a non-clinical sample. As part of a mass testing session,

  14. A statistical toolbox for metagenomics: assessing functional diversity in microbial communities

    Directory of Open Access Journals (Sweden)

    Handelsman Jo

    2008-01-01

    Full Text Available Abstract Background The 99% of bacteria in the environment that are recalcitrant to culturing have spurred the development of metagenomics, a culture-independent approach to sample and characterize microbial genomes. Massive datasets of metagenomic sequences have been accumulated, but analysis of these sequences has focused primarily on the descriptive comparison of the relative abundance of proteins that belong to specific functional categories. More robust statistical methods are needed to make inferences from metagenomic data. In this study, we developed and applied a suite of tools to describe and compare the richness, membership, and structure of microbial communities using peptide fragment sequences extracted from metagenomic sequence data. Results Application of these tools to acid mine drainage, soil, and whale fall metagenomic sequence collections revealed groups of peptide fragments with a relatively high abundance and no known function. When combined with analysis of 16S rRNA gene fragments from the same communities these tools enabled us to demonstrate that although there was no overlap in the types of 16S rRNA gene sequence observed, there was a core collection of operational protein families that was shared among the three environments. Conclusion The results of comparisons between the three habitats were surprising considering the relatively low overlap of membership and the distinctively different characteristics of the three habitats. These tools will facilitate the use of metagenomics to pursue statistically sound genome-based ecological analyses.

  15. An integrated metagenome and -proteome analysis of the microbial community residing in a biogas production plant.

    Science.gov (United States)

    Ortseifen, Vera; Stolze, Yvonne; Maus, Irena; Sczyrba, Alexander; Bremges, Andreas; Albaum, Stefan P; Jaenicke, Sebastian; Fracowiak, Jochen; Pühler, Alfred; Schlüter, Andreas

    2016-08-10

    To study the metaproteome of a biogas-producing microbial community, fermentation samples were taken from an agricultural biogas plant for microbial cell and protein extraction and corresponding metagenome analyses. Based on metagenome sequence data, taxonomic community profiling was performed to elucidate the composition of bacterial and archaeal sub-communities. The community's cytosolic metaproteome was represented in a 2D-PAGE approach. Metaproteome databases for protein identification were compiled based on the assembled metagenome sequence dataset for the biogas plant analyzed and non-corresponding biogas metagenomes. Protein identification results revealed that the corresponding biogas protein database facilitated the highest identification rate followed by other biogas-specific databases, whereas common public databases yielded insufficient identification rates. Proteins of the biogas microbiome identified as highly abundant were assigned to the pathways involved in methanogenesis, transport and carbon metabolism. Moreover, the integrated metagenome/-proteome approach enabled the examination of genetic-context information for genes encoding identified proteins by studying neighboring genes on the corresponding contig. Exemplarily, this approach led to the identification of a Methanoculleus sp. contig encoding 16 methanogenesis-related gene products, three of which were also detected as abundant proteins within the community's metaproteome. Thus, metagenome contigs provide additional information on the genetic environment of identified abundant proteins. PMID:27312700

  16. MBMC: An Effective Markov Chain Approach for Binning Metagenomic Reads from Environmental Shotgun Sequencing Projects.

    Science.gov (United States)

    Wang, Ying; Hu, Haiyan; Li, Xiaoman

    2016-08-01

    Metagenomics is a next-generation omics field currently impacting postgenomic life sciences and medicine. Binning metagenomic reads is essential for the understanding of microbial function, compositions, and interactions in given environments. Despite the existence of dozens of computational methods for metagenomic read binning, it is still very challenging to bin reads. This is especially true for reads from unknown species, from species with similar abundance, and/or from low-abundance species in environmental samples. In this study, we developed a novel taxonomy-dependent and alignment-free approach called MBMC (Metagenomic Binning by Markov Chains). Different from all existing methods, MBMC bins reads by measuring the similarity of reads to the trained Markov chains for different taxa instead of directly comparing reads with known genomic sequences. By testing on more than 24 simulated and experimental datasets with species of similar abundance, species of low abundance, and/or unknown species, we report here that MBMC reliably grouped reads from different species into separate bins. Compared with four existing approaches, we demonstrated that the performance of MBMC was comparable with existing approaches when binning reads from sequenced species, and superior to existing approaches when binning reads from unknown species. MBMC is a pivotal tool for binning metagenomic reads in the current era of Big Data and postgenomic integrative biology. The MBMC software can be freely downloaded at http://hulab.ucf.edu/research/projects/metagenomics/MBMC.html . PMID:27447888

  17. Use of the experience sampling method in the context of clinical trials

    Science.gov (United States)

    Verhagen, Simone J W; Hasmi, Laila; Drukker, Marjan; van Os, J; Delespaul, Philippe A E G

    2016-01-01

    Objective The experience sampling method (ESM) is a structured diary technique to appraise subjective experiences in daily life. It is applied in psychiatric patients, as well as in patients with somatic illness. Despite the potential of ESM assessment, the improved logistics and its increased administration in research, its use in clinical trials remains limited. This paper introduces ESM for clinical trials in psychiatry and beyond. Methods ESM is an ecologically valid method that yields a comprehensive view of an individual's daily life. It allows the assessment of various constructs (eg, quality of life, psychopathology) and psychological mechanisms (eg, stress-sensitivity, coping). These constructs are difficult to assess using cross-sectional questionnaires. ESM can be applied in treatment monitoring, as an ecological momentary intervention, in clinical trials, or in single case clinical trials. Technological advances (eg, smartphone applications) make its implementation easier. Results Advantages of ESM are highlighted and disadvantages are discussed. Furthermore, the ecological nature of ESM data and its consequences are explored, including the potential pitfalls of ambiguously formulated research questions and the specificities of ESM in statistical analyses. The last section focuses on ESM in relation to clinical trials and discusses its future use in optimising clinical decision-making. Conclusions ESM can be a valuable asset in clinical trial research and should be used more often to study the benefits of treatment in psychiatry and somatic health. PMID:27443678

  18. Buffer AVL Alone Does Not Inactivate Ebola Virus in a Representative Clinical Sample Type.

    Science.gov (United States)

    Smither, Sophie J; Weller, Simon A; Phelps, Amanda; Eastaugh, Lin; Ngugi, Sarah; O'Brien, Lyn M; Steward, Jackie; Lonsdale, Steve G; Lever, Mark S

    2015-10-01

    Rapid inactivation of Ebola virus (EBOV) is crucial for high-throughput testing of clinical samples in low-resource, outbreak scenarios. The EBOV inactivation efficacy of Buffer AVL (Qiagen) was tested against marmoset serum (EBOV concentration of 1 × 10(8) 50% tissue culture infective dose per milliliter [TCID50 · ml(-1)]) and murine blood (EBOV concentration of 1 × 10(7) TCID50 · ml(-1)) at 4:1 vol/vol buffer/sample ratios. Posttreatment cell culture and enzyme-linked immunosorbent assay (ELISA) analysis indicated that treatment with Buffer AVL did not inactivate EBOV in 67% of samples, indicating that Buffer AVL, which is designed for RNA extraction and not virus inactivation, cannot be guaranteed to inactivate EBOV in diagnostic samples. Murine blood samples treated with ethanol (4:1 [vol/vol] ethanol/sample) or heat (60°C for 15 min) also showed no viral inactivation in 67% or 100% of samples, respectively. However, combined Buffer AVL and ethanol or Buffer AVL and heat treatments showed total viral inactivation in 100% of samples tested. The Buffer AVL plus ethanol and Buffer AVL plus heat treatments were also shown not to affect the extraction of PCR quality RNA from EBOV-spiked murine blood samples. PMID:26179307

  19. Buffer AVL Alone Does Not Inactivate Ebola Virus in a Representative Clinical Sample Type.

    Science.gov (United States)

    Smither, Sophie J; Weller, Simon A; Phelps, Amanda; Eastaugh, Lin; Ngugi, Sarah; O'Brien, Lyn M; Steward, Jackie; Lonsdale, Steve G; Lever, Mark S

    2015-10-01

    Rapid inactivation of Ebola virus (EBOV) is crucial for high-throughput testing of clinical samples in low-resource, outbreak scenarios. The EBOV inactivation efficacy of Buffer AVL (Qiagen) was tested against marmoset serum (EBOV concentration of 1 × 10(8) 50% tissue culture infective dose per milliliter [TCID50 · ml(-1)]) and murine blood (EBOV concentration of 1 × 10(7) TCID50 · ml(-1)) at 4:1 vol/vol buffer/sample ratios. Posttreatment cell culture and enzyme-linked immunosorbent assay (ELISA) analysis indicated that treatment with Buffer AVL did not inactivate EBOV in 67% of samples, indicating that Buffer AVL, which is designed for RNA extraction and not virus inactivation, cannot be guaranteed to inactivate EBOV in diagnostic samples. Murine blood samples treated with ethanol (4:1 [vol/vol] ethanol/sample) or heat (60°C for 15 min) also showed no viral inactivation in 67% or 100% of samples, respectively. However, combined Buffer AVL and ethanol or Buffer AVL and heat treatments showed total viral inactivation in 100% of samples tested. The Buffer AVL plus ethanol and Buffer AVL plus heat treatments were also shown not to affect the extraction of PCR quality RNA from EBOV-spiked murine blood samples.

  20. Undergraduate Urban Metagenomics Research Module †

    OpenAIRE

    Muth, Theodore R.; McEntee, Catherine M.

    2014-01-01

    The undergraduate urban metagenomics research module is an adaptable and tractable project that can be incorporated into exisiting microbiology, ecology, bioinformatics, introductory biology or other courses.  The module takes advantage of recent advances in metagenomics and next generation sequencing, giving students an opportunity to apply these current technologies to novel questions regarding microbial community diversity and dynamics.

  1. Metagenomic data analysis : computational methods and applications

    NARCIS (Netherlands)

    Gori, F.

    2013-01-01

    Metagenomics is the study of the genomic content of microbial communities, acquired through DNA sequencing technology. The main advantage of metagenomics is that it can overcome the limitations of individual genome sequencing, that can work only on the few culturable microbes. Unfortunately, the an

  2. Clinical, radiological and molecular diagnosis correlation in serum samples from patients with osteoarticular tuberculosis

    Institute of Scientific and Technical Information of China (English)

    Guadalupe Garca-Elorriaga; Olga Martnez-Elizondo; Guillermo del Rey-Pineda; Csar Gonzlez-Bonilla

    2014-01-01

    Objective: To assess the role of polymerase chain reaction (PCR) in serum samples, in the diagnosis of osteoarticular tuberculosis (OTB) in a setting where only clinical and imaging diagnoses determine the treatment.Methods:A total of 44 consecutive serum specimens were collected from clinically suspected OTB patients, based on clinical and radiological [X-ray or magnetic resonance imaging/computed tomography] features. They were screened by in-house nested PCR. In addition, a few specimens were examined by Gram stain, acid-fast bacilli stain, histopathology and routine bacterial culture. A total of 39 specimens were collected from patients suffering from other bone diseases of nontuberculous origin and included as negative controls.Results:Of the 44 clinically suspected OTB patients, in-house nested PCR was positive in 40 (91%) cases; PCR was negative in 38 (97%) negative controls. Sensitivity and specificity of our in-house nested PCR was 90.9% and 97.4%, respectively. The PCR report was available within 48 h. It was possible to standardize serum PCR technique and in positive cases, a good correlation was observed in terms of an adequate treatment response.Conclusions:Nested PCR in serum samples is a rapid, highly sensitive and specific modality for OTB detection. PCR should be performed in addition to clinical evaluation, imaging studies, acid-fast bacilli staining, culture and histopathology diagnosis, if possible.

  3. Routine application of the polymerase chain reaction for detection of Mycobacterium tuberculosis in clinical samples.

    Science.gov (United States)

    Noordhoek, G T; Kaan, J A; Mulder, S; Wilke, H; Kolk, A H

    1995-01-01

    AIM--To investigate the use of the polymerase chain reaction (PCR) in the routine laboratory for the detection of Mycobacterium tuberculosis in clinical samples. METHODS--Samples were divided and processed separately for the detection of M tuberculosis by microscopy, culture and PCR. After DNA extraction, PCR was performed with primers specific for the insertion element IS6110 and the product was analysed by agarose gel electrophoresis, Southern blotting or dot blotting and hybridisation with a digoxigenin labelled internal probe. Each sample was tested for inhibitors of Taq polymerase with the aid of an internal control. Multiple negative and positive controls were used to monitor each step of the procedure. RESULTS--The data from two laboratories, using the same operating procedures, were combined. Of 1957 specimens, 79 (4%) were culture and PCR positive, while 1839 (93.9%) were negative in both tests. Thirty specimens (1.5%) were PCR positive only and nine (0.5%) were culture positive but PCR negative. CONCLUSION--Using culture and clinical history as the gold standard, sensitivity and specificity for PCR were 92.1% and 99.8%, respectively. With elaborate precautions, PCR is a suitable and reliable method for the detection of M tuberculosis in clinical samples in a routine microbiology laboratory. Images PMID:7490312

  4. Comparison of clinical samples for visceral Leishmaniasis diagnosis in asymptomatic dogs by PCR hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Ferreira, Sidney A.; Ituassu, Leonardo T.; Melo, Maria N. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Parasitologia], e-mail: saninoalmeida@gmail.com, e-mail: Itituassu@yahoo.com.br, e-mail: melo@icb.ufmg.br; Leite, Rodrigo S.; Andrade, Antero S.R. [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN-CNEN/MG), Belo Horizonte, MG (Brazil)], e-mail: rleite2005@gmail.com, e-mail: antero@cdtn.br

    2009-07-01

    The canine visceral leishmaniasis (CVL) diagnosis still represents a challenge because of complexity of this disease. The aim of present study was to compare different clinical samples for diagnosis of CVL by Polymerase Chain Reaction (PCR) combined with hybridization of {sup 32}P labeled probes. Bone marrow (BM), skin biopsy (SB), peripheral blood (PB) and conjunctival swab (CS) were used in this work. With this purpose 40 asymptomatic dogs, all positive by parasitological test, were obtained. From each animal were collected SB with sterile punches from ear internal surface, 1.0 mL of PB, BM aspirates from sternum and CS from both lower eyelid. Each clinical sample was submitted to suitable DNA purification process and PCR-hybridization assays. The positive results obtained with PCR were 55%, 25%, 30% and 22.5% for CS, BM, SB and PB respectively while the PCR followed by hybridization showed a positivity of 87.5%, 50%, 45% and 27.5% respectively. The hybridization assay was able to increase the PCR positivity in all kinds of clinical samples. The best performance was obtained using CS samples. We concluded that the PCR associated with DNA radioactive probes was a very sensitive tool for diagnosis of CVL in asymptomatic dogs and the CS has an important potential for regular screening of dogs. (author)

  5. Comparison of clinical samples for visceral Leishmaniasis diagnosis in asymptomatic dogs by PCR hybridization

    International Nuclear Information System (INIS)

    The canine visceral leishmaniasis (CVL) diagnosis still represents a challenge because of complexity of this disease. The aim of present study was to compare different clinical samples for diagnosis of CVL by Polymerase Chain Reaction (PCR) combined with hybridization of 32P labeled probes. Bone marrow (BM), skin biopsy (SB), peripheral blood (PB) and conjunctival swab (CS) were used in this work. With this purpose 40 asymptomatic dogs, all positive by parasitological test, were obtained. From each animal were collected SB with sterile punches from ear internal surface, 1.0 mL of PB, BM aspirates from sternum and CS from both lower eyelid. Each clinical sample was submitted to suitable DNA purification process and PCR-hybridization assays. The positive results obtained with PCR were 55%, 25%, 30% and 22.5% for CS, BM, SB and PB respectively while the PCR followed by hybridization showed a positivity of 87.5%, 50%, 45% and 27.5% respectively. The hybridization assay was able to increase the PCR positivity in all kinds of clinical samples. The best performance was obtained using CS samples. We concluded that the PCR associated with DNA radioactive probes was a very sensitive tool for diagnosis of CVL in asymptomatic dogs and the CS has an important potential for regular screening of dogs. (author)

  6. Culture-independent discovery of natural products from soil metagenomes.

    Science.gov (United States)

    Katz, Micah; Hover, Bradley M; Brady, Sean F

    2016-03-01

    Bacterial natural products have proven to be invaluable starting points in the development of many currently used therapeutic agents. Unfortunately, traditional culture-based methods for natural product discovery have been deemphasized by pharmaceutical companies due in large part to high rediscovery rates. Culture-independent, or "metagenomic," methods, which rely on the heterologous expression of DNA extracted directly from environmental samples (eDNA), have the potential to provide access to metabolites encoded by a large fraction of the earth's microbial biosynthetic diversity. As soil is both ubiquitous and rich in bacterial diversity, it is an appealing starting point for culture-independent natural product discovery efforts. This review provides an overview of the history of soil metagenome-driven natural product discovery studies and elaborates on the recent development of new tools for sequence-based, high-throughput profiling of environmental samples used in discovering novel natural product biosynthetic gene clusters. We conclude with several examples of these new tools being employed to facilitate the recovery of novel secondary metabolite encoding gene clusters from soil metagenomes and the subsequent heterologous expression of these clusters to produce bioactive small molecules. PMID:26586404

  7. Culture-independent discovery of natural products from soil metagenomes.

    Science.gov (United States)

    Katz, Micah; Hover, Bradley M; Brady, Sean F

    2016-03-01

    Bacterial natural products have proven to be invaluable starting points in the development of many currently used therapeutic agents. Unfortunately, traditional culture-based methods for natural product discovery have been deemphasized by pharmaceutical companies due in large part to high rediscovery rates. Culture-independent, or "metagenomic," methods, which rely on the heterologous expression of DNA extracted directly from environmental samples (eDNA), have the potential to provide access to metabolites encoded by a large fraction of the earth's microbial biosynthetic diversity. As soil is both ubiquitous and rich in bacterial diversity, it is an appealing starting point for culture-independent natural product discovery efforts. This review provides an overview of the history of soil metagenome-driven natural product discovery studies and elaborates on the recent development of new tools for sequence-based, high-throughput profiling of environmental samples used in discovering novel natural product biosynthetic gene clusters. We conclude with several examples of these new tools being employed to facilitate the recovery of novel secondary metabolite encoding gene clusters from soil metagenomes and the subsequent heterologous expression of these clusters to produce bioactive small molecules.

  8. Metagenomic applications in environmental monitoring and bioremediation.

    Science.gov (United States)

    Techtmann, Stephen M; Hazen, Terry C

    2016-10-01

    With the rapid advances in sequencing technology, the cost of sequencing has dramatically dropped and the scale of sequencing projects has increased accordingly. This has provided the opportunity for the routine use of sequencing techniques in the monitoring of environmental microbes. While metagenomic applications have been routinely applied to better understand the ecology and diversity of microbes, their use in environmental monitoring and bioremediation is increasingly common. In this review we seek to provide an overview of some of the metagenomic techniques used in environmental systems biology, addressing their application and limitation. We will also provide several recent examples of the application of metagenomics to bioremediation. We discuss examples where microbial communities have been used to predict the presence and extent of contamination, examples of how metagenomics can be used to characterize the process of natural attenuation by unculturable microbes, as well as examples detailing the use of metagenomics to understand the impact of biostimulation on microbial communities. PMID:27558781

  9. [Isolation of Sporothrix pallida complex in clinical and environmental samples from Chile].

    Science.gov (United States)

    Cruz Choappa, Rodrigo M; Vieille Oyarzo, Peggy I; Carvajal Silva, Laura C

    2014-01-01

    The isolation of S. pallida complex from medical samples and home garden soil of a patient in Chile is here in reported. Fungi of the Sporothrix schenckii complex can cause various infections. In Chile, the medical and environmental isolates of these this complex are rare. The aim of this study was to identify an unusual agent in a case of onychomycosis and to detect its presence in the patient's home garden. For this purpose, clinical samples were obtained by scraping the patient's subungueal first right toe nail as well as by taking soil samples from different areas of her home garden. Species identification was performed by morphophysiology and one of the strains isolated from the patient's toe nail was sent to CBS for molecular confirmation (14.062). S. pallida complex was identified both from the patient's toe nail and samples taken from her home garden.

  10. Sampling

    CERN Document Server

    Thompson, Steven K

    2012-01-01

    Praise for the Second Edition "This book has never had a competitor. It is the only book that takes a broad approach to sampling . . . any good personal statistics library should include a copy of this book." —Technometrics "Well-written . . . an excellent book on an important subject. Highly recommended." —Choice "An ideal reference for scientific researchers and other professionals who use sampling." —Zentralblatt Math Features new developments in the field combined with all aspects of obtaining, interpreting, and using sample data Sampling provides an up-to-date treat

  11. Diversity of Bipolaris species in clinical samples in the United States and their antifungal susceptibility profiles.

    Science.gov (United States)

    da Cunha, K C; Sutton, D A; Fothergill, A W; Cano, J; Gené, J; Madrid, H; De Hoog, S; Crous, P W; Guarro, J

    2012-12-01

    A set of 104 isolates from human clinical samples from the United States, morphologically compatible with Bipolaris, were morphologically and molecularly identified through the sequence analysis of the internal transcribed space (ITS) region of the nuclear ribosomal DNA (rDNA). The predominant species was Bipolaris spicifera (67.3%), followed by B. hawaiiensis (18.2%), B. cynodontis (8.6%), B. micropus (2.9%), B. australiensis (2%), and B. setariae (1%). Bipolaris cynodontis, B. micropus, and B. setariae represent new records from clinical samples. The most common anatomical sites where isolates were recovered were the nasal region (30.7%), skin (19.2%), lungs (14.4%), and eyes (12.5%). The antifungal susceptibilities of 5 species of Bipolaris to 9 drugs are provided. With the exception of fluconazole and flucytosine, the antifungals tested showed good activity. PMID:23052310

  12. Validity Evidences for the Dimensional Clinical Personality Inventory in Outpatient Psychiatric Sample

    Directory of Open Access Journals (Sweden)

    Roberta Katz Abela

    2015-08-01

    Full Text Available The Dimensional Clinical Personality Inventory (IDCP was developed in Brazil for the assessment of pathological personality traits. This study aimed to seek validity evidence for the dimensions of IDCP based on external criteria, psychiatric diagnosis. We examined the profile in IDCP of 105 psychotherapy outpatients, previously diagnosed with personality disorders. The profiles were compared with the profile of the normative non-clinical sample and we conducted the repeated measures analysis to investigate whether the IDCP is able to discriminate consistent profiles for different diagnoses and compared the general population. The results suggest validity evidence based on external criteria for the IDCP dimensions and points to the clinical effectiveness of the instrument.

  13. A Simplified Method to Recover Urinary Vesicles for Clinical Applications, and Sample Banking

    OpenAIRE

    Musante, Luca; Tataruch, Dorota; Gu, Dongfeng; Benito-Martin, Alberto; Calzaferri, Giulio; Aherne, Sinead; Holthofer, Harry

    2014-01-01

    Urinary extracellular vesicles provide a novel source for valuable biomarkers for kidney and urogenital diseases: Current isolation protocols include laborious, sequential centrifugation steps which hampers their widespread research and clinical use. Furthermore, large individual urine sample volumes or sizable target cohorts are to be processed (e.g. for biobanking), the storage capacity is an additional problem. Thus, alternative methods are necessary to overcome such limitations. We have d...

  14. Vibrio parahaemolyticus Strains of Pandemic Serotypes Identified from Clinical and Environmental Samples from Jiangsu, China

    OpenAIRE

    Jingjiao eLi; Feng eXue; Zhenquan eYang; Xiaoping eZhang; Dexin eZeng; Guoxiang eChao; Yuan eJiang; Baoguang eLi

    2016-01-01

    Vibrio parahaemolyticus has emerged as a major foodborne pathogen in China, Japan, Thailand and other Asian countries. In this study, 72 strains of V. parahaemolyticus were isolated from clinical and environmental samples between 2006 and 2014 in Jiangsu, China. The serotypes and six virulence genes including thermostable direct hemolysin (TDR) and TDR-related hemolysin (TRH) genes were assessed among the isolates. Twenty five serotypes were identified and O3:K6 was one of the dominant seroty...

  15. Diversity of Bipolaris species in clinical samples in the United States and their antifungal susceptibility profiles

    OpenAIRE

    Cunha, DA; Sutton, D.A.; Fothergill, A. W.; Cano, J.; Gene, J.; Madrid, H.; Hoog, de, G.S.; Crous, P.W.; Guarro, J

    2012-01-01

    A set of 104 isolates from human clinical samples from the United States, morphologically compatible with Bipolaris, were morphologically and molecularly identified through the sequence analysis of the internal transcribed space (ITS) region of the nuclear ribosomal DNA (rDNA). The predominant species was Bipolaris spicifera (67.3%), followed by B. hawaiiensis (18.2%), B. cynodontis (8.6%), B. micropus (2.9%), B. australiensis (2%), and B. setariae (1%). Bipolaris cynodontis, B. micropus, and...

  16. Clinical features and prognosis of a sample of patients with trisomy 13 (Patau syndrome) from Brazil.

    Science.gov (United States)

    Petry, Patrícia; Polli, Janaina B; Mattos, Vinícius F; Rosa, Rosana C M; Zen, Paulo R G; Graziadio, Carla; Paskulin, Giorgio A; Rosa, Rafael F M

    2013-06-01

    Trisomy 13 or Patau syndrome (PS) is a chromosomal disorder characterized by a well known presentation of multiple congenital anomalies. Our objective was to determine the clinical features and prognosis observed in a sample of patients with PS. The series was composed of patients with diagnosis of PS consecutively evaluated by a Clinical Genetics Service from a reference hospital of southern Brazil, in the period between 1975 and 2012. Statistical analysis was performed using PEPI program (version 4.0), with two-tailed Fisher's exact test for comparison of frequencies (P<0.05). The sample consisted of 30 patients, 60% male, median age at first evaluation of 9 days. Full trisomy of chromosome 13 was the main cytogenetic alteration (73%). The major clinical findings included: cryptorchidism (78%), abnormal auricles (77%), congenital heart defects (76%), polydactyly (63%), microphthalmia (60%) and micrognathia (50%). Four patients (13%) simultaneously had micro/anophthalmia, oral clefts and polydactyly. Some findings were only observed in our sample and included, among others, preauricular tags (10%), duplication of the hallux (3%) and spots following the lines of Blaschko (3%). Mosaicism (20% of cases) had a statistically significant association only with absence of cryptorchidism. The median of survival was 26 days. Patients with and without mosaicism had similar median of survival. Our findings, in agreement with the literature, show that the anomalies in patients with PS can be quite variable, sometimes even atypical. There is no pathognomonic finding, which may make the early identification of these patients challenging. PMID:23613355

  17. Significant Decline in Galactomannan Signal during Storage of Clinical Serum Samples

    Directory of Open Access Journals (Sweden)

    Samir G. Agrawal

    2013-06-01

    Full Text Available Galactomannan (GM is widely used for detection of invasive aspergillosis in high-risk haemato-oncology patients. Recent publications have reported a lack of repeatability of GM detection. The objective of this retrospective study was to assess the repeatability of GM levels during storage of clinical samples. In a GM screening strategy, positive sera were repeat tested as per manufacturer’s recommendations. Short-term (ST storage of samples was at +4 °C while long-term (LT storage was at −80 °C. Bronchoalveolar (BAL fluid was also repeating tested after ST storage and LT storage. Wilcoxon Signed Ranks Test was employed to assess the repeatability of GM levels. In a subset of 14 GM positive sera, repeat testing was performed on both the original serum and ethylenediaminetetraacetic acid (EDTA pre-treated sample. There was a significant reduction in GM signals on repeat testing following ST storage (median GM index: 0.65 vs. 0.19; p < 0.001 and LT storage (median GM index: 0.56 vs. 0.10; p < 0.001 of serum samples. Of samples that were initially GM positive, an average GM index reduction of 50% was seen, with approximately two-thirds becoming GM negative on repeat testing of the same sample. In contrast, GM signal loss was not seen on repeat testing of BAL fluid following ST or LT storage. When GM positive serum samples were repeat tested using EDTA pre-treated serum from the first step of the testing protocol, all samples remained GM positive. In contrast, when the same samples were repeat tested from the original collected serum, 9 samples (64% became GM negative. The significant reduction in GM signals during ST and LT storage of serum samples has implications for clinical management. Although the reasons for GM decline are unknown, they occur prior to the EDTA pre-treatment stage, indicating that the time from phlebotomy to testing should be minimized. BAL fluid GM index values remain stable.

  18. Metagenome reveals potential microbial degradation of hydrocarbon coupled with sulfate reduction in an oil-immersed chimney from Guaymas Basin

    Directory of Open Access Journals (Sweden)

    Ying eHe

    2013-06-01

    Full Text Available Deep-sea hydrothermal vent chimneys contain a high diversity of microorganisms, yet the metabolic activity and the ecological functions of the microbial communities remain largely unexplored. In this study, a metagenomic approach was applied to characterize the metabolic potential in a Guaymas hydrothermal vent chimney and to conduct comparative genomic analysis among a variety of environments with sequenced metagenomes. Complete clustering of functional gene categories with a comparative metagenomic approach showed that this Guaymas chimney metagenome was clustered most closely with a chimney metagenome from Juan de Fuca. All chimney samples were enriched with genes involved in recombination and repair, chemotaxis and flagellar assembly, highlighting their roles in coping with the fluctuating extreme deep-sea environments. A high proportion of transposases was observed in all the metagenomes from deep-sea chimneys, supporting the previous hypothesis that horizontal gene transfer may be common in the deep-sea vent chimney biosphere. In the Guaymas chimney metagenome, thermophilic sulfate reducing microorganisms including bacteria and archaea were found predominant, and genes coding for the degradation of refractory organic compounds such as cellulose, lipid, pullullan, as well as a few hydrocarbons including toluene, ethylbenzene and o-xylene were identified. Therefore, this oil-immersed chimney supported a thermophilic microbial community capable of oxidizing a range of hydrocarbons that served as electron donors for sulphate reduction under anaerobic conditions.

  19. Clinical,radiological and molecular diagnosis correlation in serum samples from patients with osteoarticular tuberculosis

    Institute of Scientific and Technical Information of China (English)

    Guadalupe; Garcia-Elorriaga; Olga; Martinez-Elizondo; Guillermo; del; Rey-Pineda; Cesar; Gonzalez-Bonilla

    2014-01-01

    Objective:To assess the role of polymerase chain reaction(PCR)in serum sauples,in the diagnosis of osteoarticular tuberculosis(OTB)in a setting where only clinical and imaging diagnoses determine the treatment.Methods:A total of 44 consecutive serum specimens were collected from clinically suspected OTB patients,based on clinical and radiological[X-ray or magnetic resonance imagng/computecl tomography]features.They were scrcened by in-house nested PCR.In addition,a few specimens were examined by Gram stain,acid-fast bacilli stain,histand routine bacterial culture.A total of 39 specimens were collected from patients suffering from other bone diseases of nontuberculous origin and included as negative controls.Results:of the 44 clinically suspected OTB patients,in-house nested PCR was positive in 40(91%)cases;PCR was negative in 38(97%)negative controls.Sensitivity and specificity of our in—house nested PCR was 90.3%and 97.4%,respectively.The PCR report was available within 48 h.It was possible to standardize serum PCR technique and in positive cases,a good n was observed in terms of an adequate treatment response.Conclusions:Nested PCR in serum samples is a rapid,highly sensitive and specific modality for OTB detection,PCR should be performed in addition to clinical evaluation,imaging studies,acidfast bacilli staining,culture and histopathology diagnosis,if possible.

  20. Prevalence of Aspergillus species in clinical samples isolated in an Indian tertiary care hospital

    Directory of Open Access Journals (Sweden)

    Xess Immaculata

    2004-12-01

    Full Text Available CONTEXT (BACKGROUND: In recent times, it has become important to determine the prevalence of different Aspergillus species in clinical samples in view of difference in antifungal susceptibility noted in some species. AIMS: To determine the species prevalence of Aspergillus isolates in various clinical samples received in the Mycology Laboratory at our institute. METHOD: Over a period of 4-years, a total of 18,731 samples were processed, and species identification carried out by standard microbiological methods. RESULTS: Four hundred and fifty six samples (2.43% were culture positive for Aspergillus species. A.flavus (46.93% was the most common isolate, followed by A.fumigatus (37.72% and A.niger (15.35%. It was observed that A.fumigatus was the predominant species isolated from blood and respiratory specimens, A.flavus was predominantly isolated from nasal polyps whereas A.niger predominated in nail specimens. Culture positivity was highest in the age group 12-65 years and in males. Sixty-nine patients (15.13% were admitted to the intensive care unit. CONCLUSIONS: The study highlights the diverse manifestations caused by Aspergillus species in human beings and also throws light on the different species prevalent locally. The knowledge would prove useful in selecting empirical antifungal therapy and formulating prophylactic and pre-emptive strategies.

  1. Vibrio parahaemolyticus in shellfish and clinical samples during two large epidemics of diarrhoea in southern Chile.

    Science.gov (United States)

    Fuenzalida, Loreto; Hernández, Cristina; Toro, Jessica; Rioseco, M Luisa; Romero, Jaime; Espejo, Romilio T

    2006-04-01

    Large epidemics of diarrhoea associated with seafood consumption and Vibrio parahaemolyticus occurred during the austral summers of 2004 and 2005 in the environs of Puerto Montt, Chile (41 degrees 29'S 72 degrees 24'W). There are no reports of V. parahaemolyticus infections before 2004 in this region, their absence being explained by the low ocean temperatures which seldom reach 16 degrees C. We analysed V. parahaemolyticus obtained from shellfish and clinical samples during epidemics. Isolates were examined using conventional protocols and an improved method for restriction enzyme analysis using total bacterial DNA which permits direct genome restriction enzyme analysis by conventional gel electrophoresis (DGREA) with a similar discrimination index as restriction fragment length polymorphism-pulsed field gel electrophoresis (RFLP-PFGE). Analysis of clinical samples showed that the epidemics were caused by the V. parahaemolyticus O3:K6 pandemic clonal group. On the other hand, analysis of shellfish samples during both epidemics showed that 53% contained V. parahaemolyticus (3-93 g(-1)). Detailed analysis of 50 positive shellfish samples showed that only three contained detectable levels of the pandemic clone. Most V. parahaemolyticus isolates obtained from shellfish corresponded to non-pandemic clones differentiated into 14 groups by DGREA. In summary, the causative agent during epidemics was only a minor component of a small but diverse population of V. parahaemolyticus in shellfish. PMID:16584479

  2. [Sample size calculation in clinical post-marketing evaluation of traditional Chinese medicine].

    Science.gov (United States)

    Fu, Yingkun; Xie, Yanming

    2011-10-01

    In recent years, as the Chinese government and people pay more attention on the post-marketing research of Chinese Medicine, part of traditional Chinese medicine breed has or is about to begin after the listing of post-marketing evaluation study. In the post-marketing evaluation design, sample size calculation plays a decisive role. It not only ensures the accuracy and reliability of post-marketing evaluation. but also assures that the intended trials will have a desired power for correctly detecting a clinically meaningful difference of different medicine under study if such a difference truly exists. Up to now, there is no systemic method of sample size calculation in view of the traditional Chinese medicine. In this paper, according to the basic method of sample size calculation and the characteristic of the traditional Chinese medicine clinical evaluation, the sample size calculation methods of the Chinese medicine efficacy and safety are discussed respectively. We hope the paper would be beneficial to medical researchers, and pharmaceutical scientists who are engaged in the areas of Chinese medicine research. PMID:22292397

  3. New viruses in veterinary medicine, detected by metagenomic approaches.

    Science.gov (United States)

    Belák, Sándor; Karlsson, Oskar E; Blomström, Anne-Lie; Berg, Mikael; Granberg, Fredrik

    2013-07-26

    In our world, which is faced today with exceptional environmental changes and dramatically intensifying globalisation, we are encountering challenges due to many new factors, including the emergence or re-emergence of novel, so far "unknown" infectious diseases. Although a broad arsenal of diagnostic methods is at our disposal, the majority of the conventional diagnostic tests is highly virus-specific or is targeted entirely towards a limited group of infectious agents. This specificity complicates or even hinders the detection of new or unexpected pathogens, such as new, emerging or re-emerging viruses or novel viral variants. The recently developed approaches of viral metagenomics provide an effective novel way to screen samples and detect viruses without previous knowledge of the infectious agent, thereby enabling a better diagnosis and disease control, in line with the "One World, One Health" principles (www.oneworldonehealth.org). Using metagenomic approaches, we have recently identified a broad variety of new viruses, such as novel bocaviruses, Torque Teno viruses, astroviruses, rotaviruses and kobuviruses in porcine disease syndromes, new virus variants in honeybee populations, as well as a range of other infectious agents in further host species. These findings indicate that the metagenomic detection of viral pathogens is becoming now a powerful, cultivation-independent, and useful novel diagnostic tool in veterinary diagnostic virology. PMID:23428379

  4. New viruses in veterinary medicine, detected by metagenomic approaches.

    Science.gov (United States)

    Belák, Sándor; Karlsson, Oskar E; Blomström, Anne-Lie; Berg, Mikael; Granberg, Fredrik

    2013-07-26

    In our world, which is faced today with exceptional environmental changes and dramatically intensifying globalisation, we are encountering challenges due to many new factors, including the emergence or re-emergence of novel, so far "unknown" infectious diseases. Although a broad arsenal of diagnostic methods is at our disposal, the majority of the conventional diagnostic tests is highly virus-specific or is targeted entirely towards a limited group of infectious agents. This specificity complicates or even hinders the detection of new or unexpected pathogens, such as new, emerging or re-emerging viruses or novel viral variants. The recently developed approaches of viral metagenomics provide an effective novel way to screen samples and detect viruses without previous knowledge of the infectious agent, thereby enabling a better diagnosis and disease control, in line with the "One World, One Health" principles (www.oneworldonehealth.org). Using metagenomic approaches, we have recently identified a broad variety of new viruses, such as novel bocaviruses, Torque Teno viruses, astroviruses, rotaviruses and kobuviruses in porcine disease syndromes, new virus variants in honeybee populations, as well as a range of other infectious agents in further host species. These findings indicate that the metagenomic detection of viral pathogens is becoming now a powerful, cultivation-independent, and useful novel diagnostic tool in veterinary diagnostic virology.

  5. The future of skin metagenomics.

    Science.gov (United States)

    Mathieu, Alban; Vogel, Timothy M; Simonet, Pascal

    2014-01-01

    Metagenomics, the direct exploitation of environmental microbial DNA, is complementary to traditional culture-based approaches for deciphering taxonomic and functional microbial diversity in a plethora of ecosystems, including those related to the human body such as the mouth, saliva, teeth, gut or skin. DNA extracted from human skin analyzed by sequencing the PCR-amplified rrs gene has already revealed the taxonomic diversity of microbial communities colonizing the human skin ("skin microbiome"). Each individual possesses his/her own skin microbial community structure, with marked taxonomic differences between different parts of the body and temporal evolution depending on physical and chemical conditions (sweat, washing etc.). However, technical limitations due to the low bacterial density at the surface of the human skin or contamination by human DNA still has inhibited extended use of the metagenomic approach for investigating the skin microbiome at a functional level. These difficulties have been overcome in part by the new generation of sequencing platforms that now provide sequences describing the genes and functions carried out by skin bacteria. These methodological advances should help us understand the mechanisms by which these microorganisms adapt to the specific chemical composition of each skin and thereby lead to a better understanding of bacteria/human host interdependence. This knowledge will pave the way for more systemic and individualized pharmaceutical and cosmetic applications.

  6. A Delphi Technology Foresight Study: Mapping Social Construction of Scientific Evidence on Metagenomics Tests for Water Safety

    OpenAIRE

    Stanislav Birko; Dove, Edward S.; Vural Özdemir

    2015-01-01

    Access to clean water is a grand challenge in the 21st century. Water safety testing for pathogens currently depends on surrogate measures such as fecal indicator bacteria (e.g., E. coli). Metagenomics concerns high-throughput, culture-independent, unbiased shotgun sequencing of DNA from environmental samples that might transform water safety by detecting waterborne pathogens directly instead of their surrogates. Yet emerging innovations such as metagenomics are often fiercely contested. Inno...

  7. Identification of novel glycosyl hydrolases with cellulolytic activity against crystalline cellulose from metagenomic libraries constructed from bacterial enrichment cultures

    OpenAIRE

    Mori, Toshio; Kamei, Ichiro; Hirai, Hirofumi; Kondo, Ryuichiro

    2014-01-01

    To obtain cellulases that are capable of degrading crystalline cellulose and cedar wood, metagenomic libraries were constructed from raw soil sample which was covered to pile of cedar wood sawdust or from its enrichment cultures. The efficiency of screening of metagenomic library was improved more than 3 times by repeating enrichment cultivation using crystalline cellulose as a carbon source, compared with the library constructed from raw soil. Four cellulase genes were obtained from the meta...

  8. Acquiring Reference Genomes from Uncultured Microbes by Micromanipulation and Low-complexity Metagenomics

    DEFF Research Database (Denmark)

    Karst, Søren Michael; Albertsen, Mads; Nielsen, Jeppe Lund;

    in order to obtain low complexity metagenomes from which high quality draft genomes could be assembled. Microorganisms were visualized with FISH in samples from Danish wastewater treatment plants and single filaments and microcolonies were isolated using an epifluorescense microscope equipped...... with a Skerman micromanipulator mounted with µm sized glass hooks. The isolated cells were lysed and genomic DNA was amplified using multiple displacement amplification. The amplified DNA was validated by 16/18S PCR and Sanger sequencing before being sequenced on an Illumina platform. Metagenome assembly...... analysis several samples showed promising assembly statistics, low metagenome complexity and good binning capabilities. Species obtained belong to a number phylogenetic groups which are highly relevant in wastewater treatment: Chloroflexi, Thiothrix and Microthrix....

  9. MG-RAST, a Metagenomics Service for Analysis of Microbial Community Structure and Function.

    Science.gov (United States)

    Keegan, Kevin P; Glass, Elizabeth M; Meyer, Folker

    2016-01-01

    Approaches in molecular biology, particularly those that deal with high-throughput sequencing of entire microbial communities (the field of metagenomics), are rapidly advancing our understanding of the composition and functional content of microbial communities involved in climate change, environmental pollution, human health, biotechnology, etc. Metagenomics provides researchers with the most complete picture of the taxonomic (i.e., what organisms are there) and functional (i.e., what are those organisms doing) composition of natively sampled microbial communities, making it possible to perform investigations that include organisms that were previously intractable to laboratory-controlled culturing; currently, these constitute the vast majority of all microbes on the planet. All organisms contained in environmental samples are sequenced in a culture-independent manner, most often with 16S ribosomal amplicon methods to investigate the taxonomic or whole-genome shotgun-based methods to investigate the functional content of sampled communities. Metagenomics allows researchers to characterize the community composition and functional content of microbial communities, but it cannot show which functional processes are active; however, near parallel developments in transcriptomics promise a dramatic increase in our knowledge in this area as well. Since 2008, MG-RAST (Meyer et al., BMC Bioinformatics 9:386, 2008) has served as a public resource for annotation and analysis of metagenomic sequence data, providing a repository that currently houses more than 150,000 data sets (containing 60+ tera-base-pairs) with more than 23,000 publically available. MG-RAST, or the metagenomics RAST (rapid annotation using subsystems technology) server makes it possible for users to upload raw metagenomic sequence data in (preferably) fastq or fasta format. Assessments of sequence quality, annotation with respect to multiple reference databases, are performed automatically with minimal

  10. EBI metagenomics--a new resource for the analysis and archiving of metagenomic data.

    Science.gov (United States)

    Hunter, Sarah; Corbett, Matthew; Denise, Hubert; Fraser, Matthew; Gonzalez-Beltran, Alejandra; Hunter, Christopher; Jones, Philip; Leinonen, Rasko; McAnulla, Craig; Maguire, Eamonn; Maslen, John; Mitchell, Alex; Nuka, Gift; Oisel, Arnaud; Pesseat, Sebastien; Radhakrishnan, Rajesh; Rocca-Serra, Philippe; Scheremetjew, Maxim; Sterk, Peter; Vaughan, Daniel; Cochrane, Guy; Field, Dawn; Sansone, Susanna-Assunta

    2014-01-01

    Metagenomics is a relatively recently established but rapidly expanding field that uses high-throughput next-generation sequencing technologies to characterize the microbial communities inhabiting different ecosystems (including oceans, lakes, soil, tundra, plants and body sites). Metagenomics brings with it a number of challenges, including the management, analysis, storage and sharing of data. In response to these challenges, we have developed a new metagenomics resource (http://www.ebi.ac.uk/metagenomics/) that allows users to easily submit raw nucleotide reads for functional and taxonomic analysis by a state-of-the-art pipeline, and have them automatically stored (together with descriptive, standards-compliant metadata) in the European Nucleotide Archive.

  11. Genotypic characterization, invasion index and antimicrobial resistance pattern in Listeria monocytogenes strains isolated from clinical samples

    Institute of Scientific and Technical Information of China (English)

    Behrooz Sadeghi Kalani; Abazar Pournajaf; Mansour Sedighi; Abbas Bahador; Gholamreza Irajian; Firuzeh Valian

    2015-01-01

    Objective: To evaluate antimicrobial resistance, invasion index and genetic profile in Listeriamonocytogenes isolated from clinical samples. Methods: At all, 170 clinical samples were collected from patients with spontaneous abortions hospitalized in Shariati hospital in Tehran during June 2010 to August 2013. Invasion index was determined using HeLa cells. The multiple-locus variable-number tandem-repeats analysis (MLVA) was used for evaluation of genetic relatedness. Results: Out of 14 L. monocytogenes isolates, 4 (28.57%), 2 (14.28%), 0 (0%), 5 (35.71%) and 3 (21.42%) were isolated from placental tissue, urine, blood, vaginal and rectal swabs, respectively. High resistance to penicillin and multidrug resistant were found amongst isolates. The invasion index was in the range of 0.001-0.007. Seven different types were obtained by MLVA assay and type 2 and 3 with 4 strains were the most frequent type. Strains isolated from the vagina and the placenta of the same type were also more resistant to penicillin. Conclusions: Since MLVA is a high-throughput screening method that is fairly inexpensive, easy to accomplish, rapid, and trustworthy, it is well suited to interlaboratory comparisons during epidemiological investigations. Also further studies of larger samples from a variety of sources such as food and animal specimens recommended comparing by MLVA method.

  12. Prevalence and Reasons for Tooth Loss in a Sample from a Dental Clinic in Brazil

    Directory of Open Access Journals (Sweden)

    Andréia Affonso Barretto Montandon

    2012-01-01

    Full Text Available Purpose. To evaluate the prevalence and reasons for teeth extractions in a sample from a dental clinic in Brazil. Methods. The prevalence of teeth mortality was analyzed by gender, age, tooth type and reasons for extraction on 800 teeth of 439 subjects, whose data was collected in clinical records in a convenience sample. Results. The groups with range from 35 to 44 years, 45 to 54 years and 55 to 64 years revealed significantly greater number of teeth extractions than other age groups (P<0.0001. The anterior teeth loss increased significantly with aging, while the tooth mortality of premolar and molar were higher in younger people. The caries was the more prevalent reason for tooth mortality among young and adults up to 44 years old, while the periodontal disease was the main reason for extractions from 45 years old until range of 81 years (P<0.0001. Conclusions. It can be suggested that some reasons for tooth loss were age-dependent, but the caries and the periodontal diseases were the main reasons for tooth mortality in this Brazilian sample.

  13. Gender Differences in Borderline Personality Disorder: Results From a Multinational, Clinical Trial Sample.

    Science.gov (United States)

    Silberschmidt, Amy; Lee, Susanne; Zanarini, Mary; Schulz, S Charles

    2015-12-01

    This study aims to extend previous research by considering gender differences in borderline personality (BPD) using both dimensional self-reported and clinical measures of symptomatology. Drawing from a cross-cultural, clinical trial sample, the authors compared female and male BPD subjects (N = 770; 211 male) between the ages of 18 and 65 using diagnostic and self-report data. The authors found that women with BPD have greater hostility and relationship disruption than men. Gender differences in eating disorders, particularly bulimia, are more divergent than in the general population. Generally, gender differences in BPD in this sample are consistent with known general population differences. Women show greater overall symptomatology, including depressive, anxious, and somatic symptoms. Men have higher rates of antisocial personality disorder and a trend toward higher rates of narcissistic personality disorder. However, several gender differences consistently found in the general population are not present in this BPD sample. There are no differences in aggression, suicidality, substance abuse, panic disorder, or obsessive-compulsive disorder. Gender differences in major depression and posttraumatic stress disorder are attenuated. These findings support the conclusion that BPD may diminish normal gender differences. PMID:25562535

  14. Resolving prokaryotic taxonomy without rRNA: longer oligonucleotide word lengths improve genome and metagenome taxonomic classification.

    Directory of Open Access Journals (Sweden)

    Eric B Alsop

    Full Text Available Oligonucleotide signatures, especially tetranucleotide signatures, have been used as method for homology binning by exploiting an organism's inherent biases towards the use of specific oligonucleotide words. Tetranucleotide signatures have been especially useful in environmental metagenomics samples as many of these samples contain organisms from poorly classified phyla which cannot be easily identified using traditional homology methods, including NCBI BLAST. This study examines oligonucleotide signatures across 1,424 completed genomes from across the tree of life, substantially expanding upon previous work. A comprehensive analysis of mononucleotide through nonanucleotide word lengths suggests that longer word lengths substantially improve the classification of DNA fragments across a range of sizes of relevance to high throughput sequencing. We find that, at present, heptanucleotide signatures represent an optimal balance between prediction accuracy and computational time for resolving taxonomy using both genomic and metagenomic fragments. We directly compare the ability of tetranucleotide and heptanucleotide world lengths (tetranucleotide signatures are the current standard for oligonucleotide word usage analyses for taxonomic binning of metagenome reads. We present evidence that heptanucleotide word lengths consistently provide more taxonomic resolving power, particularly in distinguishing between closely related organisms that are often present in metagenomic samples. This implies that longer oligonucleotide word lengths should replace tetranucleotide signatures for most analyses. Finally, we show that the application of longer word lengths to metagenomic datasets leads to more accurate taxonomic binning of DNA scaffolds and have the potential to substantially improve taxonomic assignment and assembly of metagenomic data.

  15. The impact of drug samples on clinical recommendations in dental education.

    Science.gov (United States)

    Hujoel, Philippe P; Gillette, Jane

    2011-10-01

    The distribution of branded drug samples in an educational setting may obscure the line between an evidence-based and a marketing-based education. The aim of this study was to evaluate the impact of branded drug samples on clinical recommendations in a dental education setting. Survey respondents exposed (n=95) and unexposed (n=80) to a specific branded drug sample program containing non-patented ingredients responded to a brief questionnare on recommendations for dentinal hypersensitivity. The results showed that an exposure to the branded drug sample was associated with twelve-fold increased odds for recommending the brand name (95 percent confidence interval [CI]: 5.8-24.5; precommending the therapeutic class to which the branded drug sample belonged (95 percent CI: 1.1-6.0; p=0.02); a 66 percent reduction in odds for considering other therapeutic classes (95 percent CI: 0.18-0.63; p=0.00015); and an 84 percent reduction in considering etiology (95 percent CI: 0.08-0.35; precommendation, narrowing therapeutic thinking, and decreased consideration for removing causes. PMID:22012775

  16. Quantitation of sulfate and thiosulfate in clinical samples by ion chromatography.

    Science.gov (United States)

    Cole, D E; Evrovski, J

    1997-11-21

    For assay of serum sulfate, quantitation by ion conductimetry after separation by anion-exchange chromatography is the method of choice. In comparison to classical barium precipitation methods, chromatographic methods demonstrate increased precision, specificity and sensitivity, and they may be superior to spectrophotometric methods that rely on organic cation precipitation of sulfate. The increased sensitivity and specificity, as well as the inherent capacity of chromatographic methods for simultaneous determination of other anions, has led to its increasing use in the determination of excreted sulfate in clinical profiles of urinary anion composition. Ion chromatography can also be used to quantitate free sulfate in other clinical samples, including cerebrospinal fluid, sweat, saliva, breast milk and human tissues. Finally, ion chromatography shows promise as a more precise and sensitive method for measurement of total acid-labile sulfoesters and thiosulfate.

  17. Antibiotic Selection Pressure Determination through Sequence-Based Metagenomics.

    Science.gov (United States)

    Willmann, Matthias; El-Hadidi, Mohamed; Huson, Daniel H; Schütz, Monika; Weidenmaier, Christopher; Autenrieth, Ingo B; Peter, Silke

    2015-12-01

    The human gut forms a dynamic reservoir of antibiotic resistance genes (ARGs). Treatment with antimicrobial agents has a significant impact on the intestinal resistome and leads to enhanced horizontal transfer and selection of resistance. We have monitored the development of intestinal ARGs over a 6-day course of ciprofloxacin (Cp) treatment in two healthy individuals by using sequenced-based metagenomics and different ARG quantification methods. Fixed- and random-effect models were applied to determine the change in ARG abundance per defined daily dose of Cp as an expression of the respective selection pressure. Among various shifts in the composition of the intestinal resistome, we found in one individual a strong positive selection for class D beta-lactamases which were partly located on a mobile genetic element. Furthermore, a trend to a negative selection has been observed with class A beta-lactamases (-2.66 hits per million sample reads/defined daily dose; P = 0.06). By 4 weeks after the end of treatment, the composition of ARGs returned toward their initial state but to a different degree in both subjects. We present here a novel analysis algorithm for the determination of antibiotic selection pressure which can be applied in clinical settings to compare therapeutic regimens regarding their effect on the intestinal resistome. This information is of critical importance for clinicians to choose antimicrobial agents with a low selective force on their patients' intestinal ARGs, likely resulting in a diminished spread of resistance and a reduced burden of hospital-acquired infections with multidrug-resistant pathogens. PMID:26369961

  18. Antibiotic Selection Pressure Determination through Sequence-Based Metagenomics.

    Science.gov (United States)

    Willmann, Matthias; El-Hadidi, Mohamed; Huson, Daniel H; Schütz, Monika; Weidenmaier, Christopher; Autenrieth, Ingo B; Peter, Silke

    2015-12-01

    The human gut forms a dynamic reservoir of antibiotic resistance genes (ARGs). Treatment with antimicrobial agents has a significant impact on the intestinal resistome and leads to enhanced horizontal transfer and selection of resistance. We have monitored the development of intestinal ARGs over a 6-day course of ciprofloxacin (Cp) treatment in two healthy individuals by using sequenced-based metagenomics and different ARG quantification methods. Fixed- and random-effect models were applied to determine the change in ARG abundance per defined daily dose of Cp as an expression of the respective selection pressure. Among various shifts in the composition of the intestinal resistome, we found in one individual a strong positive selection for class D beta-lactamases which were partly located on a mobile genetic element. Furthermore, a trend to a negative selection has been observed with class A beta-lactamases (-2.66 hits per million sample reads/defined daily dose; P = 0.06). By 4 weeks after the end of treatment, the composition of ARGs returned toward their initial state but to a different degree in both subjects. We present here a novel analysis algorithm for the determination of antibiotic selection pressure which can be applied in clinical settings to compare therapeutic regimens regarding their effect on the intestinal resistome. This information is of critical importance for clinicians to choose antimicrobial agents with a low selective force on their patients' intestinal ARGs, likely resulting in a diminished spread of resistance and a reduced burden of hospital-acquired infections with multidrug-resistant pathogens.

  19. Survey and Rapid detection of Bordetella pertussis in clinical samples targeting the BP485 in China

    Directory of Open Access Journals (Sweden)

    Wei eLiu

    2015-03-01

    Full Text Available Bordetella pertussis is an important human respiratory pathogen. Here, we describe a loop-mediated isothermal amplification (LAMP method for the rapid detection of B. pertussis in clinical samples based on a visual test. The LAMP assay detected the BP485 target sequence within 60 min with a detection limit of 1.3 pg/µl, a 10-fold increase in sensitivity compared with conventional PCR. All 31 non-pertussis respiratory pathogens tested were negative for LAMP detection, indicating the high specificity of the primers for B. pertussis. To evaluate the application of the LAMP assay to clinical diagnosis, of 105 sputum and nasopharyngeal samples collected from the patients with suspected respiratory infections in China, a total of 12 Bordetella pertussis isolates were identified from 33 positive samples detected by LAMP-based surveillance targeting BP485. Strikingly, a 4.5 months old baby and her mother were found to be infected with B. pertussis at the same time. All isolates belonged to different B. pertussis multilocus sequence typing (MLST groups with different alleles of the virulence-related genes including 4 alleles of ptxA, 6 of prn, 4 of tcfA, 2 of fim2 and 3 of fim3. The diversity of B. pertussis carrying toxin genes in clinical strains indicates a rapid and continuing evolution of B. pertussis. This combined with its high prevalence will make it difficult to control. In conclusion, we have developed a visual detection LAMP assay, which could be a useful tool for rapid B. pertussis detection, especially in situations where resources are poor and in point-of-care tests.

  20. Vibrio parahaemolyticus Strains of Pandemic Serotypes Identified from Clinical and Environmental Samples from Jiangsu, China

    Directory of Open Access Journals (Sweden)

    Jingjiao eLi

    2016-05-01

    Full Text Available Vibrio parahaemolyticus has emerged as a major foodborne pathogen in China, Japan, Thailand and other Asian countries. In this study, 72 strains of V. parahaemolyticus were isolated from clinical and environmental samples between 2006 and 2014 in Jiangsu, China. The serotypes and six virulence genes including thermostable direct hemolysin (TDR and TDR-related hemolysin (TRH genes were assessed among the isolates. Twenty five serotypes were identified and O3:K6 was one of the dominant serotypes. The genetic diversity was assessed by multilocus sequence typing (MLST analysis, and 48 sequence types (STs were found, suggesting this V. parahaemolyticus group is widely dispersed and undergoing rapid evolution. A total of 25 strains of pandemic serotypes such as O3:K6, O5:K17 and O1:KUT were identified. It is worth noting that the pandemic serotypes were not exclusively identified from clinical samples, rather, nine strains were also isolated from environmental samples; and some of these strains harbored several virulence genes, which may render those strains pathogenicity potential. Therefore, the emergence of these environmental pandemic V. parahaemolyticus strains may poses a new threat to the public health in China. Furthermore, six novel serotypes and 34 novel STs were identified among the 72 isolates, indicating that V. parahaemolyticus were widely distributed and fast evolving in the environment in Jiangsu, China. The findings of this study provide new insight into the phylogenic relationship between V. parahaemolyticus strains of pandemic serotypes from clinical and environmental sources and enhance the MLST database; and our proposed possible O- and K- antigen evolving paths of V. parahaemolyticus may help understand how the serotypes of this dispersed bacterial population evolve.

  1. Rapid detection of Candida albicans by polymerase spiral reaction assay in clinical blood samples

    Directory of Open Access Journals (Sweden)

    Xiaoqun eJiang

    2016-06-01

    Full Text Available Candida albicans is the most common human yeast pathogen which causes mucosal infections and invasive fungal diseases. Early detection of this pathogen is needed to guide preventative and therapeutic treatment. The aim of this study was to establish a polymerase spiral reaction (PSR assay that rapidly and accurately detects C. albicans and to assess the clinical applicability of PSR-based diagnostic testing. Internal transcribed spacer 2 (ITS2, a region between 5.8S and 28S fungal ribosomal DNA, was used as the target sequence. Four primers were designed for amplification of ITS2 with the PSR method, which was evaluated using real time turbidity monitoring and visual detection using a pH indicator. Fourteen non- C. albicans yeast strains were negative for detection, which indicated the specificity of PSR assay was 100%. A 10-fold serial dilution of C. albicans genomic DNA was subjected to PSR and conventional PCR to compare their sensitivities. The detection limit of PSR was 6.9 pg/µl within 1 h, 10-fold higher than that of PCR (69.0 pg/µl. Blood samples (n=122 were collected from intensive care unit and hematological patients with proven or suspected C. albicans infection at two hospitals in Beijing, China. Both PSR assay and the culture method were used to analyze the samples. Of the 122 clinical samples, 34 were identified as positive by PSR. The result was consistent with those obtained by the culture method. In conclusion, a novel and effective C. albicans detection assay was developed that has a great potential for clinical screening and point-of-care testing.

  2. Metagenomics and the Human Virome in Asymptomatic Individuals.

    Science.gov (United States)

    Rascovan, Nicolás; Duraisamy, Raja; Desnues, Christelle

    2016-09-01

    High-throughput sequencing technologies have revolutionized how we think about viruses. Investigators can now go beyond pathogenic viruses and have access to the thousands of viruses that inhabit our bodies without causing clinical symptoms. By studying their interactions with each other, with other microbes, and with host genetics and immune systems, we can learn how they affect health and disease. This article reviews current knowledge of the composition and diversity of the human virome in physiologically healthy individuals. It focuses on recent results from metagenomics studies and discusses the contribution of bacteriophages and eukaryotic viruses to human health. PMID:27607550

  3. Metagenomics: Application of Genomics to Uncultured Microorganisms

    OpenAIRE

    Handelsman, Jo

    2004-01-01

    Metagenomics (also referred to as environmental and community genomics) is the genomic analysis of microorganisms by direct extraction and cloning of DNA from an assemblage of microorganisms. The development of metagenomics stemmed from the ineluctable evidence that as-yet-uncultured microorganisms represent the vast majority of organisms in most environments on earth. This evidence was derived from analyses of 16S rRNA gene sequences amplified directly from the environment, an approach that ...

  4. Metagenomics: A new horizon in cancer research

    OpenAIRE

    Joyita Banerjee; Neetu Mishra; Yogita Dhas

    2015-01-01

    Metagenomics has broadened the scope of targeting microbes responsible for inducing various types of cancers. About 16.1% of cancers are associated with microbial infection. Metagenomics is an equitable way of identifying and studying micro-organisms within their habitat. In cancer research, this approach has revolutionized the way of identifying, analyzing and targeting the microbial diversity present in the tissue specimens of cancer patients. The genomic analyses of these micro-organisms t...

  5. Unreliability of results of PCR detection of Helicobacter pylori in clinical or environmental samples.

    Science.gov (United States)

    Sugimoto, Mitsushige; Wu, Jeng-Yih; Abudayyeh, Suhaib; Hoffman, Jill; Brahem, Hajer; Al-Khatib, Khaldun; Yamaoka, Yoshio; Graham, David Y

    2009-03-01

    The aim of this study was to compare published Helicobacter pylori primer pairs for their ability to reliably detect H. pylori in gastric biopsy specimens and salivary samples. Detection limits of the 26 PCR primer pairs previously described for detection of H. pylori DNA in clinical samples were determined. Sensitivity and specificity were determined using primers with detection limits of pylori-positive and -negative (by concordance by culture and histology) coded gastric biopsy specimens. These results were then confirmed with gastric biopsy specimens and saliva from patients with confirmed H. pylori status. Five of the twenty-six previously reported primer pairs (HP64-f/HP64-r, HP1/HP2, EHC-U/EHC-L, VAG-F/VAG-R, and ICT37/ICT38) had detection limits of 90% with gastric biopsy specimens. No combinations of primer pairs improved the results. Using these five primer pairs, 54% of the positive saliva samples were determined to be false positive; both the HP64-f/HP64-r and the HP1/HP2 sets produced false positives with saliva. We conclude that clinicians should not rely on results using current PCR primers alone to decide the H. pylori status of an individual patient or as a basis for treatment decisions. The results of studies based on PCR identification of H. pylori in environmental samples should be viewed with caution. Possibly, specific primers sets can be identified based on the presence of multiple putative virulence factor genes. PMID:19129407

  6. Clinical utility and performance of sock sampling in weaner pig diarrhoea

    DEFF Research Database (Denmark)

    Pedersen, Ken Steen; Okholm, Elisabeth; Johansen, Markku;

    2015-01-01

    , agreement between three consecutive herd examinations from the same herd and agreement between quantitative PCR results from pooled faecal samples and sock samples.Twenty-four veterinarians submitted faecal and sock samples for quantitative PCR testing from outbreaks of diarrhoea in nursery pigs (n=38 herds......’ clinical aetiological diagnosis and the pooled faecal sample was 0.18 (95% CL: 0.08–0.34), and Cohen’s Kappa was 0.03 (95% CL: −0.08 to 0.14). Antibiotic treatment or prevention strategies were changed in 0.63 (95% CL: 0.46–0.78) of the herds, and the veterinarians indicated that, for 0.32 (95% CL: 0......, and Cohen’s Kappa was 0.61 (95% CI: 0.26–0.95). In relation to detection of the individual infections, agreement was 0.63 (95% CI: 0.46–0.78), and Cohen’s Kappa was 0.53 (95% CI: 0.34–0.71).In conclusion, low pathogen diarrhoea is a common finding amongst diarrhoea outbreaks that are subjected to antibiotic...

  7. Challenges and Opportunities of Airborne Metagenomics

    KAUST Repository

    Behzad, H.

    2015-05-06

    Recent metagenomic studies of environments, such as marine and soil, have significantly enhanced our understanding of the diverse microbial communities living in these habitats and their essential roles in sustaining vast ecosystems. The increase in the number of publications related to soil and marine metagenomics is in sharp contrast to those of air, yet airborne microbes are thought to have significant impacts on many aspects of our lives from their potential roles in atmospheric events such as cloud formation, precipitation, and atmospheric chemistry to their major impact on human health. In this review, we will discuss the current progress in airborne metagenomics, with a special focus on exploring the challenges and opportunities of undertaking such studies. The main challenges of conducting metagenomic studies of airborne microbes are as follows: 1) Low density of microorganisms in the air, 2) efficient retrieval of microorganisms from the air, 3) variability in airborne microbial community composition, 4) the lack of standardized protocols and methodologies, and 5) DNA sequencing and bioinformatics-related challenges. Overcoming these challenges could provide the groundwork for comprehensive analysis of airborne microbes and their potential impact on the atmosphere, global climate, and our health. Metagenomic studies offer a unique opportunity to examine viral and bacterial diversity in the air and monitor their spread locally or across the globe, including threats from pathogenic microorganisms. Airborne metagenomic studies could also lead to discoveries of novel genes and metabolic pathways relevant to meteorological and industrial applications, environmental bioremediation, and biogeochemical cycles.

  8. Challenges and opportunities of airborne metagenomics.

    Science.gov (United States)

    Behzad, Hayedeh; Gojobori, Takashi; Mineta, Katsuhiko

    2015-05-06

    Recent metagenomic studies of environments, such as marine and soil, have significantly enhanced our understanding of the diverse microbial communities living in these habitats and their essential roles in sustaining vast ecosystems. The increase in the number of publications related to soil and marine metagenomics is in sharp contrast to those of air, yet airborne microbes are thought to have significant impacts on many aspects of our lives from their potential roles in atmospheric events such as cloud formation, precipitation, and atmospheric chemistry to their major impact on human health. In this review, we will discuss the current progress in airborne metagenomics, with a special focus on exploring the challenges and opportunities of undertaking such studies. The main challenges of conducting metagenomic studies of airborne microbes are as follows: 1) Low density of microorganisms in the air, 2) efficient retrieval of microorganisms from the air, 3) variability in airborne microbial community composition, 4) the lack of standardized protocols and methodologies, and 5) DNA sequencing and bioinformatics-related challenges. Overcoming these challenges could provide the groundwork for comprehensive analysis of airborne microbes and their potential impact on the atmosphere, global climate, and our health. Metagenomic studies offer a unique opportunity to examine viral and bacterial diversity in the air and monitor their spread locally or across the globe, including threats from pathogenic microorganisms. Airborne metagenomic studies could also lead to discoveries of novel genes and metabolic pathways relevant to meteorological and industrial applications, environmental bioremediation, and biogeochemical cycles.

  9. Coping behavior in normal and clinical samples: more similarities than differences?

    Science.gov (United States)

    Seiffge-Krenke, I

    1993-09-01

    In our studies we tried to integrate a developmental and a clinical perspective on coping and adaptation in adolescence. Starting with a review of the author's own research, involving over 3000 12- to 20-year-olds from various cultures, the problems typical of this developmental phase and the ways of coping with these normative demands are presented. The results show that coping skills of young people in dealing with age-specific problems have so far been considerably underestimated. Their response to problems stemming from different developmental fields such as parents, peers, school or future involved three main modes of coping: Active Coping, Internal Coping and Withdrawal. Withdrawal was employed very rarely and only for certain types of problems. Age, gender and problem-specific effects in coping were found. Whereas normal adolescents most frequently choose to cope with difficulties actively by means of social resources and to think out possible solutions, risk populations appear to have a more ambivalent pattern of coping strategies with high functionality and high dysfunctionality. Even their appraisal of problems is already disturbed; they feel more readily threatened by everyday problem situations and respond more uniformly with withdrawal. Finally, similarities between the female coping style in normal samples and the more pronounced ambivalent pattern in clinical samples were discussed and related to psychopathology. PMID:8282899

  10. Cognitive Predictors of Verbal Memory in a Mixed Clinical Pediatric Sample

    Directory of Open Access Journals (Sweden)

    Shelley C. Heaton

    2013-08-01

    Full Text Available Verbal memory problems, along with other cognitive difficulties, are common in children diagnosed with neurological and/or psychological disorders. Historically, these “memory problems” have been poorly characterized and often present with a heterogeneous pattern of performance across memory processes, even within a specific diagnostic group. The current study examined archival neuropsychological data from a large mixed clinical pediatric sample in order to understand whether functioning in other cognitive areas (i.e., verbal knowledge, attention, working memory, executive functioning may explain some of the performance variability seen across verbal memory tasks of the Children’s Memory Scale (CMS. Multivariate analyses revealed that among the cognitive functions examined, only verbal knowledge explained a significant amount of variance in overall verbal memory performance. Further univariate analyses examining the component processes of verbal memory indicated that verbal knowledge is specifically related to encoding, but not the retention or retrieval stages. Future research is needed to replicate these findings in other clinical samples, to examine whether verbal knowledge predicts performance on other verbal memory tasks and to explore whether these findings also hold true for visual memory tasks. Successful replication of the current study findings would indicate that interventions targeting verbal encoding deficits should include efforts to improve verbal knowledge.

  11. Childhood trauma associates with clinical features of schizophrenia in a sample of Chinese inpatients.

    Science.gov (United States)

    Li, Xian-Bin; Li, Qi-Yong; Liu, Jin-Tong; Zhang, Liang; Tang, Yi-Lang; Wang, Chuan-Yue

    2015-08-30

    This study examined the association between childhood trauma and clinical features, comorbid anxiety and post-traumatic stress disorder (PTSD) symptoms, and suicidal and aggressive behaviors in Chinese patients with schizophrenia. The Childhood Trauma Questionnaire - Short Form (CTQ-SF), the Impact of Events Scale - Revised (IES-R), and the State-Trait Anxiety Inventory (STAI) were administered to 182 Chinese inpatients with schizophrenia. The relationship between the severity and the number of traumic experiences and clinical features were analyzed. Physical neglect (PN) in childhood was reported in 71.7% of this sample, followed by emotional neglect (EN, 58.6%), sexual abuse (SA, 39.9%), emotional abuse (EA, 31.7%) and physical abuse (PA, 22.2%). Significant negative correlations existed between age of onset and the EA scores. Significant positive correlations were found between the subscores of IES-R, STAI and CTQ-SF. Patients with history of suicidal or aggressive behaviors had significantly higher trauma scores than patients without such behaviors. Exposure to childhood trauma is associated with early age of onset, more PTSD and anxiety symptoms, and history of suicidal and aggressive behaviors. A dose-effect may exist between severity, number of trauma experiences, and clinical features. PMID:26096662

  12. A review on the use of NEO-PI-R validity scales in normative, job selection, and clinical samples

    Directory of Open Access Journals (Sweden)

    Angel Blanch

    2009-06-01

    Full Text Available Background and Objectives: In this study we review the use of the Positive Presentation Management (PPM and Negative Presentation Management (NPM scales, two NEO-PI-R derived measures originally devised to control for biased and distorted responses. These scales have been used with normative, job selection and clinical samples, in cross-sectional and experimental studies. Methods: Web-based and manual searches in personality and psychological assessment journals were conducted, and information on the PPM and NPM scales was systematically recorded. Means, standard deviations and reliability coefficients were summarized and compared between three types of samples: normative, job selection and clinical. Results: Five studies were performed with normative samples (33%, 3 with employment samples (20% and 7 with clinical samples (47%. Cross-sectional designs were most common (60%, although there were also experimental studies (40%. Reported reliability coefficients were lower than usually accepted. There were differences in mean PPM and NPM scores in regard to the study sample background. Conclusions: There were some discrepancies when reporting PPM and NPM results across the reviewed studies. Normative and employment samples scored higher in PPM than clinical samples. Clinical samples scored higher in NPM than normative and employment samples The PPM and NPM scales could be useful in applied situations, although parallel sources of information should be taken into account to detect distorted responses to the questionnaire. However, the results on these scales should be systematically reported in future studies.

  13. Clinical illnesses associated with isolation of dysgonic fermenter 3 from stool samples.

    Science.gov (United States)

    Blum, R N; Berry, C D; Phillips, M G; Hamilos, D L; Koneman, E W

    1992-02-01

    The clinical significance of the fastidious organism DF-3 isolated from stool cultures is unclear. We sought to improve our understanding of this organism and to further define its association with human disease. Stool cultures for DF-3 were obtained from three sources: an ongoing study of enteric pathogens in patients infected with the human immunodeficiency virus, a screening procedure in which all stool samples submitted for Clostridium difficile toxin assay were cultured for DF-3, and stool samples submitted specifically for DF-3 culture. Retrospective clinical data were obtained from chart reviews of patients with positive cultures. Antimicrobial susceptibility testing and cell wall fatty acid analysis were performed for each DF-3 isolated. Eight isolates of DF-3 were obtained over a period of 8 months. All patients either had severe underlying disease or were immunocompromised, including three patients coinfected with human immunodeficiency virus and two patients with inflammatory bowel disease. The spectrum of clinical disease ranged from chronic diarrhea with a well-defined response to therapy for DF-3 to an asymptomatic carrier state. Cell wall fatty acid analysis of these isolates demonstrated a consistent pattern with a large peak of 12-methyltetradecanoate. DF-3, a fastidious gram-negative coccobacillus, can be recovered from stool cultures of immunocompromised patients by using selective media. The presence of 12-methyltetradecanoate in cell wall fatty acid analysis assists in identification. The increased use of a selective medium-(cefoperazone-vancomycin-amphotericin B) in the evaluation of diarrhea in immunocompromised hosts, including persons with inflammatory bowel disease, may better define the association of DF-3 with human gastrointestinal disease.

  14. Evaluation of dengue NS1 antigen rapid tests and ELISA kits using clinical samples.

    Directory of Open Access Journals (Sweden)

    Subhamoy Pal

    Full Text Available Early diagnosis of dengue virus (DENV infection can improve clinical outcomes by ensuring close follow-up, initiating appropriate supportive therapies and raising awareness to the potential of hemorrhage or shock. Non-structural glycoprotein-1 (NS1 has proven to be a useful biomarker for early diagnosis of dengue. A number of rapid diagnostic tests (RDTs and enzyme-linked immunosorbent assays (ELISAs targeting NS1 antigen (Ag are now commercially available. Here we evaluated these tests using a well-characterized panel of clinical samples to determine their effectiveness for early diagnosis.Retrospective samples from South America were used to evaluate the following tests: (i "Dengue NS1 Ag STRIP" and (ii "Platelia Dengue NS1 Ag ELISA" (Bio-Rad, France, (iii "Dengue NS1 Detect Rapid Test (1st Generation" and (iv "DENV Detect NS1 ELISA" (InBios International, United States, (v "Panbio Dengue Early Rapid (1st generation" (vi "Panbio Dengue Early ELISA (2nd generation" and (vii "SD Bioline Dengue NS1 Ag Rapid Test" (Alere, United States. Overall, the sensitivity of the RDTs ranged from 71.9%-79.1% while the sensitivity of the ELISAs varied between 85.6-95.9%, using virus isolation as the reference method. Most tests had lower sensitivity for DENV-4 relative to the other three serotypes, were less sensitive in detecting secondary infections, and appeared to be most sensitive on Day 3-4 post symptom onset. The specificity of all evaluated tests ranged from 95%-100%.ELISAs had greater overall sensitivity than RDTs. In conjunction with other parameters, the performance data can help determine which dengue diagnostics should be used during the first few days of illness, when the patients are most likely to present to a clinic seeking care.

  15. Prevalence of Extended –Spectrum-Beta-Lactamase-Producing Klebsiella Pneumonia Isolates from Clinical Samples

    Directory of Open Access Journals (Sweden)

    Alizade, H. (MSc

    2014-06-01

    Full Text Available Background and Objective: Klebsiella pneumonia (K.pneumonia is one of the common causes of nosocomial infections. The aim of this research was to determine the prevalence of beta-lactamase genes and phenotypic confirmation of extended–spectrum-beta-lactamase (ESBL producing K.pneumonia isolates from clinical samples. Material and Methods: In this study, 122 K.pneumonia were isolated from clinical specimens of Khoramabad city and were confirmed by standard bacteriological tests. The presence of ESBL enzymes was detected by combined disk diffusion method. PCR assay with specific primers was used to determine blaSHV, blaTEM, blaCTX-15 and blaCTX-M genes in the confirmed isolates. Results: of 122 K.pneumonia isolates, 78 (64.18% were positive for ESBL, using disk diffusion method. According to antibiogram results, 10.65% of isolates were resistant to cefotaxime, 3.27% to ceftazidime and 68.03% to both antibiotics. Ninety isolates (64.18% considered as ESBLs isolates, at the same time, with being resistant to cefotaxime and ceftazidime were also sensitive to cefotaxime-clavulanic acid and ceftazidime-clavulanic acid. In PCR assays, blaCTX-15, blaSHV, blaCTX-M and blaTEM genes were detected in 78.68%, 40.16%, 26.22% and 22.13% of isolates, respectively. Ten resistant patterns of genes were detected. Conclusion: The significance percentage of antibiotic resistant genes of K.pneumonia isolates from clinical samples in Khoramabad city had ESBLs genes; CTX-M category was the most prevalent encoding genes of these enzymes. Keywords: Klebsiella Pneumonia, Extended-Spectrum Beta-Lactamase, Antibiotic Resistance

  16. Amplicon-based metagenomics identified candidate organisms in soils that caused yield decline in strawberry

    OpenAIRE

    Xiangming Xu; Thomas Passey; Feng Wei; Robert Saville; Harrison, Richard J.

    2015-01-01

    A phenomenon of yield decline due to weak plant growth in strawberry was recently observed in non-chemo-fumigated soils, which was not associated with the soil fungal pathogen Verticillium dahliae, the main target of fumigation. Amplicon-based metagenomics was used to profile soil microbiota in order to identify microbial organisms that may have caused the yield decline. A total of 36 soil samples were obtained in 2013 and 2014 from four sites for metagenomic studies; two of the four sites ha...

  17. Technical Report: Benchmarking for Quasispecies Abundance Inference with Confidence Intervals from Metagenomic Sequence Data

    Energy Technology Data Exchange (ETDEWEB)

    McLoughlin, K. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2016-01-22

    The software application “MetaQuant” was developed by our group at Lawrence Livermore National Laboratory (LLNL). It is designed to profile microbial populations in a sample using data from whole-genome shotgun (WGS) metagenomic DNA sequencing. Several other metagenomic profiling applications have been described in the literature. We ran a series of benchmark tests to compare the performance of MetaQuant against that of a few existing profiling tools, using real and simulated sequence datasets. This report describes our benchmarking procedure and results.

  18. Autotrophic microbe metagenomes and metabolic pathways differentiate adjacent red sea brine pools

    KAUST Repository

    Wang, Yong

    2013-04-29

    In the Red Sea, two neighboring deep-sea brine pools, Atlantis II and Discovery, have been studied extensively, and the results have shown that the temperature and concentrations of metal and methane in Atlantis II have increased over the past decades. Therefore, we investigated changes in the microbial community and metabolic pathways. Here, we compared the metagenomes of the two pools to each other and to those of deep-sea water samples. Archaea were generally absent in the Atlantis II metagenome; Bacteria in the metagenome were typically heterotrophic and depended on aromatic compounds and other extracellular organic carbon compounds as indicated by enrichment of the related metabolic pathways. In contrast, autotrophic Archaea capable of CO2 fixation and methane oxidation were identified in Discovery but not in Atlantis II. Our results suggest that hydrothermal conditions and metal precipitation in the Atlantis II pool have resulted in elimination of the autotrophic community and methanogens.

  19. A metagenomic study of methanotrophic microorganisms in Coal Oil Point seep sediments

    Directory of Open Access Journals (Sweden)

    Haverkamp Thomas HA

    2011-10-01

    Full Text Available Abstract Background Methane oxidizing prokaryotes in marine sediments are believed to function as a methane filter reducing the oceanic contribution to the global methane emission. In the anoxic parts of the sediments, oxidation of methane is accomplished by anaerobic methanotrophic archaea (ANME living in syntrophy with sulphate reducing bacteria. This anaerobic oxidation of methane is assumed to be a coupling of reversed methanogenesis and dissimilatory sulphate reduction. Where oxygen is available aerobic methanotrophs take part in methane oxidation. In this study, we used metagenomics to characterize the taxonomic and metabolic potential for methane oxidation at the Tonya seep in the Coal Oil Point area, California. Two metagenomes from different sediment depth horizons (0-4 cm and 10-15 cm below sea floor were sequenced by 454 technology. The metagenomes were analysed to characterize the distribution of aerobic and anaerobic methanotrophic taxa at the two sediment depths. To gain insight into the metabolic potential the metagenomes were searched for marker genes associated with methane oxidation. Results Blast searches followed by taxonomic binning in MEGAN revealed aerobic methanotrophs of the genus Methylococcus to be overrepresented in the 0-4 cm metagenome compared to the 10-15 cm metagenome. In the 10-15 cm metagenome, ANME of the ANME-1 clade, were identified as the most abundant methanotrophic taxon with 8.6% of the reads. Searches for particulate methane monooxygenase (pmoA and methyl-coenzyme M reductase (mcrA, marker genes for aerobic and anaerobic oxidation of methane respectively, identified pmoA in the 0-4 cm metagenome as Methylococcaceae related. The mcrA reads from the 10-15 cm horizon were all classified as originating from the ANME-1 clade. Conclusions Most of the taxa detected were present in both metagenomes and differences in community structure and corresponding metabolic potential between the two samples were mainly

  20. OTU analysis using metagenomic shotgun sequencing data.

    Directory of Open Access Journals (Sweden)

    Xiaolin Hao

    Full Text Available Because of technological limitations, the primer and amplification biases in targeted sequencing of 16S rRNA genes have veiled the true microbial diversity underlying environmental samples. However, the protocol of metagenomic shotgun sequencing provides 16S rRNA gene fragment data with natural immunity against the biases raised during priming and thus the potential of uncovering the true structure of microbial community by giving more accurate predictions of operational taxonomic units (OTUs. Nonetheless, the lack of statistically rigorous comparison between 16S rRNA gene fragments and other data types makes it difficult to interpret previously reported results using 16S rRNA gene fragments. Therefore, in the present work, we established a standard analysis pipeline that would help confirm if the differences in the data are true or are just due to potential technical bias. This pipeline is built by using simulated data to find optimal mapping and OTU prediction methods. The comparison between simulated datasets revealed a relationship between 16S rRNA gene fragments and full-length 16S rRNA sequences that a 16S rRNA gene fragment having a length >150 bp provides the same accuracy as a full-length 16S rRNA sequence using our proposed pipeline, which could serve as a good starting point for experimental design and making the comparison between 16S rRNA gene fragment-based and targeted 16S rRNA sequencing-based surveys possible.

  1. VIP: an integrated pipeline for metagenomics of virus identification and discovery.

    Science.gov (United States)

    Li, Yang; Wang, Hao; Nie, Kai; Zhang, Chen; Zhang, Yi; Wang, Ji; Niu, Peihua; Ma, Xuejun

    2016-01-01

    Identification and discovery of viruses using next-generation sequencing technology is a fast-developing area with potential wide application in clinical diagnostics, public health monitoring and novel virus discovery. However, tremendous sequence data from NGS study has posed great challenge both in accuracy and velocity for application of NGS study. Here we describe VIP ("Virus Identification Pipeline"), a one-touch computational pipeline for virus identification and discovery from metagenomic NGS data. VIP performs the following steps to achieve its goal: (i) map and filter out background-related reads, (ii) extensive classification of reads on the basis of nucleotide and remote amino acid homology, (iii) multiple k-mer based de novo assembly and phylogenetic analysis to provide evolutionary insight. We validated the feasibility and veracity of this pipeline with sequencing results of various types of clinical samples and public datasets. VIP has also contributed to timely virus diagnosis (~10 min) in acutely ill patients, demonstrating its potential in the performance of unbiased NGS-based clinical studies with demand of short turnaround time. VIP is released under GPLv3 and is available for free download at: https://github.com/keylabivdc/VIP.

  2. Efficient adaptive designs with mid-course sample size adjustment in clinical trials

    CERN Document Server

    Bartroff, Jay

    2011-01-01

    Adaptive designs have been proposed for clinical trials in which the nuisance parameters or alternative of interest are unknown or likely to be misspecified before the trial. Whereas most previous works on adaptive designs and mid-course sample size re-estimation have focused on two-stage or group sequential designs in the normal case, we consider here a new approach that involves at most three stages and is developed in the general framework of multiparameter exponential families. Not only does this approach maintain the prescribed type I error probability, but it also provides a simple but asymptotically efficient sequential test whose finite-sample performance, measured in terms of the expected sample size and power functions, is shown to be comparable to the optimal sequential design, determined by dynamic programming, in the simplified normal mean case with known variance and prespecified alternative, and superior to the existing two-stage designs and also to adaptive group sequential designs when the al...

  3. The frequency of Listeria monocytogenes strains recovered from clinical and non-clinical samples using phenotypic methods and confirmed by PCR

    OpenAIRE

    abazar pournajaf; lida lotfollahi; gholamreza irajian; abdollah ardebili; Behrooz Sadeghi kalani; mojtabata Taghizadeh armaki

    2013-01-01

    Background: Listeria monocytogenes is a facultative intracellular pathogen that causes listeriosis which has extensive clinical manifestations. Infections with L. monocytogenes are a serious threat to immunocompromised persons. The aim of this study was to determine the frequency of L. monocytogenes strains recovered from clinical and non-clinical samples using phenotypic methods and confirmed by PCR. Materials and Methods: In this study, 617 specimens were analyzed. All specimens were cu...

  4. Microbial food safety: Potential of DNA extraction methods for use in diagnostic metagenomics

    DEFF Research Database (Denmark)

    Josefsen, Mathilde Hasseldam; Andersen, Sandra Christine; Christensen, Julia;

    2015-01-01

    The efficiency of ten widely applied DNA extraction protocols was evaluated for suitability for diagnostic metagenomics. The protocols were selected based on a thorough literature study. Chicken fecal samples inoculated with about 1×103 and 1×106CFU/g Campylobacter jejuni were used as a model...

  5. myPhyloDB: a local web server for the storage and analysis of metagenomics data

    Science.gov (United States)

    myPhyloDB is a user-friendly personal database with a browser-interface designed to facilitate the storage, processing, analysis, and distribution of metagenomics data. MyPhyloDB archives raw sequencing files, and allows for easy selection of project(s)/sample(s) of any combination from all availab...

  6. Screening and Cloning of IMP Cyclohydrolase from a Metagenomic Cosmid Library of a Deep-sea Sediment Sample%深海沉积物微生物宏基因组文库中IMP环水解酶的筛选及基因克隆

    Institute of Scientific and Technical Information of China (English)

    陈兴麟; 赵晶; 徐宪忠; 曾润颖

    2011-01-01

    为了解嘌呤合成途径的关键酶——IMP环水解酶的多样性,从深海沉积物样品中提取总DNA构建Cosmid宏基因组文库,从中筛选出一个具有IMP环水解酶活性的克隆(CAIMP1).通过基因步移的方法从CAIMP1的约35 kb的插入片段中扩增出长度为l 599 bp的完整的IMP环水解酶基因,该基因包含MGS和AICARFT_IMPCHas两个保守结构域.活性检测结果表明CAIMP1克隆的发酵液上清具有较强的IMP环水解酶活性.该酶与数据库中收录的IMP环水解酶的氨基酸序列同源性较低,系统发育分析也表明该酶与其它参照序列亲缘关系较远,表明该序列可能为一个新的IMP环水解酶基因.%IMP cyclohydrolase is a key enzyme in the purine synthesis pathway. A clone (CAIMP1) producing IMP cyclohydrolase activity was isolated from a metagenomic library which was constructed from metagenomic DNA of a deep-sea sediment sample. The gene coding for the IMP cyclohydrolase with the size of 1 599 bp was subcloned from the 35 kb inserted fragment of CAIMP1 by gene walking. Two conserved domains, MGS and AICARFT_IMPCHas, was found in the gene. The CAIMP1 exhibited high activity towards 5-formaminoimidazole-4-carboxamide ribonucleotide (FAICAR). The alignment and phylogenetic analysis of amino acid sequence of the CAIMP1 IMP cyclohydrolase showed that it had low homology with other referenced sequences in GenBank, indicating that it might be a new sequence. Fig 5, Tab 3, Ref 17

  7. Psychometric properties of the child and parent versions of Spence children's anxiety scale in a Danish community and clinical sample.

    Science.gov (United States)

    Arendt, Kristian; Hougaard, Esben; Thastum, Mikael

    2014-12-01

    This study examined the psychometric properties and norms of the Spence Children's Anxiety Scale (SCAS) and the associated parent version (SCAS-P) in a Danish community and a clinical sample. The total sample consisted of 1240 children (972 from community sample), age 7-17 years, and 805 parents (537 from community sample). Results indicated that SCAS and SCAS-P had good internal consistency on the total scale and all subscales, with exception of the subscale for fear of physical injury. Both scales showed satisfactory 2-week and 3-month retest stability. All subscales and total scales of the SCAS and SCAS-P discriminated between the clinical and community sample. A comparison with the Beck Youth Inventories and the Strength and Difficulty Questionnaire in the clinical sample supported the scales' convergent and divergent validity. Results of confirmatory factor analyses for SCAS and SCAS-P were in favor of the original model with six correlated factors.

  8. Metagenomics of Glassy-Winged Sharpshooter, Homalodisca vitripennis (Hemiptera: Cicadellidae)

    Science.gov (United States)

    A Metagenomics approach was used to identify unknown organisms which live in association with the glassy-winged sharpshooter, Homalodisca vitripennis (Hemiptera: Cicadellidae). Metagenomics combines molecular biology and genetics to identify, and characterize genetic material from unique biological ...

  9. Evaluation of Inhibitor-Resistant Real-Time PCR Methods for Diagnostics in Clinical and Environmental Samples

    OpenAIRE

    Adrienne Trombley Hall; Ashley McKay Zovanyi; Deanna Rose Christensen; Jeffrey William Koehler; Timothy Devins Minogue

    2013-01-01

    Polymerase chain reaction (PCR) is commonly used for pathogen detection in clinical and environmental samples. These sample matrices often contain inhibitors of PCR, which is a primary reason for sample processing; however, the purification process is highly inefficient, becoming unacceptable at lower signature concentrations. One potential solution is direct PCR assessment without sample processing. Here, we evaluated nine inhibitor-resistant PCR reagents for direct detection of Francisella ...

  10. Interactive metagenomic visualization in a Web browser

    Directory of Open Access Journals (Sweden)

    Phillippy Adam M

    2011-09-01

    Full Text Available Abstract Background A critical output of metagenomic studies is the estimation of abundances of taxonomical or functional groups. The inherent uncertainty in assignments to these groups makes it important to consider both their hierarchical contexts and their prediction confidence. The current tools for visualizing metagenomic data, however, omit or distort quantitative hierarchical relationships and lack the facility for displaying secondary variables. Results Here we present Krona, a new visualization tool that allows intuitive exploration of relative abundances and confidences within the complex hierarchies of metagenomic classifications. Krona combines a variant of radial, space-filling displays with parametric coloring and interactive polar-coordinate zooming. The HTML5 and JavaScript implementation enables fully interactive charts that can be explored with any modern Web browser, without the need for installed software or plug-ins. This Web-based architecture also allows each chart to be an independent document, making them easy to share via e-mail or post to a standard Web server. To illustrate Krona's utility, we describe its application to various metagenomic data sets and its compatibility with popular metagenomic analysis tools. Conclusions Krona is both a powerful metagenomic visualization tool and a demonstration of the potential of HTML5 for highly accessible bioinformatic visualizations. Its rich and interactive displays facilitate more informed interpretations of metagenomic analyses, while its implementation as a browser-based application makes it extremely portable and easily adopted into existing analysis packages. Both the Krona rendering code and conversion tools are freely available under a BSD open-source license, and available from: http://krona.sourceforge.net.

  11. The phenomenology of the first panic attack in clinical and community-based samples.

    Science.gov (United States)

    Pané-Farré, Christiane A; Stender, Jan P; Fenske, Kristin; Deckert, Jürgen; Reif, Andreas; John, Ulrich; Schmidt, Carsten Oliver; Schulz, Andrea; Lang, Thomas; Alpers, Georg W; Kircher, Tilo; Vossbeck-Elsebusch, Anna N; Grabe, Hans J; Hamm, Alfons O

    2014-08-01

    The purpose of the study was to contrast first panic attacks (PAs) of patients with panic disorder (PD) with vs. without agoraphobia and to explore differences between first PAs leading to the development of PD and those that remain isolated. Data were drawn from a community survey (N=2259 including 88 isolated PAs and 75 PD cases). An additional sample of 234 PD patients was recruited in a clinical setting. A standardized interview assessed the symptoms of the first PA, context of its occurrence and subsequent coping attempts. Persons who developed PD reported more severe first PAs, more medical service utilization and exposure-limiting coping attempts than those with isolated PAs. The context of the first PA did not differ between PD and isolated PAs. PD with agoraphobia was specifically associated with greater symptom severity and occurrence of first attacks in public. Future research should validate these findings using a longitudinal approach. PMID:24973697

  12. Factor structure of the SOCRATES in a clinical sample of adolescents.

    Science.gov (United States)

    Maisto, Stephen A; Chung, Tammy A; Cornelius, Jack R; Martin, Christopher S

    2003-06-01

    This study investigated the Stages of Change Readiness and Treatment Eagerness Scale (SOCRATES; W. R. Miller & J. S. Tonigan, 1996) in adolescents presenting for treatment of alcohol use disorder (AUD). The participants were 80 males and 43 females (mean age = 16.8 years) who presented for AUD treatment (95.1% outpatient, 4.9% inpatient). Participants completed assessments at baseline and 1 year and provided information on alcohol use and related variables monthly between these 2 assessments. Principal-components and confirmatory factor analyses of the baseline SOCRATES identified 2 factors, Taking Steps and Recognition, which showed good internal consistency and concurrent and predictive evidence of validity. The results were interpreted as supporting the use of the SOCRATES with clinical samples of adolescents.

  13. Self-esteem in a clinical sample of morbidly obese children and adolescents

    DEFF Research Database (Denmark)

    Nowicka, P; Höglund, P; Birgerstam, P;

    2009-01-01

    AIM: To study self-esteem in clinical sample of obese children and adolescents. METHODS: Obese children and adolescents aged 8-19 years (n = 107, mean age 13.2 years, mean BMI 32.5 [range 22.3-50.6], mean BMI z-score 3.22 [range 2.19-4.79]; 50 boys and 57 girls) were referred for treatment...... of primary obesity. Self-esteem was measured with a validated psychological test with five subscales: physical characteristics, talents and skills, psychological well-being, relations with the family and relations with others. A linear mixed effect model used the factors gender and adolescence group......, and the continuous covariates: BMI z-scores, and BMI for the parents as fixed effects and subjects as random effects. RESULTS: Age and gender, but neither the child's BMI z-score nor the BMI of the parents were significant covariates. Self-esteem decreased (p

  14. PNA-based fluorescence in situ hybridization for identification of bacteria in clinical samples

    DEFF Research Database (Denmark)

    Fazli, Mustafa; Bjarnsholt, Thomas; Høiby, Niels;

    2014-01-01

    Fluorescence in situ hybridization with PNA probes (PNA-FISH) that target specific bacterial ribosomal RNA sequences is a powerful and rapid tool for identification of bacteria in clinical samples. PNA can diffuse readily through the bacterial cell wall due to its uncharged backbone, and PNA......-FISH can be performed with high specificity due to the extraordinary thermal stability of RNA-PNA hybrid complexes. We describe a PNA-FISH procedure and provide examples of the application of PNA-FISH for the identification of bacteria in chronic wounds, cystic fibrosis lungs, and soft tissue fillers....... In all these cases, bacteria can be identified in biofilm aggregates, which may explain their recalcitrance to antibiotic treatment....

  15. Evaluation of Microarray Preprocessing Algorithms Based on Concordance with RT-PCR in Clinical Samples

    DEFF Research Database (Denmark)

    Hansen, Kasper Lage; Szallasi, Zoltan Imre; Eklund, Aron Charles;

    2009-01-01

    Several preprocessing algorithms for Affymetrix gene expression microarrays have been developed, and their performance on spike-in data sets has been evaluated previously. However, a comprehensive comparison of preprocessing algorithms on samples taken under research conditions has not been...... that were most consistent with RT-PCR measurements, although the difference in performance between most of the algorithms was not statistically significant. CONCLUSIONS/SIGNIFICANCE: Our results support the choice of PLIER+16 for the preprocessing of clinical Affymetrix microarray data. However, other...... performed. METHODOLOGY/PRINCIPAL FINDINGS: We used TaqMan RT-PCR arrays as a reference to evaluate the accuracy of expression values from Affymetrix microarrays in two experimental data sets: one comprising 84 genes in 36 colon biopsies, and the other comprising 75 genes in 29 cancer cell lines. We...

  16. Diel Metagenomics and Metatranscriptomics of Elkhorn Slough Hypersaline Microbial Mat

    Science.gov (United States)

    Lee, J.; Detweiler, A. M.; Everroad, R. C.; Bebout, L. E.; Weber, P. K.; Pett-Ridge, J.; Bebout, B.

    2014-12-01

    To understand the variation in gene expression associated with the daytime oxygenic phototrophic and nighttime fermentation regimes seen in hypersaline microbial mats, a contiguous mat piece was subjected to sampling at regular intervals over a 24-hour diel period. Additionally, to understand the impact of sulfate reduction on biohydrogen consumption, molybdate was added to a parallel experiment in the same run. 4 metagenome and 12 metatranscriptome Illumina HiSeq lanes were completed over day / night, and control / molybdate experiments. Preliminary comparative examination of noon and midnight metatranscriptomic samples mapped using bowtie2 to reference genomes has revealed several notable results about the dominant mat-building cyanobacterium Microcoleus chthonoplastes PCC 7420. Dominant cyanobacterium M. chthonoplastes PCC 7420 shows expression in several pathways for nitrogen scavenging, including nitrogen fixation. Reads mapped to M. chthonoplastes PCC 7420 shows expression of two starch storage and utilization pathways, one as a starch-trehalose-maltose-glucose pathway, another through UDP-glucose-cellulose-β-1,4 glucan-glucose pathway. The overall trend of gene expression was primarily light driven up-regulation followed by down-regulation in dark, while much of the remaining expression profile appears to be constitutive. Co-assembly of quality-controlled reads from 4 metagenomes was performed using Ray Meta with progressively smaller K-mer sizes, with bins identified and filtered using principal component analysis of coverages from all libraries and a %GC filter, followed by reassembly of the remaining co-assembly reads and binned reads. Despite having relatively similar abundance profiles in each metagenome, this binning approach was able to distinctly resolve bins from dominant taxa, but also sulfate reducing bacteria that are desired for understanding molybdate inhibition. Bins generated from this iterative assembly process will be used for downstream

  17. Metagenomic reconstructions of bacterial CRISPR loci constrain population histories.

    Science.gov (United States)

    Sun, Christine L; Thomas, Brian C; Barrangou, Rodolphe; Banfield, Jillian F

    2016-04-01

    Bacterial CRISPR-Cas systems provide insight into recent population history because they rapidly incorporate, in a unidirectional manner, short fragments (spacers) from coexisting infective virus populations into host chromosomes. Immunity is achieved by sequence identity between transcripts of spacers and their targets. Here, we used metagenomics to study the stability and dynamics of the type I-E CRISPR-Cas locus of Leptospirillum group II bacteria in biofilms sampled over 5 years from an acid mine drainage (AMD) system. Despite recovery of 452,686 spacers from CRISPR amplicons and metagenomic data, rarefaction curves of spacers show no saturation. The vast repertoire of spacers is attributed to phage/plasmid population diversity and retention of old spacers, despite rapid evolution of the targeted phage/plasmid genome regions (proto-spacers). The oldest spacers (spacers found at the trailer end) are conserved for at least 5 years, and 12% of these retain perfect or near-perfect matches to proto-spacer targets. The majority of proto-spacer regions contain an AAG proto-spacer adjacent motif (PAM). Spacers throughout the locus target the same phage population (AMDV1), but there are blocks of consecutive spacers without AMDV1 target sequences. Results suggest long-term coexistence of Leptospirillum with AMDV1 and periods when AMDV1 was less dominant. Metagenomics can be applied to millions of cells in a single sample to provide an extremely large spacer inventory, allow identification of phage/plasmids and enable analysis of previous phage/plasmid exposure. Thus, this approach can provide insights into prior bacterial environment and genetic interplay between hosts and their viruses. PMID:26394009

  18. Preliminary Examination of the Interpersonal Psychological Theory of Suicide in an Adolescent Clinical Sample.

    Science.gov (United States)

    Horton, Sarah E; Hughes, Jennifer L; King, Jessica D; Kennard, Betsy D; Westers, Nicholas J; Mayes, Taryn L; Stewart, Sunita M

    2016-08-01

    This study offers a preliminary examination of the Interpersonal-Psychological Theory of Suicide (IPTS; Joiner 2005) in an adolescent clinical sample. The IPTS offers a nuanced framework that has many conceptual and practical merits. Although this theory has a growing base of evidence among adults, it has yet to be tested in adolescents using direct measures of its central constructs. Participants were 147 adolescents (76.2 % girls) on an inpatient psychiatric unit, who completed measures of key IPTS constructs of thwarted belongingness, perceived burdensomeness, acquired capability for suicide, as well as depression severity, hopelessness, and severity of suicidal symptoms. Our findings were largely consistent with hypotheses derived from the IPTS: perceived burdensomeness, and at a marginal level, thwarted belongingness, were independently associated with current suicidal ideation. The thwarted belongingness by perceived burdensomeness interaction marginally distinguished between adolescents with passive and active suicidal ideation. Acquired capability for suicide was associated with recent suicidal intent. Examination of all three IPTS constructs simultaneously revealed main effects of each construct (with a marginal effect of thwarted belongingness), and interaction effects for thwarted belongingness by perceived burdensomeness, and thwarted belongingness by perceived burdensomeness by acquired capability for suicide in association with suicidal symptom severity. Sex, age, depression severity, and hopelessness were controlled in all analyses. This study offers strong, albeit preliminary, support of the IPTS in a clinical adolescent sample. Assessment of IPTS constructs may be useful in determining persistent risk for suicide attempt. Prospective tests of the theory, and extensions to intervention and prevention should be considered in future IPTS research. PMID:26667025

  19. [Histological view of ethics in medicine and handling of residual samples in clinical laboratories].

    Science.gov (United States)

    Yoshida, Hiroshi

    2004-03-01

    One of the important ethical issues in clinical laboratory medicine is whether organs and/or specimens should belong to the examinees. Tracing back to ancient Greece, an episode of the death of Asklepios, killed by Zeus to revive the dead, and the great contribution of Hippocrates to medicine including the vow and ethics of medicine, have been described. In the relationship between doctors and patients, the former had been superior to the latter for more than 2400 years, however, the situation has been changing from that to the same position since 1960th, along with the development of bioethics from medical ethics. For the promotion of bioethics, world medical associations have contributed declarations and continuous discussion. The declarations are based on the avoidance of actions detrimental to the life, health, privacy or dignity of examinees. On the medical use of human organs and specimens in relation to human rights, the mind and the body, discussion has continued, however, a consensus on the details has not been reached. A view on the use of residual samples for methodological study, teaching and research in the clinical laboratory was proposed by the Japanese Society of Laboratory Medicine in 2002. Briefly, it included confidentiality of the laboratory staff, responsibility of the laboratory director, the absence of a necessity to obtain consent for the use of residual samples for methodological study when they are made anonymous or pooled, and the recommendation to obtain a judgement by an ethics committee for research use. The background and discussion for the proposal and the current situation on how to obtain consent from patients in Japan are mentioned.

  20. [Occurrence and antimicrobial susceptibility of Morganella morganii strains isolated from clinical samples].

    Science.gov (United States)

    Zalas-Wiecek, Patrycja; Gospodarek, Eugenia; Wróblewska, Joanna

    2012-01-01

    The aim of this study was the evaluation of occurrence and antimicrobial susceptibility of M morganii rods isolated from clinical samples. This study included 201 strains isolated in the Clinical Microbiology Department of Dr. A. Jurasz University Hospital in 2008-2010. Identification to species was carried out on the basis of the results of biochemical reactions included in the tests ID 32E and VITEK2 GN. Antimicrobial susceptibility of M. morganii rods was determined by the disk-diffusion method on Mueller-Hinton II Agar. Strains of M morganii most commonly isolated from skin and soft tissue, and material taken from the urinary tract, mainly from patients of Anesthesiology and Intensive Care Unit, Department of General and Vascular Surgery and Department of General Surgery and Endocrinology. All of M morganii strains isolated during the three years were susceptible to carbapenems. We reported decrease of strains susceptible to piperacillin and chloramphenicol. In 2010 we showed a higher percentage of strains intermediate to tigecycline, compared with 2009. We observed increase in the percentage of strains resistant to cefoperazone with sulbactam and reported decrease in the percentage of strains resistant and intermediate to aminoglycosides. Extended Spectrum Beta-Lactamases were produced by 13 (6,5%) of M morganii strains.

  1. IDENTIFICATION OF CANDIDA SPECIES FROM CLINICAL SAMPLES AND THEIR ANTIFUNGAL SUSCEPTIBILITY PATTERNS

    Directory of Open Access Journals (Sweden)

    Bhaskar

    2015-09-01

    Full Text Available OBJECTIVE AND BACKGROUND : The incidence of Candida infections has increased dramatically over the past few decades due to increase in the number of population susceptible to fungal infections. With multiple antifungal ag ents that are available and recovery of clinical isolates that exhibit inherent or developed resistance to commonly used antifungal agents, it has become imperative to do susceptibility testing routinely. The study was done to determine the predisposing fa ctors, species incidence and susceptibility pattern of Candida isolates to commonly use d antifungal agents. METHODS: A total of 108 Candida species were recovered from symptomatic clinical cases. Candida isolates were speciated by germ tube test, chlamydospore formation on corn meal agar and color produced on chromogenic media. Antifungal susceptibility test was done by disk diffusion method for nystatin, fluconazole, itraconazole, voriconazole and amphotericin - B. RESULTS: Candida albicans is the m ost frequently isolated species. However, non - albicans Candida species, taken as a group has predominated in clinical samples. Chromogenic agar medium showed good correlation in species identification in comparison with conventional germ tube test and chla mydospore formation on corn meal agar. C. albicans (41, C. tropicalis (33, C. krusei (30 and C. glabrata (04 were isolated. Candida species showed 95.4% susceptibility to amphotericin - B, 77.8% to voriconazol e, 69.4% to nystatin, 64.1% to f luconazole an d 63.9% to itraconazole. CONCLUSION : Increasing incidence of non - albicans species infection. Chromogenic medium can be used for species identification. Increasing resistance of Candida species to commonly used antifungal agents.

  2. Soil Metagenomes from Different Pristine Environments of Northwest Argentina

    OpenAIRE

    McCarthy, Christina B.; Colman, Déborah I.

    2015-01-01

    This is the first study to use a high-throughput metagenomic shotgun approach to explore the biosynthetic potential of soil metagenomes from different pristine environments of northwest Argentina. Our data sets characterize these metagenomes and provide information on the possible effect these ecosystems have on their diversity and biosynthetic potential.

  3. Improved metagenome assemblies and taxonomic binning using long-read circular consensus sequence data.

    Science.gov (United States)

    Frank, J A; Pan, Y; Tooming-Klunderud, A; Eijsink, V G H; McHardy, A C; Nederbragt, A J; Pope, P B

    2016-05-09

    DNA assembly is a core methodological step in metagenomic pipelines used to study the structure and function within microbial communities. Here we investigate the utility of Pacific Biosciences long and high accuracy circular consensus sequencing (CCS) reads for metagenomic projects. We compared the application and performance of both PacBio CCS and Illumina HiSeq data with assembly and taxonomic binning algorithms using metagenomic samples representing a complex microbial community. Eight SMRT cells produced approximately 94 Mb of CCS reads from a biogas reactor microbiome sample that averaged 1319 nt in length and 99.7% accuracy. CCS data assembly generated a comparative number of large contigs greater than 1 kb, to those assembled from a ~190x larger HiSeq dataset (~18 Gb) produced from the same sample (i.e approximately 62% of total contigs). Hybrid assemblies using PacBio CCS and HiSeq contigs produced improvements in assembly statistics, including an increase in the average contig length and number of large contigs. The incorporation of CCS data produced significant enhancements in taxonomic binning and genome reconstruction of two dominant phylotypes, which assembled and binned poorly using HiSeq data alone. Collectively these results illustrate the value of PacBio CCS reads in certain metagenomics applications.

  4. Improved metagenome assemblies and taxonomic binning using long-read circular consensus sequence data.

    Science.gov (United States)

    Frank, J A; Pan, Y; Tooming-Klunderud, A; Eijsink, V G H; McHardy, A C; Nederbragt, A J; Pope, P B

    2016-01-01

    DNA assembly is a core methodological step in metagenomic pipelines used to study the structure and function within microbial communities. Here we investigate the utility of Pacific Biosciences long and high accuracy circular consensus sequencing (CCS) reads for metagenomic projects. We compared the application and performance of both PacBio CCS and Illumina HiSeq data with assembly and taxonomic binning algorithms using metagenomic samples representing a complex microbial community. Eight SMRT cells produced approximately 94 Mb of CCS reads from a biogas reactor microbiome sample that averaged 1319 nt in length and 99.7% accuracy. CCS data assembly generated a comparative number of large contigs greater than 1 kb, to those assembled from a ~190x larger HiSeq dataset (~18 Gb) produced from the same sample (i.e approximately 62% of total contigs). Hybrid assemblies using PacBio CCS and HiSeq contigs produced improvements in assembly statistics, including an increase in the average contig length and number of large contigs. The incorporation of CCS data produced significant enhancements in taxonomic binning and genome reconstruction of two dominant phylotypes, which assembled and binned poorly using HiSeq data alone. Collectively these results illustrate the value of PacBio CCS reads in certain metagenomics applications. PMID:27156482

  5. A statistical framework for accurate taxonomic assignment of metagenomic sequencing reads.

    Directory of Open Access Journals (Sweden)

    Hongmei Jiang

    Full Text Available The advent of next-generation sequencing technologies has greatly promoted the field of metagenomics which studies genetic material recovered directly from an environment. Characterization of genomic composition of a metagenomic sample is essential for understanding the structure of the microbial community. Multiple genomes contained in a metagenomic sample can be identified and quantitated through homology searches of sequence reads with known sequences catalogued in reference databases. Traditionally, reads with multiple genomic hits are assigned to non-specific or high ranks of the taxonomy tree, thereby impacting on accurate estimates of relative abundance of multiple genomes present in a sample. Instead of assigning reads one by one to the taxonomy tree as many existing methods do, we propose a statistical framework to model the identified candidate genomes to which sequence reads have hits. After obtaining the estimated proportion of reads generated by each genome, sequence reads are assigned to the candidate genomes and the taxonomy tree based on the estimated probability by taking into account both sequence alignment scores and estimated genome abundance. The proposed method is comprehensively tested on both simulated datasets and two real datasets. It assigns reads to the low taxonomic ranks very accurately. Our statistical approach of taxonomic assignment of metagenomic reads, TAMER, is implemented in R and available at http://faculty.wcas.northwestern.edu/hji403/MetaR.htm.

  6. A Multitrait-Multimethod Analysis of the Construct Validity of Child Anxiety Disorders in a Clinical Sample

    Science.gov (United States)

    Langer, David A.; Wood, Jeffrey J.; Bergman, R. Lindsey; Piacentini, John C.

    2010-01-01

    The present study examines the construct validity of separation anxiety disorder (SAD), social phobia (SoP), panic disorder (PD), and generalized anxiety disorder (GAD) in a clinical sample of children. Participants were 174 children, 6 to 17 years old (94 boys) who had undergone a diagnostic evaluation at a university hospital based clinic.…

  7. Preliminary High-Throughput Metagenome Assembly

    Energy Technology Data Exchange (ETDEWEB)

    Dusheyko, Serge; Furman, Craig; Pangilinan, Jasmyn; Shapiro, Harris; Tu, Hank

    2007-03-26

    Metagenome data sets present a qualitatively different assembly problem than traditional single-organism whole-genome shotgun (WGS) assembly. The unique aspects of such projects include the presence of a potentially large number of distinct organisms and their representation in the data set at widely different fractions. In addition, multiple closely related strains could be present, which would be difficult to assemble separately. Failure to take these issues into account can result in poor assemblies that either jumble together different strains or which fail to yield useful results. The DOE Joint Genome Institute has sequenced a number of metagenomic projects and plans to considerably increase this number in the coming year. As a result, the JGI has a need for high-throughput tools and techniques for handling metagenome projects. We present the techniques developed to handle metagenome assemblies in a high-throughput environment. This includes a streamlined assembly wrapper, based on the JGI?s in-house WGS assembler, Jazz. It also includes the selection of sensible defaults targeted for metagenome data sets, as well as quality control automation for cleaning up the raw results. While analysis is ongoing, we will discuss preliminary assessments of the quality of the assembly results (http://fames.jgi-psf.org).

  8. Rapid Isolation of Staphylococcus aureus Pathogens from Infected Clinical Samples Using Magnetic Beads Coated with Fc-Mannose Binding Lectin.

    Science.gov (United States)

    Bicart-See, A; Rottman, M; Cartwright, M; Seiler, B; Gamini, N; Rodas, M; Penary, M; Giordano, G; Oswald, E; Super, M; Ingber, D E

    2016-01-01

    Here we describe how Staphylococcus aureus bacteria can be rapidly isolated from clinical samples of articular fluid and synovial tissue using magnetic beads coated with the engineered chimeric human opsonin protein, Fc-mannose-binding lectin (FcMBL). The FcMBL-beads were used to capture and magnetically remove bacteria from purified cultures of 12 S. aureus strains, and from 8 articular fluid samples and 4 synovial tissue samples collected from patients with osteoarthritis or periprosthetic infections previously documented by positive S. aureus cultures. While the capture efficiency was high (85%) with purified S. aureus strains grown in vitro, direct FcMBL-bead capture from the clinical samples was initially disappointing (viscosity of these complex biological samples. Importantly, capture of pathogens using the FcMBL-beads was increased to 76% efficiency by pretreating clinical specimens with hypotonic washes, hyaluronidase and a protease cocktail. Using this approach, S. aureus bacteria could be isolated from infected osteoarthritic tissues within 2 hours after sample collection. This FcMBL-enabled magnetic method for rapid capture and concentration of pathogens from clinical samples could be integrated upstream of current processes used in clinical microbiology laboratories to identify pathogens and perform antibiotic sensitivity testing when bacterial culture is not possible or before colonies can be detected. PMID:27275840

  9. Rapid Isolation of Staphylococcus aureus Pathogens from Infected Clinical Samples Using Magnetic Beads Coated with Fc-Mannose Binding Lectin.

    Directory of Open Access Journals (Sweden)

    A Bicart-See

    Full Text Available Here we describe how Staphylococcus aureus bacteria can be rapidly isolated from clinical samples of articular fluid and synovial tissue using magnetic beads coated with the engineered chimeric human opsonin protein, Fc-mannose-binding lectin (FcMBL. The FcMBL-beads were used to capture and magnetically remove bacteria from purified cultures of 12 S. aureus strains, and from 8 articular fluid samples and 4 synovial tissue samples collected from patients with osteoarthritis or periprosthetic infections previously documented by positive S. aureus cultures. While the capture efficiency was high (85% with purified S. aureus strains grown in vitro, direct FcMBL-bead capture from the clinical samples was initially disappointing (< 5% efficiency. Further analysis revealed that inhibition of FcMBL binding was due to coating of the bacteria by immunoglobulins and immune cells that masked FcMBL binding sites, and to the high viscosity of these complex biological samples. Importantly, capture of pathogens using the FcMBL-beads was increased to 76% efficiency by pretreating clinical specimens with hypotonic washes, hyaluronidase and a protease cocktail. Using this approach, S. aureus bacteria could be isolated from infected osteoarthritic tissues within 2 hours after sample collection. This FcMBL-enabled magnetic method for rapid capture and concentration of pathogens from clinical samples could be integrated upstream of current processes used in clinical microbiology laboratories to identify pathogens and perform antibiotic sensitivity testing when bacterial culture is not possible or before colonies can be detected.

  10. Viral metagenomics and blood safety.

    Science.gov (United States)

    Sauvage, V; Eloit, M

    2016-02-01

    The characterization of the human blood-associated viral community (also called blood virome) is essential for epidemiological surveillance and to anticipate new potential threats for blood transfusion safety. Currently, the risk of blood-borne agent transmission of well-known viruses (HBV, HCV, HIV and HTLV) can be considered as under control in high-resource countries. However, other viruses unknown or unsuspected may be transmitted to recipients by blood-derived products. This is particularly relevant considering that a significant proportion of transfused patients are immunocompromised and more frequently subjected to fatal outcomes. Several measures to prevent transfusion transmission of unknown viruses have been implemented including the exclusion of at-risk donors, leukocyte reduction of donor blood, and physicochemical treatment of the different blood components. However, up to now there is no universal method for pathogen inactivation, which would be applicable for all types of blood components and, equally effective for all viral families. In addition, among available inactivation procedures of viral genomes, some of them are recognized to be less effective on non-enveloped viruses, and inadequate to inactivate higher viral titers in plasma pools or derivatives. Given this, there is the need to implement new methodologies for the discovery of unknown viruses that may affect blood transfusion. Viral metagenomics combined with High Throughput Sequencing appears as a promising approach for the identification and global surveillance of new and/or unexpected viruses that could impair blood transfusion safety. PMID:26778104

  11. Complete Genome Sequences of Three African Foot-and-Mouth Disease Viruses from Clinical Samples Isolated in 2009 and 2010.

    Science.gov (United States)

    Van Borm, Steven; Rosseel, Toon; Haegeman, Andy; Fana, Mpolokang Elliot; Seoke, Latoa; Hyera, Joseph; Matlho, George; Vandenbussche, Frank; De Clercq, Kris

    2016-01-01

    The complete genome sequences of three foot-and-mouth disease viruses (one virus of each serotype SAT1, SAT2 and O) were directly sequenced from RNA extracted from clinical bovine samples, demonstrating the feasibility of full-genome sequencing from strong positive samples taken from symptomatic animals. PMID:27151795

  12. The Appraisal of Social Concerns Scale: Psychometric Validation with a Clinical Sample of Patients with Social Anxiety Disorder

    Science.gov (United States)

    Schultz, Luke T.; Heimberg, Richard G.; Rodebaugh, Thomas L.; Schneier, Franklin R.; Liebowitz, Michael R.; Telch, Michael J.

    2006-01-01

    The Appraisal of Social Concerns (ASC) Scale was created by Telch et al. (2004) to improve upon existing self-report measures of social anxiety-related cognition. In a largely nonclinical sample, the ASC was found to possess three factors and was psychometrically sound. In a smaller clinical sample, the ASC demonstrated sensitivity to the effects…

  13. Metagenomic exploration of antibiotic resistance in soil.

    Science.gov (United States)

    Monier, Jean-Michel; Demanèche, Sandrine; Delmont, Tom O; Mathieu, Alban; Vogel, Timothy M; Simonet, Pascal

    2011-06-01

    The ongoing development of metagenomic approaches is providing the means to explore antibiotic resistance in nature and address questions that could not be answered previously with conventional culture-based strategies. The number of available environmental metagenomic sequence datasets is rapidly expanding and henceforth offer the ability to gain a more comprehensive understanding of antibiotic resistance at the global scale. Although there is now evidence that the environment constitutes a vast reservoir of antibiotic resistance gene determinants (ARGDs) and that the majority of ARGDs acquired by human pathogens may have an environmental origin, a better understanding of their diversity, prevalence and ecological significance may help predict the emergence and spreading of newly acquired resistances. Recent applications of metagenomic approaches to the study of ARGDs in natural environments such as soil should help overcome challenges concerning expanding antibiotic resistances. PMID:21601510

  14. Pathway-Based Functional Analysis of Metagenomes

    Science.gov (United States)

    Bercovici, Sivan; Sharon, Itai; Pinter, Ron Y.; Shlomi, Tomer

    Metagenomic data enables the study of microbes and viruses through their DNA as retrieved directly from the environment in which they live. Functional analysis of metagenomes explores the abundance of gene families, pathways, and systems, rather than their taxonomy. Through such analysis researchers are able to identify those functional capabilities most important to organisms in the examined environment. Recently, a statistical framework for the functional analysis of metagenomes was described that focuses on gene families. Here we describe two pathway level computational models for functional analysis that take into account important, yet unaddressed issues such as pathway size, gene length and overlap in gene content among pathways. We test our models over carefully designed simulated data and propose novel approaches for performance evaluation. Our models significantly improve over current approach with respect to pathway ranking and the computations of relative abundance of pathways in environments.

  15. Assembling the marine metagenome, one cell at a time.

    Directory of Open Access Journals (Sweden)

    Tanja Woyke

    Full Text Available The difficulty associated with the cultivation of most microorganisms and the complexity of natural microbial assemblages, such as marine plankton or human microbiome, hinder genome reconstruction of representative taxa using cultivation or metagenomic approaches. Here we used an alternative, single cell sequencing approach to obtain high-quality genome assemblies of two uncultured, numerically significant marine microorganisms. We employed fluorescence-activated cell sorting and multiple displacement amplification to obtain hundreds of micrograms of genomic DNA from individual, uncultured cells of two marine flavobacteria from the Gulf of Maine that were phylogenetically distant from existing cultured strains. Shotgun sequencing and genome finishing yielded 1.9 Mbp in 17 contigs and 1.5 Mbp in 21 contigs for the two flavobacteria, with estimated genome recoveries of about 91% and 78%, respectively. Only 0.24% of the assembling sequences were contaminants and were removed from further analysis using rigorous quality control. In contrast to all cultured strains of marine flavobacteria, the two single cell genomes were excellent Global Ocean Sampling (GOS metagenome fragment recruiters, demonstrating their numerical significance in the ocean. The geographic distribution of GOS recruits along the Northwest Atlantic coast coincided with ocean surface currents. Metabolic reconstruction indicated diverse potential energy sources, including biopolymer degradation, proteorhodopsin photometabolism, and hydrogen oxidation. Compared to cultured relatives, the two uncultured flavobacteria have small genome sizes, few non-coding nucleotides, and few paralogous genes, suggesting adaptations to narrow ecological niches. These features may have contributed to the abundance of the two taxa in specific regions of the ocean, and may have hindered their cultivation. We demonstrate the power of single cell DNA sequencing to generate reference genomes of uncultured

  16. Assembling The Marine Metagenome, One Cell At A Time

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Gang [Los Alamos National Laboratory; Han, Shunsheng [Los Alamos National Laboratory; Kiss, Hajnalka [Los Alamos National Laboratory; Saw, Jimmy [Los Alamos National Laboratory; Senin, Pavel [Los Alamos National Laboratory; Woyke, Tanja [DOE JOINT GENOME INAT.; Copeland, Alex [DOE JOINT GENSOME INST.; Gonzalez, Jose [UNIV OF LAGUNA, SPAIN; Chatterji, Sourav [DOE JOINT GENSOME INST.; Cheng, Jan - Fang [DOE JOINT GENSOME INST.; Eisen, Jonathan A [DOE JOINT GENOME INST.; Sieracki, Michael E [UNIV OF CA-DAVIS; Stepanauskas, Ramunas [BIGELOW LAB

    2008-01-01

    The difficulty associated with the cultivation of most microorganisms and the complexity of natural microbial assemblages, such as marine plankton or human microbiome, hinder genome reconstruction of representative taxa using cultivation or metagenomic approaches. Here we used an alternative, single cell sequencing approach to obtain high-quality genome assemblies of two uncultured, numerically significant marine microorganisms. We employed fluorescence-activated cell sorting and multiple displacement amplification to obtain hundreds of micrograms of genomic DNA from individual, uncultured cells of two marine flavobacteria from the Gulf of Maine that were phylogenetically distant from existing cultured strains. Shotgun sequencing and genome finishing yielded 1.9 Mbp in 17 contigs and 1.5 Mbp in 21 contigs for the two flavobacteria, with estimated genome recoveries of about 91% and 78%, respectively. Only 0.24% of the assembling sequences were contaminants and were removed from further analysis using rigorous quality control. In contrast to all cultured strains of marine flavobacteria, the two single cell genomes were excellent Global Ocean Sampling (GOS) metagenome fragment recruiters, demonstrating their numerical significance in the ocean. The geographic distribution of GOS recruits along the Northwest Atlantic coast coincided with ocean surface currents. Metabolic reconstruction indicated diverse potential energy sources, including biopolymer degradation, proteorhodopsin photometabolism, and hydrogen oxidation. Compared to cultured relatives, the two uncultured flavobacteria have small genome sizes, few non-coding nucleotides, and few paralogous genes, suggesting adaptations to narrow ecological niches. These features may have contributed to the abundance of the two taxa in specific regions of the ocean, and may have hindered their cultivation. We demonstrate the power of single cell DNA sequencing to generate reference genomes of uncultured taxa from a complex

  17. Assembling the Marine Metagenome, One Cell at a Time

    Energy Technology Data Exchange (ETDEWEB)

    Woyke, Tanja; Xie, Gary; Copeland, Alex; Gonzalez, Jose M.; Han, Cliff; Kiss, Hajnalka; Saw, Jimmy H.; Senin, Pavel; Yang, Chi; Chatterji, Sourav; Cheng, Jan-Fang; Eisen, Jonathan A.; Sieracki, Michael E.; Stepanauskas, Ramunas

    2010-06-24

    The difficulty associated with the cultivation of most microorganisms and the complexity of natural microbial assemblages, such as marine plankton or human microbiome, hinder genome reconstruction of representative taxa using cultivation or metagenomic approaches. Here we used an alternative, single cell sequencing approach to obtain high-quality genome assemblies of two uncultured, numerically significant marine microorganisms. We employed fluorescence-activated cell sorting and multiple displacement amplification to obtain hundreds of micrograms of genomic DNA from individual, uncultured cells of two marine flavobacteria from the Gulf of Maine that were phylogenetically distant from existing cultured strains. Shotgun sequencing and genome finishing yielded 1.9 Mbp in 17 contigs and 1.5 Mbp in 21 contigs for the two flavobacteria, with estimated genome recoveries of about 91percent and 78percent, respectively. Only 0.24percent of the assembling sequences were contaminants and were removed from further analysis using rigorous quality control. In contrast to all cultured strains of marine flavobacteria, the two single cell genomes were excellent Global Ocean Sampling (GOS) metagenome fragment recruiters, demonstrating their numerical significance in the ocean. The geographic distribution of GOS recruits along the Northwest Atlantic coast coincided with ocean surface currents. Metabolic reconstruction indicated diverse potential energy sources, including biopolymer degradation, proteorhodopsin photometabolism, and hydrogen oxidation. Compared to cultured relatives, the two uncultured flavobacteria have small genome sizes, few non-coding nucleotides, and few paralogous genes, suggesting adaptations to narrow ecological niches. These features may have contributed to the abundance of the two taxa in specific regions of the ocean, and may have hindered their cultivation. We demonstrate the power of single cell DNA sequencing to generate reference genomes of uncultured

  18. Evaluation of the DSM-5 severity indicator for binge eating disorder in a clinical sample

    Science.gov (United States)

    Grilo, Carlos M.; Ivezaj, Valentina; White, Marney A.

    2015-01-01

    Objective This study tested the new DSM-5 severity criterion for binge eating disorder (BED) based on frequency of binge-eating in a clinical sample. This study also tested overvaluation of shape/weight as an alternative severity specifier. Method Participants were 834 treatment-seeking adults diagnosed with DSM-5 BED using semistructured diagnostic and eating-disorder interviews. Participants sub-grouped based on DSM-5 severity levels and on overvaluation of shape/weight were compared on demographic and clinical variables. Results Based on DSM-5 severity definitions, 331 (39.7%) participants were categorized as mild, 395 (47.5%) as moderate, 83 (10.0%) as severe, and 25 (3.0%) as extreme. Analyses comparing three (mild, moderate, and severe/extreme) severity groups revealed no significant differences in demographic variables or body mass index (BMI). Analyses revealed significantly higher eating-disorder psychopathology in the severe/extreme than moderate and mild groups and higher depression in moderate and severe/extreme groups than the mild group; effect sizes were small. Participants characterized with overvaluation (N = 449; 54%) versus without overvaluation (N = 384; 46%) did not differ significantly in age, sex, BMI, or binge-eating frequency, but had significantly greater eating-disorder psychopathology and depression. The robustly greater eating-disorder psychopathology and depression levels (medium-to-large effect sizes) in the overvaluation group was observed without attenuation of effect sizes after adjusting for ethnicity/race and binge-eating severity/frequency. Conclusions Our findings provide support for overvaluation of shape/weight as a severity specifier for BED as it provides stronger information about the severity of homogeneous groupings of patients than the DSM-5 rating based on binge-eating. PMID:26114779

  19. Functionalized graphene oxide for clinical glucose biosensing in urine and serum samples

    Directory of Open Access Journals (Sweden)

    Veerapandian M

    2012-12-01

    Full Text Available Murugan Veerapandian,1 Yeong-Tai Seo,2 Hyunkyung Shin,3 Kyusik Yun,1 Min-Ho Lee41Department of Bionanotechnology, Gachon University, Gyeonggi-Do, 2School of Electrical Engineering and Computer Science, Seoul National University, Seoul, 3Department of Mathematics and Information, Gachon University, Gyeonggi-Do, 4Korea Electronics Technology Institute, Medical IT Technology, Gyeonggi-Do, Republic of KoreaAbstract: A novel clinical glucose biosensor fabricated using functionalized metalloid-polymer (silver-silica coated with polyethylene glycol hybrid nanoparticles on the surface of a graphene oxide nanosheet is reported. The cyclic voltammetric response of glucose oxidase modification on the surface of a functionalized graphene oxide electrode showed a surface-confined reaction and an effective redox potential near zero volts, with a wide linearity of 0.1–20 mM and a sensitivity of 7.66 µA mM-1 cm-2. The functionalized graphene oxide electrode showed a better electrocatalytic response toward oxidation of H2O2 and reduction of oxygen. The practical applicability of the functionalized graphene oxide electrode was demonstrated by measuring the peak current against multiple urine and serum samples from diabetic patients. This new hybrid nanoarchitecture combining a three-dimensional metalloid-polymer hybrid and two-dimensional graphene oxide provided a thin solid laminate on the electrode surface. The easy fabrication process and retention of bioactive immobilized enzymes on the functionalized graphene oxide electrode could potentially be extended to detection of other biomolecules, and have broad applications in electrochemical biosensing.Keywords: clinical diagnostics, glucose biosensor, metalloid-polymer nanoparticles, glucose oxidase, cyclic voltammetry

  20. Levels and types of alcohol biomarkers in DUI and clinic samples for estimating workplace alcohol problems.

    Science.gov (United States)

    Marques, Paul R

    2012-02-01

    Widespread concern about illicit drugs as an aspect of workplace performance potentially diminishes attention on employee alcohol use. Alcohol is the dominant drug contributing to poor job performance; it also accounts for a third of the worldwide public health burden. Evidence from public roadways--a workplace for many--provides an example of work-related risk exposure and performance lapses. In most developed countries, alcohol is involved in 20-35% of fatal crashes; drugs other than alcohol are less prominently involved in fatalities. Alcohol biomarkers can improve detection by extending the timeframe for estimating problematic exposure levels and thereby provide better information for managers. But what levels and which markers are right for the workplace? In this paper, an established high-sensitivity proxy for alcohol-driving risk proclivity is used: an average eight months of failed blood alcohol concentration (BAC) breath tests from alcohol ignition interlock devices. Higher BAC test fail rates are known to presage higher rates of future impaired-driving convictions (driving under the influence; DUI). Drivers in alcohol interlock programmes log 5-7 daily BAC tests; in 12 months, this yields thousands of samples. Also, higher programme entry levels of alcohol biomarkers predict a higher likelihood of failed interlock BAC tests during subsequent months. This paper summarizes the potential of selected biomarkers for workplace screening. Markers include phosphatidylethanol (PEth), percent carbohydrate deficient transferrin (%CDT), gammaglutamyltransferase (GGT), gamma %CDT (γ%CDT), and ethylglucuronide (EtG) in hair. Clinical cut-off levels and median/mean levels of these markers in abstinent people, the general population, DUI drivers, and rehabilitation clinics are summarized for context. PMID:22311827

  1. Levels and Types of Alcohol Biomarkers in DUI and Clinic Samples for Estimating Workplace Alcohol Problemsa

    Science.gov (United States)

    Marques, Paul R

    2013-01-01

    Widespread concern about illicit drugs as an aspect of workplace performance potentially diminishes attention on employee alcohol use. Alcohol is the dominant drug contributing to poor job performance; it also accounts for a third of the worldwide public health burden. Evidence from public roadways – a workplace for many – provides an example for work-related risk exposure and performance lapses. In most developed countries, alcohol is involved in 20-35% of fatal crashes; drugs other than alcohol are less prominently involved in fatalities. Alcohol biomarkers can improve detection by extending the timeframe for estimating problematic exposure levels and thereby provide better information for managers. But what levels and which markers are right for the workplace? In this report, an established high-sensitivity proxy for alcohol-driving risk proclivity is used: an average 8 months of failed blood alcohol concentration (BAC) breath tests from alcohol ignition interlock devices. Higher BAC test fail rates are known to presage higher rates of future impaired-driving convictions (DUI). Drivers in alcohol interlock programs log 5-7 daily BAC tests; in 12 months, this yields thousands of samples. Also, higher program entry levels of alcohol biomarkers predict a higher likelihood of failed interlock BAC tests during subsequent months. This report summarizes selected biomarkers’ potential for workplace screening. Markers include phosphatidylethanol (PEth), percent carbohydrate deficient transferrin (%CDT), gammaglutamyltransferase (GGT), gamma %CDT (γ%CDT), and ethylglucuronide (EtG) in hair. Clinical cutoff levels and median/mean levels of these markers in abstinent people, the general population, DUI drivers, and rehabilitation clinics are summarized for context. PMID:22311827

  2. Eu-Detect: An algorithm for detecting eukaryotic sequences in metagenomic data sets

    Indian Academy of Sciences (India)

    Monzoorul Haque Mohammed; Sudha Chadaram Dinakar; Dinakar Komanduri; Tarini Shankar Ghosh; Sharmila S Mande

    2011-09-01

    Physical partitioning techniques are routinely employed (during sample preparation stage) for segregating the prokaryotic and eukaryotic fractions of metagenomic samples. In spite of these efforts, several metagenomic studies focusing on bacterial and archaeal populations have reported the presence of contaminating eukaryotic sequences inmetagenomic data sets. Contaminating sequences originate not only from genomes of micro-eukaryotic species but also from genomes of (higher) eukaryotic host cells. The latter scenario usually occurs in the case of host-associatedmetagenomes. Identification and removal of contaminating sequences is important, since these sequences not only impact estimates of microbial diversity but also affect the accuracy of several downstream analyses. Currently, the computational techniques used for identifying contaminating eukaryotic sequences, being alignment based, are slow, inefficient, and require huge computing resources. In this article, we present Eu-Detect, an alignment-free algorithm that can rapidly identify eukaryotic sequences contaminating metagenomic data sets. Validation results indicate that on a desktop with modest hardware specifications, the Eu-Detect algorithm is able to rapidly segregate DNA sequence fragments of prokaryotic and eukaryotic origin, with high sensitivity. A Web server for the Eu-Detect algorithm is available at http://metagenomics.atc.tcs.com/Eu-Detect/.

  3. Metagenomic survey for viruses in Western Arctic caribou, Alaska, through iterative assembly of taxonomic units.

    Directory of Open Access Journals (Sweden)

    Anita C Schürch

    Full Text Available Pathogen surveillance in animals does not provide a sufficient level of vigilance because it is generally confined to surveillance of pathogens with known economic impact in domestic animals and practically nonexistent in wildlife species. As most (re-emerging viral infections originate from animal sources, it is important to obtain insight into viral pathogens present in the wildlife reservoir from a public health perspective. When monitoring living, free-ranging wildlife for viruses, sample collection can be challenging and availability of nucleic acids isolated from samples is often limited. The development of viral metagenomics platforms allows a more comprehensive inventory of viruses present in wildlife. We report a metagenomic viral survey of the Western Arctic herd of barren ground caribou (Rangifer tarandus granti in Alaska, USA. The presence of mammalian viruses in eye and nose swabs of 39 free-ranging caribou was investigated by random amplification combined with a metagenomic analysis approach that applied exhaustive iterative assembly of sequencing results to define taxonomic units of each metagenome. Through homology search methods we identified the presence of several mammalian viruses, including different papillomaviruses, a novel parvovirus, polyomavirus, and a virus that potentially represents a member of a novel genus in the family Coronaviridae.

  4. [Metagenomics in studying gastrointestinal tract microorganism].

    Science.gov (United States)

    Xu, Bo; Yang, Yunjuan; Li, Junjun; Tang, Xianghua; Mu, Yuelin; Huang, Zunxi

    2013-12-01

    Animal gastrointestinal tract contains a complex community of microbes, whose composition ultimately reflects the co-evolution of microorganisms with their animal host. The gut microbial community of humans and animals has received significant attention from researchers because of its association with health and disease. The application of metagenomics technology enables researchers to study not only the microbial composition but also the function of microbes in the gastrointestinal tract. In this paper, combined with our own findings, we summarized advances in studying gastrointestinal tract microorganism with metagenomics and the bioinformatics technology.

  5. Structured Interview of Personality Organization (STIPO): preliminary psychometrics in a clinical sample.

    Science.gov (United States)

    Stern, Barry L; Caligor, Eve; Clarkin, John F; Critchfield, Kenneth L; Horz, Susanne; MacCornack, Verna; Lenzenweger, Mark F; Kernberg, Otto F

    2010-01-01

    In this article, we describe the development and preliminary psychometric properties of the Structured Interview of Personality Organization (STIPO), a semistructured interview designed for the dimensional assessment of identity, primitive defenses, and reality testing, the three primary content domains in the model of personality health and disorder elaborated by Kernberg (1984; Kernberg & Caligor, 2005). Results of this investigation, conducted in a clinical sample representing a broad range of personality pathology, indicate that identity and primitive defenses as operationalized in the STIPO are internally consistent and that interrater reliability for all 3 content domains is adequate. Validity findings suggest that the assessment of one's sense of self and significant others (Identity) is predictive of measures of positive and negative affect, whereas the maladaptive ways in which the subject uses his or her objects for purposes of regulating one's self experience (Primitive Defenses) is predictive of measures of aggression and personality disorder traits associated with cluster B personality disorders. We discuss implications of these findings in terms of the theory-driven and trait-based assessment of personality pathology. PMID:20013454

  6. Functionalized graphene oxide for clinical glucose biosensing in urine and serum samples.

    Science.gov (United States)

    Veerapandian, Murugan; Seo, Yeong-Tai; Shin, Hyunkyung; Yun, Kyusik; Lee, Min-Ho

    2012-01-01

    A novel clinical glucose biosensor fabricated using functionalized metalloid-polymer (silver-silica coated with polyethylene glycol) hybrid nanoparticles on the surface of a graphene oxide nanosheet is reported. The cyclic voltammetric response of glucose oxidase modification on the surface of a functionalized graphene oxide electrode showed a surface-confined reaction and an effective redox potential near zero volts, with a wide linearity of 0.1-20 mM and a sensitivity of 7.66 μA mM(-1) cm(-2). The functionalized graphene oxide electrode showed a better electrocatalytic response toward oxidation of H(2)O(2) and reduction of oxygen. The practical applicability of the functionalized graphene oxide electrode was demonstrated by measuring the peak current against multiple urine and serum samples from diabetic patients. This new hybrid nanoarchitecture combining a three-dimensional metalloid-polymer hybrid and two-dimensional graphene oxide provided a thin solid laminate on the electrode surface. The easy fabrication process and retention of bioactive immobilized enzymes on the functionalized graphene oxide electrode could potentially be extended to detection of other biomolecules, and have broad applications in electrochemical biosensing. PMID:23269871

  7. Turkish adaptation of the Fear of Spiders Questionnaire: Reliability and validity in non-clinical samples

    Directory of Open Access Journals (Sweden)

    Robert W. Booth

    2016-12-01

    Full Text Available The rapid, objective measurement of spider fear is important for clinicians, and for researchers studying fear. To facilitate this, we adapted the Fear of Spiders Questionnaire (FSQ into Turkish. The FSQ is quick to complete and easy to understand. Compared to the commonly used Spider Phobia Questionnaire, it has shown superior test-retest reliability and better discrimination of lower levels of spider fear, facilitating fear research in non-clinical samples. In two studies, with 137 and 105 undergraduates and unselected volunteers, our adapted FSQ showed excellent internal consistency (Cronbach’s α = .95 and .96 and test-retest reliability (r = .90, and good discriminant validity against the State–Trait Anxiety Inventory—Trait (r = .23 and Beck Anxiety Inventory—Trait (r = .07. Most importantly, our adapted FSQ significantly predicted 26 students’ self-reported discomfort upon approaching a caged tarantula; however, a measure of behavioural avoidance of the tarantula yielded little variability, so a more sensitive task will be required for future behavioural testing. Based on this initial testing, we recommend our adapted FSQ for research use. Further research is required to verify that our adapted FSQ discriminates individuals with and without phobia effectively. A Turkish-language report of the studies is included as supplementary material.

  8. Emotional processing in a non-clinical psychosis-prone sample.

    Science.gov (United States)

    van 't Wout, Mascha; Aleman, André; Kessels, Roy P C; Larøi, Frank; Kahn, René S

    2004-06-01

    Symptoms of psychosis have been proposed to form part of a continuous distribution of experiences in the general population rather than being an all-or-nothing phenomenon. Indeed, schizotypal signs have been reported in subjects from non-clinical samples. Emotional processing has been documented to be deficient in schizophrenia. In the present study, we tested the hypothesis whether putatively psychosis-prone subjects would show abnormalities in emotion processing. Based on the extremes of Launay-Slade Hallucination Scale (LSHS) ratings of 200 undergraduate students, two groups of subjects (total N=40) were selected. All 40 participants filled in the Schizotypal Personality Questionnaire (SPQ). We compared both groups on an alexithymia questionnaire and on four behavioral emotional information processing tasks. Hallucination-proneness was associated with an increased subjective emotional arousal and fantasy-proneness. Although no differences between the high and low group were observed on three behavioral emotion processing tasks, on the affective word-priming task presentation of emotional stimuli was associated with longer reactions times to neutral words in high schizotypal subjects. Also, SPQ scores correlated with several emotion processing tasks. We conclude that these findings lend partial support to the hypothesis of continuity between symptoms characteristic of schizophrenia and psychosis-related phenomena in the normal population. PMID:15099609

  9. Metagenomics as a Tool for Enzyme Discovery: Hydrolytic Enzymes from Marine-Related Metagenomes.

    Science.gov (United States)

    Popovic, Ana; Tchigvintsev, Anatoly; Tran, Hai; Chernikova, Tatyana N; Golyshina, Olga V; Yakimov, Michail M; Golyshin, Peter N; Yakunin, Alexander F

    2015-01-01

    This chapter discusses metagenomics and its application for enzyme discovery, with a focus on hydrolytic enzymes from marine metagenomic libraries. With less than one percent of culturable microorganisms in the environment, metagenomics, or the collective study of community genetics, has opened up a rich pool of uncharacterized metabolic pathways, enzymes, and adaptations. This great untapped pool of genes provides the particularly exciting potential to mine for new biochemical activities or novel enzymes with activities tailored to peculiar sets of environmental conditions. Metagenomes also represent a huge reservoir of novel enzymes for applications in biocatalysis, biofuels, and bioremediation. Here we present the results of enzyme discovery for four enzyme activities, of particular industrial or environmental interest, including esterase/lipase, glycosyl hydrolase, protease and dehalogenase.

  10. Binning sequences using very sparse labels within a metagenome

    Directory of Open Access Journals (Sweden)

    Halgamuge Saman K

    2008-04-01

    Full Text Available Abstract Background In metagenomic studies, a process called binning is necessary to assign contigs that belong to multiple species to their respective phylogenetic groups. Most of the current methods of binning, such as BLAST, k-mer and PhyloPythia, involve assigning sequence fragments by comparing sequence similarity or sequence composition with already-sequenced genomes that are still far from comprehensive. We propose a semi-supervised seeding method for binning that does not depend on knowledge of completed genomes. Instead, it extracts the flanking sequences of highly conserved 16S rRNA from the metagenome and uses them as seeds (labels to assign other reads based on their compositional similarity. Results The proposed seeding method is implemented on an unsupervised Growing Self-Organising Map (GSOM, and called Seeded GSOM (S-GSOM. We compared it with four well-known semi-supervised learning methods in a preliminary test, separating random-length prokaryotic sequence fragments sampled from the NCBI genome database. We identified the flanking sequences of the highly conserved 16S rRNA as suitable seeds that could be used to group the sequence fragments according to their species. S-GSOM showed superior performance compared to the semi-supervised methods tested. Additionally, S-GSOM may also be used to visually identify some species that do not have seeds. The proposed method was then applied to simulated metagenomic datasets using two different confidence threshold settings and compared with PhyloPythia, k-mer and BLAST. At the reference taxonomic level Order, S-GSOM outperformed all k-mer and BLAST results and showed comparable results with PhyloPythia for each of the corresponding confidence settings, where S-GSOM performed better than PhyloPythia in the ≥ 10 reads datasets and comparable in the ≥ 8 kb benchmark tests. Conclusion In the task of binning using semi-supervised learning methods, results indicate S-GSOM to be the best of

  11. Separating metagenomic short reads into genomes via clustering

    Directory of Open Access Journals (Sweden)

    Tanaseichuk Olga

    2012-09-01

    Full Text Available Abstract Background The metagenomics approach allows the simultaneous sequencing of all genomes in an environmental sample. This results in high complexity datasets, where in addition to repeats and sequencing errors, the number of genomes and their abundance ratios are unknown. Recently developed next-generation sequencing (NGS technologies significantly improve the sequencing efficiency and cost. On the other hand, they result in shorter reads, which makes the separation of reads from different species harder. Among the existing computational tools for metagenomic analysis, there are similarity-based methods that use reference databases to align reads and composition-based methods that use composition patterns (i.e., frequencies of short words or l-mers to cluster reads. Similarity-based methods are unable to classify reads from unknown species without close references (which constitute the majority of reads. Since composition patterns are preserved only in significantly large fragments, composition-based tools cannot be used for very short reads, which becomes a significant limitation with the development of NGS. A recently proposed algorithm, AbundanceBin, introduced another method that bins reads based on predicted abundances of the genomes sequenced. However, it does not separate reads from genomes of similar abundance levels. Results In this work, we present a two-phase heuristic algorithm for separating short paired-end reads from different genomes in a metagenomic dataset. We use the observation that most of the l-mers belong to unique genomes when l is sufficiently large. The first phase of the algorithm results in clusters of l-mers each of which belongs to one genome. During the second phase, clusters are merged based on l-mer repeat information. These final clusters are used to assign reads. The algorithm could handle very short reads and sequencing errors. It is initially designed for genomes with similar abundance levels and then

  12. A comparative analysis of the intestinal metagenomes present in guinea pigs (Cavia porcellus) and humans (Homo sapiens)

    DEFF Research Database (Denmark)

    Hildebrand, Falk; Ebersbach, Tine; Nielsen, Henrik Bjørn;

    2012-01-01

    Background: Guinea pig (Cavia porcellus) is an important model for human intestinal research. We have characterized the faecal microbiota of 60 guinea pigs using Illumina shotgun metagenomics, and used this data to compile a gene catalogue of its prevalent microbiota. Subsequently, we compared...... the guinea pig microbiome to existing human gut metagenome data from the MetaHIT project. Results: We found that the bacterial richness obtained for human samples was lower than for guinea pig samples. The intestinal microbiotas of both species were dominated by the two phyla Bacteroidetes and Firmicutes...

  13. A tale of two approaches: how metagenomics and proteomics are shaping the future of encephalitis diagnostics

    OpenAIRE

    Schubert, RD; Wilson

    2015-01-01

    © 2015 Wolters Kluwer Health, Inc. We highlight how metagenomics and proteomics-based approaches are being applied to the problem of diagnosis in idiopathic encephalitis. Recent findings Low cost, high-throughput next-generation sequencing platforms have enabled unbiased sequencing of biological samples. Rapid sequence-based computational algorithms then determine the source of all the nonhost (e.g., pathogen-derived) nucleic acids in a sample. This approach recently identified a case of neur...

  14. A new approach of food microbiology with the metagenomic tools: an application on fish

    OpenAIRE

    Delhalle, Laurent; Taminiau, Bernard; Nezer, Carine; Darcis, Amélie; Daube, Georges

    2012-01-01

    Metagenomics has appeared as a powerful tool to study bacterial composition of various environmental samples. Its interest has started to appear in food microbiology but only on the study of very particular bacterial populations of fermented food. This work describes the application of this technique to study the bacterial population of two fresh fish filets. The two fish species are from freshwater (pangasius) and seawater (haddock), respectively. Samples where directly analyzed the day of r...

  15. Metagenomic analysis of the turkey gut RNA virus community

    Directory of Open Access Journals (Sweden)

    Scheffler Brian E

    2010-11-01

    Full Text Available Abstract Viral enteric disease is an ongoing economic burden to poultry producers worldwide, and despite considerable research, no single virus has emerged as a likely causative agent and target for prevention and control efforts. Historically, electron microscopy has been used to identify suspect viruses, with many small, round viruses eluding classification based solely on morphology. National and regional surveys using molecular diagnostics have revealed that suspect viruses continuously circulate in United States poultry, with many viruses appearing concomitantly and in healthy birds. High-throughput nucleic acid pyrosequencing is a powerful diagnostic technology capable of determining the full genomic repertoire present in a complex environmental sample. We utilized the Roche/454 Life Sciences GS-FLX platform to compile an RNA virus metagenome from turkey flocks experiencing enteric disease. This approach yielded numerous sequences homologous to viruses in the BLAST nr protein database, many of which have not been described in turkeys. Our analysis of this turkey gut RNA metagenome focuses in particular on the turkey-origin members of the Picornavirales, the Caliciviridae, and the turkey Picobirnaviruses.

  16. Compressed sensing methods for DNA microarrays, RNA interference, and metagenomics.

    Science.gov (United States)

    Rao, Aditya; P, Deepthi; Renumadhavi, C H; Chandra, M Girish; Srinivasan, Rajgopal

    2015-02-01

    Compressed sensing (CS) is a sparse signal sampling methodology for efficiently acquiring and reconstructing a signal from relatively few measurements. Recent work shows that CS is well-suited to be applied to problems in genomics, including probe design in microarrays, RNA interference (RNAi), and taxonomic assignment in metagenomics. The principle of using different CS recovery methods in these applications has thus been established, but a comprehensive study of using a wide range of CS methods has not been done. For each of these applications, we apply three hitherto unused CS methods, namely, l1-magic, CoSaMP, and l1-homotopy, in conjunction with CS measurement matrices such as randomly generated CS m matrix, Hamming matrix, and projective geometry-based matrix. We find that, in RNAi, the l1-magic (the standard package for l1 minimization) and l1-homotopy methods show significant reduction in reconstruction error compared to the baseline. In metagenomics, we find that l1-homotopy as well as CoSaMP estimate concentration with significantly reduced time when compared to the GPSR and WGSQuikr methods.

  17. Applications of metagenomics for industrial bioproducts

    Science.gov (United States)

    Recent progress in mining the rich genetic resource of non-culturable microbes has led to the discovery of new genes, enzymes, and natural products. The impact of metagenomics is witnessed in the development of commodity and fine chemicals, agrochemicals and pharmaceuticals where the benefit of enz...

  18. Towards a more complete metagenomics toolkit

    Science.gov (United States)

    The emerging scientific discipline of metagenomics has not only created a myriad of opportunities for biologists to reveal new insights into the microbial underpinnings of our environment, but has also presented a number of interesting challenges for bioinformatics algorithms and software developers...

  19. Snowball: Strain aware gene assembly of Metagenomes

    NARCIS (Netherlands)

    Gregor, I.; Schoenhuth, A.; McHardy, A.C.

    2015-01-01

    Gene assembly is an important step in functional analysis of shotgun metagenomic data. Nonetheless, strain aware assembly remains a challenging task, as current assembly tools often fail to distinguish among strain variants or require closely related reference genomes of the studied species to be av

  20. Biomolecular and metagenomic analyses of biofouling communities

    Science.gov (United States)

    Despite the decades of research that have focused on understanding the formation of biofouling communities, relatively little is known about the soft fouling consortia that are responsible for their formation and function. In this study, we used PhyloChip microbial profiling, metagenomic DNA sequenc...

  1. Reference databases for taxonomic assignment in metagenomics.

    Science.gov (United States)

    Santamaria, Monica; Fosso, Bruno; Consiglio, Arianna; De Caro, Giorgio; Grillo, Giorgio; Licciulli, Flavio; Liuni, Sabino; Marzano, Marinella; Alonso-Alemany, Daniel; Valiente, Gabriel; Pesole, Graziano

    2012-11-01

    Metagenomics is providing an unprecedented access to the environmental microbial diversity. The amplicon-based metagenomics approach involves the PCR-targeted sequencing of a genetic locus fitting different features. Namely, it must be ubiquitous in the taxonomic range of interest, variable enough to discriminate between different species but flanked by highly conserved sequences, and of suitable size to be sequenced through next-generation platforms. The internal transcribed spacers 1 and 2 (ITS1 and ITS2) of the ribosomal DNA operon and one or more hyper-variable regions of 16S ribosomal RNA gene are typically used to identify fungal and bacterial species, respectively. In this context, reliable reference databases and taxonomies are crucial to assign amplicon sequence reads to the correct phylogenetic ranks. Several resources provide consistent phylogenetic classification of publicly available 16S ribosomal DNA sequences, whereas the state of ribosomal internal transcribed spacers reference databases is notably less advanced. In this review, we aim to give an overview of existing reference resources for both types of markers, highlighting strengths and possible shortcomings of their use for metagenomics purposes. Moreover, we present a new database, ITSoneDB, of well annotated and phylogenetically classified ITS1 sequences to be used as a reference collection in metagenomic studies of environmental fungal communities. ITSoneDB is available for download and browsing at http://itsonedb.ba.itb.cnr.it/.

  2. Functional metagenomics: recent advances and future challenges.

    Science.gov (United States)

    Chistoserdovai, Ludmila

    2010-01-01

    Metagenomics is a relatively new but fast growing field within environmental biology directed at obtaining knowledge on genomes of environmental microbes as well as of entire microbial communities. With the sequencing technologies improving steadily, generating large amounts of sequence is becoming routine. However, it remains difficult to connect specific microbial phyla to specific functions in the environment. A number of 'functional metagenomics' approaches have been implemented in the recent years that allow high-resolution genomic analysis of uncultivated microbes, connecting them to specific functions in the environment. These include analysis of niche-specialized low complexity communities, reactor enrichments, and the use labeling technologies. Metatranscriptomics and metaproteomics are the newest sub-disciplines within the metagenomics field that provide further levels of resolution for functional analysis of uncultivated microbes and communities. The recent emergence of new (next generation) sequencing technologies, resulting in higher sequence output and dramatic drop in the price of sequencing, will be defining a new era in metagenomics. At this time the sequencing effort will be taken to a new level to allow addressing new, previously unattainable biological questions as well as accelerating genome-based discovery for medical and biotechnological applications.

  3. A Multitrait–Multimethod Analysis of the Construct Validity of Child Anxiety Disorders in a Clinical Sample

    OpenAIRE

    Wood, Jeffrey J.; Bergman, R. Lindsey; Piacentini, John C.; Langer, David Adam

    2010-01-01

    The present study examines the construct validity of separation anxiety disorder (SAD), social phobia (SoP), panic disorder (PD), and generalized anxiety disorder (GAD) in a clinical sample of children. Participants were 174 children, 6 to 17 years old (94 boys) who had undergone a diagnostic evaluation at a university hospital based clinic. Parent and child ratings of symptom severity were assessed using the Multidimensional Anxiety Scale for Children (MASC). Diagnostician ratings were obtai...

  4. Detection of Shiga toxins genes by Multiplex PCR in clinical samples

    Directory of Open Access Journals (Sweden)

    2013-09-01

    Full Text Available Background: Different methods have been used for detection of shiga toxins; such as,  cell culture, ELISA, and RFPLA. However, all of these methods suffer from high cost, time-consumption and relatively low sensitivity. In this study we used Multiplex PCR method for detection of genes encoding shiga toxins. Material and Methods: In this study, 63 clinical samples were obtained from positive cultures of Shigella and E. coli O157, from Bahman 1391 until Ordibehesht 1392 in Mazandaran province. Initial confirmation of shiga toxins producing bacteria was performed by biochemical and serological methods. After DNA extraction, detection of stx1 and stx2 genes was accomplished by multiplex PCR.  For confirmation of the PCR amplicon, DNA sequencing was used. Antibiotic sensitivity tests were performed by disk diffusion method. Results:  Among the positive strains, 13 strains contained stx2 genes, 4 strains contained Stx/Stx1 genes and 4 strains harbored both Stx/Stx1 and Stx2. The DNA extracted from other Gram-negative bacteria was not protected by the relevant parts of these toxins. Sequencing of the amplified fragments indicated the correct toxin sequences.  The sensitivity for identification of Stx/Stx1 gene was 1.56 pg/ µl and for Stx2 was 1.08 pg/µl. The toxin positive strains were all sensitive to Cefixime, Gentamicin, Amikacin, Ceftriaxone, and Nitrofurantoin. Conclusion: This method is fast and accurate for detection of bacteria producing shiga toxin and can be used to identify different types of shiga toxin.

  5. Clinical validation of three short forms of the Dutch Wechsler Memory Scale – Fourth Edition (WMS-IV-NL) in a mixed clinical sample

    NARCIS (Netherlands)

    Bouman, Z.; Hendriks, M.P.H.; Veld, W.M. van der; Aldenkamp, A.P.; Kessels, R.P.C.

    2016-01-01

    The reliability and validity of three short forms of the Dutch version of the Wechsler Memory Scale–Fourth Edition (WMS-IV-NL) were evaluated in a mixed clinical sample of 235 patients. The short forms were based on the WMS-IV Flexible Approach, that is, a 3-subtest combination (Older Adult Battery

  6. The Structured Clinical Interview for DSM-IV Childhood Diagnoses (Kid-SCID): first psychometric evaluation in a Dutch sample of clinically referred youths

    NARCIS (Netherlands)

    J. Roelofs; P. Muris; C. Braet; A. Arntz; I. Beelen

    2014-01-01

    The Structured Clinical Interview for DSM-IV Childhood Disorders (Kid-SCID) is a semi-structured interview for the classification of psychiatric disorders in children and adolescents. This study presents a first evaluation of the psychometric properties of the Kid-SCID in a Dutch sample of children

  7. From cultured to uncultured genome sequences: metagenomics and modeling microbial ecosystems.

    Science.gov (United States)

    Garza, Daniel R; Dutilh, Bas E

    2015-11-01

    Microorganisms and the viruses that infect them are the most numerous biological entities on Earth and enclose its greatest biodiversity and genetic reservoir. With strength in their numbers, these microscopic organisms are major players in the cycles of energy and matter that sustain all life. Scientists have only scratched the surface of this vast microbial world through culture-dependent methods. Recent developments in generating metagenomes, large random samples of nucleic acid sequences isolated directly from the environment, are providing comprehensive portraits of the composition, structure, and functioning of microbial communities. Moreover, advances in metagenomic analysis have created the possibility of obtaining complete or nearly complete genome sequences from uncultured microorganisms, providing important means to study their biology, ecology, and evolution. Here we review some of the recent developments in the field of metagenomics, focusing on the discovery of genetic novelty and on methods for obtaining uncultured genome sequences, including through the recycling of previously published datasets. Moreover we discuss how metagenomics has become a core scientific tool to characterize eco-evolutionary patterns of microbial ecosystems, thus allowing us to simultaneously discover new microbes and study their natural communities. We conclude by discussing general guidelines and challenges for modeling the interactions between uncultured microorganisms and viruses based on the information contained in their genome sequences. These models will significantly advance our understanding of the functioning of microbial ecosystems and the roles of microbes in the environment.

  8. PCR-based detection of composite transposons and translocatable units from oral metagenomic DNA.

    Science.gov (United States)

    Tansirichaiya, Supathep; Mullany, Peter; Roberts, Adam P

    2016-09-01

    A composite transposon is a mobile genetic element consisting of two insertion sequences (ISs) flanking a segment of cargo DNA often containing antibiotic resistance (AR) genes. Composite transposons can move as a discreet unit. There have been recently several reports on a novel mechanism of movement of an IS26-based composite transposon through the formation of a translocatable unit (TU), carrying the internal DNA segment of a composite transposon and one copy of a flanking IS. In this study, we determined the presence of composite transposons and TUs in human oral metagenomic DNA using PCR primers from common IS elements. Analysis of resulting amplicons showed four different IS1216 composite transposons and one IS257 composite transposon in our metagenomic sample. As our PCR strategy would also detect TUs, PCR was carried out to detect circular TUs predicted to originate from these composite transposons. We confirmed the presence of two novel TUs, one containing an experimentally proven antiseptic resistance gene and another containing a putative universal stress response protein (UspA) encoding gene. This is the first report of a PCR strategy to amplify the DNA segment on composite transposons and TUs in metagenomic DNA. This can be used to identify AR genes associated with a variety of mobile genetic elements from metagenomes.

  9. WebCARMA: a web application for the functional and taxonomic classification of unassembled metagenomic reads

    Directory of Open Access Journals (Sweden)

    Jünemann Sebastian

    2009-12-01

    Full Text Available Abstract Background Metagenomics is a new field of research on natural microbial communities. High-throughput sequencing techniques like 454 or Solexa-Illumina promise new possibilities as they are able to produce huge amounts of data in much shorter time and with less efforts and costs than the traditional Sanger technique. But the data produced comes in even shorter reads (35-100 basepairs with Illumina, 100-500 basepairs with 454-sequencing. CARMA is a new software pipeline for the characterisation of species composition and the genetic potential of microbial samples using short, unassembled reads. Results In this paper, we introduce WebCARMA, a refined version of CARMA available as a web application for the taxonomic and functional classification of unassembled (ultra-short reads from metagenomic communities. In addition, we have analysed the applicability of ultra-short reads in metagenomics. Conclusions We show that unassembled reads as short as 35 bp can be used for the taxonomic classification of a metagenome. The web application is freely available at http://webcarma.cebitec.uni-bielefeld.de.

  10. Estimating DNA coverage and abundance in metagenomes using a gamma approximation

    Energy Technology Data Exchange (ETDEWEB)

    Hooper, Sean D; Dalevi, Daniel; Pati, Amrita; Mavromatis, Konstantinos; Ivanova, Natalia N; Kyrpides, Nikos C

    2010-01-01

    Shotgun sequencing generates large numbers of short DNA reads from either an isolated organism or, in the case of metagenomics projects, from the aggregate genome of a microbial community. These reads are then assembled based on overlapping sequences into larger, contiguous sequences (contigs). The feasibility of assembly and the coverage achieved (reads per nucleotide or distinct sequence of nucleotides) depend on several factors: the number of reads sequenced, the read length and the relative abundances of their source genomes in the microbial community. A low coverage suggests that most of the genomic DNA in the sample has not been sequenced, but it is often difficult to estimate either the extent of the uncaptured diversity or the amount of additional sequencing that would be most efficacious. In this work, we regard a metagenome as a population of DNA fragments (bins), each of which may be covered by one or more reads. We employ a gamma distribution to model this bin population due to its flexibility and ease of use. When a gamma approximation can be found that adequately fits the data, we may estimate the number of bins that were not sequenced and that could potentially be revealed by additional sequencing. We evaluated the performance of this model using simulated metagenomes and demonstrate its applicability on three recent metagenomic datasets.

  11. Construction and screening of a functional metagenomic library to identify novel enzymes produced by Antarctic bacteria

    Institute of Scientific and Technical Information of China (English)

    Ignacio Ferrés; Vanesa Amarelle; Francisco Noya; Elena Fabiano

    2015-01-01

    A metagenomic fosmid library of approximately 52 000 clones was constructed to identify functional genes encoding cold-adapted enzymes. Metagenomic DNA was extracted from a sample of glacial meltwater, collected on the Antarctic Peninsula during the ANTARKOS XXIX Expedition during the austral summer of 2012–2013. Each clone contained an insert of about 35–40 kb, so the library represented almost 2 Gb of genetic information from metagenomic DNA. Activity-driven screening was used to detect the cold-adapted functions expressed by the library. Fifty lipase/esterase and two cellulase-producing clones were isolated, and two clones able to grow on Avicel® as the sole carbon source. Interestingly, three clones formed a brown precipitate in the presence of manganese (II). Accumulation of manganese oxides was determined with a leucoberbelin blue assay, indicating that these three clones had manganese-oxidizing activity. To the best of our knowledge, this is the first report of a manganese oxidase activity detected with a functional metagenomic strategy.

  12. Global metagenomic survey reveals a new bacterial candidate phylum in geothermal springs.

    Science.gov (United States)

    Eloe-Fadrosh, Emiley A; Paez-Espino, David; Jarett, Jessica; Dunfield, Peter F; Hedlund, Brian P; Dekas, Anne E; Grasby, Stephen E; Brady, Allyson L; Dong, Hailiang; Briggs, Brandon R; Li, Wen-Jun; Goudeau, Danielle; Malmstrom, Rex; Pati, Amrita; Pett-Ridge, Jennifer; Rubin, Edward M; Woyke, Tanja; Kyrpides, Nikos C; Ivanova, Natalia N

    2016-01-27

    Analysis of the increasing wealth of metagenomic data collected from diverse environments can lead to the discovery of novel branches on the tree of life. Here we analyse 5.2 Tb of metagenomic data collected globally to discover a novel bacterial phylum ('Candidatus Kryptonia') found exclusively in high-temperature pH-neutral geothermal springs. This lineage had remained hidden as a taxonomic 'blind spot' because of mismatches in the primers commonly used for ribosomal gene surveys. Genome reconstruction from metagenomic data combined with single-cell genomics results in several high-quality genomes representing four genera from the new phylum. Metabolic reconstruction indicates a heterotrophic lifestyle with conspicuous nutritional deficiencies, suggesting the need for metabolic complementarity with other microbes. Co-occurrence patterns identifies a number of putative partners, including an uncultured Armatimonadetes lineage. The discovery of Kryptonia within previously studied geothermal springs underscores the importance of globally sampled metagenomic data in detection of microbial novelty, and highlights the extraordinary diversity of microbial life still awaiting discovery.

  13. Multidimensional metrics for estimating phage abundance, distribution, gene density, and sequence coverage in metagenomes

    Directory of Open Access Journals (Sweden)

    Ramy Karam Aziz

    2015-05-01

    Full Text Available Phages are the most abundant biological entities on Earth and play major ecological roles, yet the current sequenced phage genomes do not adequately represent their diversity, and little is known about the abundance and distribution of these sequenced genomes in nature. Although the study of phage ecology has benefited tremendously from the emergence of metagenomic sequencing, a systematic survey of phage genes and genomes in various ecosystems is still lacking, and fundamental questions about phage biology, lifestyle, and ecology remain unanswered. To address these questions and improve comparative analysis of phages in different metagenomes, we screened a core set of publicly available metagenomic samples for sequences related to completely sequenced phages using the web tool, Phage Eco-Locator. We then adopted and deployed an array of mathematical and statistical metrics for a multidimensional estimation of the abundance and distribution of phage genes and genomes in various ecosystems. Experiments using those metrics individually showed their usefulness in emphasizing the pervasive, yet uneven, distribution of known phage sequences in environmental metagenomes. Using these metrics in combination allowed us to resolve phage genomes into clusters that correlated with their genotypes and taxonomic classes as well as their ecological properties. We propose adding this set of metrics to current metaviromic analysis pipelines, where they can provide insight regarding phage mosaicism, habitat specificity, and evolution.

  14. More than a (negative) feeling: Validity of the perceived stress scale in Serbian clinical and non-clinical samples

    OpenAIRE

    Jovanović Veljko; Gavrilov-Jerković Vesna

    2015-01-01

    The goal of the present study was to test the validity of a Serbian version of the Perceived Stress Scale. The PSS was administered to 157 psychiatric outpatients, 165 adults from the non-clinical population, and 283 university students. The results of the confirmatory factor analysis supported a bifactor model of the PSS with one general factor and two specific factors reflecting perceived distress and perceived self-efficacy. Internal consistencies of the...

  15. Two Methods for High-Throughput NGS Template Preparation for Small and Degraded Clinical Samples Without Automation

    OpenAIRE

    Kamberov, E.; Tesmer, T.; Mastronardi, M.; Langmore, John

    2012-01-01

    Clinical samples are difficult to prepare for NGS, because of the small amounts or degraded states of formalin-fixed tissue, plasma, urine, and single-cell DNA. Conventional whole genome amplification methods are too biased for NGS applications, and the existing NGS preparation kits require intermediate purifications and excessive time to prepare hundreds of samples in a day without expensive automation. We have tested two 96-well manual methods to make NGS templates from FFPE tissue, plasma,...

  16. Detection and genetic characterization of foot‐and‐mouth disease viruses in samples from clinically healthy animals in endemic settings

    DEFF Research Database (Denmark)

    Jamal, Syed Muhammad; Ferrari, G.; Hussain, M.;

    2012-01-01

    in Pakistan (n = 245), one (of three) live animal market in Afghanistan (n = 61) and both the live animal markets in Tajikistan (n = 120) all tested negative. However, 2 of 129 (∼2%) samples from Gondal and 11 of 123 (9%) from Chichawatni markets in Pakistan were positive for FMDV RNA. Similarly, 12 of 81 (15......A total of 1501 oral swab samples from Pakistan, Afghanistan and Tajikistan were collected from clinically healthy animals between July 2008 and August 2009 and assayed for the presence of foot‐and‐mouth disease virus (FMDV) RNA. The oral swab samples from two (of four) live animal markets......%) samples from Kabul and 10 of 20 (50%) from Badakhshan in Afghanistan were found to be positive. Serotypes A and O of FMDV were identified within these samples. Oral swab samples were also collected from dairy colonies in Harbanspura, Lahore (n = 232) and Nagori, Karachi (n = 136), but all tested negative...

  17. Fatal Psychrobacter sp. infection in a pediatric patient with meningitis identified by metagenomic next-generation sequencing in cerebrospinal fluid.

    Science.gov (United States)

    Ortiz-Alcántara, Joanna María; Segura-Candelas, José Miguel; Garcés-Ayala, Fabiola; Gonzalez-Durán, Elizabeth; Rodríguez-Castillo, Araceli; Alcántara-Pérez, Patricia; Wong-Arámbula, Claudia; González-Villa, Maribel; León-Ávila, Gloria; García-Chéquer, Adda Jeanette; Diaz-Quiñonez, José Alberto; Méndez-Tenorio, Alfonso; Ramírez-González, José Ernesto

    2016-03-01

    The genus Psychrobacter contains environmental, psychrophilic and halotolerant gram-negative bacteria considered rare opportunistic pathogens in humans. Metagenomics was performed on the cerebrospinal fluid (CSF) of a pediatric patient with meningitis. Nucleic acids were extracted, randomly amplified, and sequenced with the 454 GS FLX Titanium next-generation sequencing (NGS) system. Sequencing reads were assembled, and potential virulence genes were predicted. Phylogenomic and phylogenetic studies were performed. Psychrobacter sp. 310 was identified, and several virulence genes characteristic of pathogenic bacteria were found. The phylogenomic study and 16S rRNA gene phylogenetic analysis showed that the closest relative of Psychrobacter sp. 310 was Psychrobacter sanguinis. To our knowledge, this is the first report of a meningitis case associated with Psychrobacter sp. identified by NGS metagenomics in CSF from a pediatric patient. The metagenomic strategy based on NGS was a powerful tool to identify a rare unknown pathogen in a clinical case.

  18. Parental Behavior and Adolescent Self-Esteem in Clinical and Nonclinical Samples.

    Science.gov (United States)

    Nielson, David M.; Metha, Arlene

    1994-01-01

    Investigated relationships between self-esteem and adolescents' perceptions of parental behaviors using nonclinical (n=119) and clinical (n=30) adolescents. Nonclinical adolescents scored higher than clinical adolescents on all self-esteem dimensions. Males scored higher than females only on dimension of Self-Esteem Competence. Perceptions of…

  19. Metagenomic Analysis of Koumiss in Kazakhstan

    Directory of Open Access Journals (Sweden)

    Samat Kozhakhmetov

    2014-12-01

    Full Text Available Introduction. Koumiss is a low-alcohol product made from fermented mare's milk, which is popular in Kazakhstan, Russia, and other countries of Central Asia, China, and Mongolia. Natural mare's milk is fermented in symbiosis of two types of microorganisms (lactobacteria and yeast. Koumiss’s microbial composition varies depending on the geographical, climatic, and cultural conditions. Based on a phenotypic characteristic from samples, Wu, R. and colleagues identified the following bacteria isolated in inner Mongolia, an autonomous region of China: L.casei, L.helveticus, L.plantarum, L.coryniformis subsp. coryniformis, L.paracasei, L.kefiranofaciens, L.curvatus, L.fermentum, and W.kandleri. Studies of the yeast composition in koumiss also showed significant variations. Thus, there were Saccharomyces unisporus related 48.3% of isolates, to Kluyveromyces marxianus (27.6%, Pichia membranaefaciens (15.0%, and Saccharomyces cerevisiae (9.2% from 87 isolated yeast cultures. The purpose of this study was to examine the bacterial composition in koumiss.Methods. To extract DNA, 1.8 ml of fermented milk was centrifuged to generate a pellet, which was suspended in 450 µl of lysis buffer P1 from the Powerfood Microbial DNA Isolation kit (MoBio Laboratories Inc, USA. Amplification of the microflora was used to determine the composition of a fragment of the gene 16S rRNA and ITS1. Plasmid library with target insertion was obtained on the basis of height copy plasmid vectors producing high pGem-T. The definition of direct nucleotide sequencing was performed by the method of Sanger using a set of "BigDye Terminanor v 3.1 Cycle sequencing Kit with automatic genetic analyzer ABI 3730xl  (Applied Biosystems, USA.  Informax Vector NTI Suite 9, Sequence Scanner v 1.0  software package used for the analysis.Results. Our studies showed that in the most samples of koumiss isolated from Akmola region (Central Kazakhstan prevailed the following bacteria species

  20. Characterization of a clinical unit for digital radiography based on irradiation side sampling technology

    Energy Technology Data Exchange (ETDEWEB)

    Rivetti, Stefano [Fisica Medica, Ospedale di Sassuolo S.p.A., 41049 Sassuolo (Italy); Lanconelli, Nico [Alma Mater Studiorum, Physics Department, University of Bologna, 40127 Bologna (Italy); Bertolini, Marco; Nitrosi, Andrea [Medical Physics Unit, Azienda Ospedaliera ASMN, Istituto di Ricovero e Cura a Carattere Scientifico, 42123 Reggio Emilia (Italy); Burani, Aldo [Ospedale di Sassuolo S.p.A., 41049 Sassuolo (Italy)

    2013-10-15

    Purpose: A characterization of a clinical unit for digital radiography (FUJIFILM FDR D-EVO) is presented. This system is based on the irradiation side sampling (ISS) technology and can be equipped with two different scintillators: one traditional gadolinium-oxysulphide phosphor (GOS) and a needle structured cesium iodide (CsI) phosphor panel.Methods: The characterization was achieved in terms of response curve, modulation transfer function (MTF), noise power spectra (NPS), detective quantum efficiency (DQE), and psychophysical parameters (contrast-detail analysis with an automatic reading of CDRAD images). For both scintillation screens the authors accomplished the measurements with four standard beam conditions: RAQ3, RQA5, RQA7, and RQA9.Results: At the Nyquist frequency (3.33 lp/mm) the MTF is about 35% and 25% for CsI and GOS detectors, respectively. The CsI scintillator has better noise properties than the GOS screen in almost all the conditions. This is particularly true for low-energy beams, where the noise for the GOS system can go up to a factor 2 greater than that found for CsI. The DQE of the CsI detector reaches a peak of 60%, 60%, 58%, and 50% for the RQA3, RQA5, RQA7, and RQA9 beams, respectively, whereas for the GOS screen the maximum DQE is 40%, 44%, 44%, and 35%. The contrast-detail analysis confirms that in the majority of cases the CsI scintillator is able to provide improved outcomes to those obtained with the GOS screen.Conclusions: The limited diffusion of light produced by the ISS reading makes possible the achievement of very good spatial resolution. In fact, the MTF of the unit with the CsI panel is only slightly lower to that achieved with direct conversion detectors. The combination of very good spatial resolution, together with the good noise properties reached with the CsI screen, allows achieving DQE on average about 1.5 times greater than that obtained with GOS. In fact, the DQE of unit equipped with CsI is comparable to the best

  1. Occurrence of ADHD in parents of ADHD children in a clinical sample

    Directory of Open Access Journals (Sweden)

    Starck M

    2016-03-01

    Full Text Available Martina Starck,1 Julia Grünwald,1 Angelika A Schlarb1,21Faculty of Science, Department of Psychology, University of Tuebingen, Tuebingen, 2Department of Psychology, Faculty for Psychology and Sport Science, University of Bielefeld, Bielefeld, GermanyBackground: Despite the fact that there is a large amount of research on childhood attention deficit hyperactivity disorder (ADHD treatment and an increasing amount of research on adult ADHD, little is known about the prevalence and influence of parental ADHD. Therefore, this study examined the frequency of parental ADHD in a clinical sample of German children suffering from ADHD. We also tried to find different levels of symptom severity for prognostic relevance. Furthermore, the association between subtypes of ADHD in children and their parents was investigated.Method: In this study, parents of 79 ADHD children were screened for ADHD according to the Diagnostic and Statistical Manual of Mental Disorders, 5th edition and International Classification of Diseases, 10th edition. The Wender Utah Rating Scale and the ADHS-Self-Report were given to 75 mothers and 49 fathers for retrospective and current symptoms. Frequency of ADHD symptoms and severity groups was calculated and relationship between parental and children’s ADHD was tested.Results: ADHD occurrence for mothers of children with ADHD was 41.3%, for fathers 51.0%. About 16.0% of the mothers had a mixed type, 9.3% had a hyperactive-impulsive subtype, and 16.0% had an inattentive subtype. Of the fathers, 18.4% had a mixed type, 10.2% had a hyperactive-impulsive subtype, and 22.4% had an inattentive subtype; 61% of the mothers and 46.9% of the fathers had low symptom severity. Medium symptom severity was reported by 37.7% mothers and 46.9% fathers, while 1.3% of the mothers and 6.2% of the fathers showed severe symptoms. No significant correlation between parental and child diagnoses was observed.Conclusion: As nearly half of the parents

  2. Comparison of Two Assays for Molecular Determination of Rifampin Resistance in Clinical Samples from Patients with Buruli Ulcer Disease

    OpenAIRE

    Jansson, M.; Beissner, M.; Phillips, R. O.; Badziklou, K.; Piten, E.; Maman, I.; Sarfo, F S; Huber, K. L.; Rhomberg, A.; Symank, D.; Wagner, M.; Wiedemann, F; Nitschke, J.; Banla Kere, A.; Herbinger, K.-H.

    2014-01-01

    This study evaluates a novel assay for detecting rifampin resistance in clinical Mycobacterium ulcerans isolates. Although highly susceptible for PCR inhibitors in 50% of the samples tested, the assay was 100% M. ulcerans specific and yielded >98% analyzable sequences with a lower limit of detection of 100 to 200 copies of the target sequence.

  3. Childhood Trauma in Substance Use Disorder and Depression: An Analysis by Gender among a Brazilian Clinical Sample

    Science.gov (United States)

    Tucci, Adriana M.; Kerr-Correa, Florence; Souza-Formigoni, Maria Lucia O.

    2010-01-01

    Objective: In this study, we compared the frequency and intensity of childhood traumas in alcohol- or other drug-dependent patients, in patients with depression, and in a control group without psychiatric diagnoses. Methods: The study had a retrospective design of a clinical sample of men and women from the groups listed above. They were evaluated…

  4. Bottom–up protein identifications from microliter quantities of individual human tear samples. Important steps towards clinical relevance.

    Directory of Open Access Journals (Sweden)

    Peter Raus

    2015-12-01

    With 375 confidently identified proteins in the healthy adult tear, the obtained results are comprehensive and in large agreement with previously published observations on pooled samples of multiple patients. We conclude that, to a limited extent, bottom–up tear protein identifications from individual patients may have clinical relevance.

  5. A Comparison of Three Self-Report Measures of the Broader Autism Phenotype in a Non-Clinical Sample

    Science.gov (United States)

    Ingersoll, Brooke; Hopwood, Christopher J.; Wainer, Allison; Donnellan, M. Brent

    2011-01-01

    Three self-report measures of the broader autism phenotype (BAP) were evaluated in terms of their internal consistency, distribution of scores, factor structure, and criterion-related validity in a non-clinical sample. All measures showed a continuous distribution. The SRS-A and BAPQ showed expected sex differences and were superior to the AQ in…

  6. MetaBoot: a machine learning framework of taxonomical biomarker discovery for different microbial communities based on metagenomic data.

    Science.gov (United States)

    Wang, Xiaojun; Su, Xiaoquan; Cui, Xinping; Ning, Kang

    2015-01-01

    As more than 90% of species in a microbial community could not be isolated and cultivated, the metagenomic methods have become one of the most important methods to analyze microbial community as a whole. With the fast accumulation of metagenomic samples and the advance of next-generation sequencing techniques, it is now possible to qualitatively and quantitatively assess all taxa (features) in a microbial community. A set of taxa with presence/absence or their different abundances could potentially be used as taxonomical biomarkers for identification of the corresponding microbial community's phenotype. Though there exist some bioinformatics methods for metagenomic biomarker discovery, current methods are not robust, accurate and fast enough at selection of non-redundant biomarkers for prediction of microbial community's phenotype. In this study, we have proposed a novel method, MetaBoot, that combines the techniques of mRMR (minimal redundancy maximal relevance) and bootstrapping, for discover of non-redundant biomarkers for microbial communities through mining of metagenomic data. MetaBoot has been tested and compared with other methods on well-designed simulated datasets considering normal and gamma distribution as well as publicly available metagenomic datasets. Results have shown that MetaBoot was robust across datasets of varied complexity and taxonomical distribution patterns and could also select discriminative biomarkers with quite high accuracy and biological consistency. Thus, MetaBoot is suitable for robustly and accurately discover taxonomical biomarkers for different microbial communities. PMID:26213658

  7. A metagenomic study of primate insect diet diversity.

    Science.gov (United States)

    Pickett, Sarah B; Bergey, Christina M; Di Fiore, Anthony

    2012-07-01

    Descriptions of primate diets are generally based on either direct observation of foraging behavior, morphological classification of food remains from feces, or analysis of the stomach contents of deceased individuals. Some diet items (e.g. insect prey), however, are difficult to identify visually, and observation conditions often do not permit adequate quantitative sampling of feeding behavior. Moreover, the taxonomically informative morphology of some food species (e.g. swallowed seeds, insect exoskeletons) may be destroyed by the digestive process. Because of these limitations, we used a metagenomic approach to conduct a preliminary, "proof of concept" study of interspecific variation in the insect component of the diets of six sympatric New World monkeys known, based on observational field studies, to differ markedly in their feeding ecology. We used generalized arthropod polymerase chain reaction (PCR) primers and cloning to sequence mitochondrial DNA (mtDNA) sequences of the arthropod cytochrome b (CYT B) gene from fecal samples of wild woolly, titi, saki, capuchin, squirrel, and spider monkeys collected from a single sampling site in western Amazonia where these genera occur sympatrically. We then assigned preliminary taxonomic identifications to the sequences by basic local alignment search tool (BLAST) comparison to arthropod CYT B sequences present in GenBank. This study is the first to use molecular techniques to identify insect prey in primate diets. The results suggest that a metagenomic approach may prove valuable in augmenting and corroborating observational data and increasing the resolution of primate diet studies, although the lack of comparative reference sequences for many South American insects limits the approach at present. As such reference data become available for more animal and plant taxa, this approach also holds promise for studying additional components of primate diets. PMID:22553123

  8. Ecogenomic perspectives on domains of unknown function: correlation-based exploration of marine metagenomes.

    Directory of Open Access Journals (Sweden)

    Pier Luigi Buttigieg

    Full Text Available BACKGROUND: The proportion of conserved DNA sequences with no clear function is steadily growing in bioinformatics databases. Studies of sequence and structural homology have indicated that many uncharacterized protein domain sequences are variants of functionally described domains. If these variants promote an organism's ecological fitness, they are likely to be conserved in the genome of its progeny and the population at large. The genetic composition of microbial communities in their native ecosystems is accessible through metagenomics. We hypothesize the co-variation of protein domain sequences across metagenomes from similar ecosystems will provide insights into their potential roles and aid further investigation. METHODOLOGY/PRINCIPAL FINDINGS: We calculated the correlation of Pfam protein domain sequences across the Global Ocean Sampling metagenome collection, employing conservative detection and correlation thresholds to limit results to well-supported hits and associations. We then examined intercorrelations between domains of unknown function (DUFs and domains involved in known metabolic pathways using network visualization and cluster-detection tools. We used a cautious "guilty-by-association" approach, referencing knowledge-level resources to identify and discuss associations that offer insight into DUF function. We observed numerous DUFs associated to photobiologically active domains and prevalent in the Cyanobacteria. Other clusters included DUFs associated with DNA maintenance and repair, inorganic nutrient metabolism, and sodium-translocating transport domains. We also observed a number of clusters reflecting known metabolic associations and cases that predicted functional reclassification of DUFs. CONCLUSION/SIGNIFICANCE: Critically examining domain covariation across metagenomic datasets can grant new perspectives on the roles and associations of DUFs in an ecological setting. Targeted attempts at DUF characterization in the

  9. Biotechnological applications of functional metagenomics in the food and pharmaceutical industries.

    Science.gov (United States)

    Coughlan, Laura M; Cotter, Paul D; Hill, Colin; Alvarez-Ordóñez, Avelino

    2015-01-01

    Microorganisms are found throughout nature, thriving in a vast range of environmental conditions. The majority of them are unculturable or difficult to culture by traditional methods. Metagenomics enables the study of all microorganisms, regardless of whether they can be cultured or not, through the analysis of genomic data obtained directly from an environmental sample, providing knowledge of the species present, and allowing the extraction of information regarding the functionality of microbial communities in their natural habitat. Function-based screenings, following the cloning and expression of metagenomic DNA in a heterologous host, can be applied to the discovery of novel proteins of industrial interest encoded by the genes of previously inaccessible microorganisms. Functional metagenomics has considerable potential in the food and pharmaceutical industries, where it can, for instance, aid (i) the identification of enzymes with desirable technological properties, capable of catalyzing novel reactions or replacing existing chemically synthesized catalysts which may be difficult or expensive to produce, and able to work under a wide range of environmental conditions encountered in food and pharmaceutical processing cycles including extreme conditions of temperature, pH, osmolarity, etc; (ii) the discovery of novel bioactives including antimicrobials active against microorganisms of concern both in food and medical settings; (iii) the investigation of industrial and societal issues such as antibiotic resistance development. This review article summarizes the state-of-the-art functional metagenomic methods available and discusses the potential of functional metagenomic approaches to mine as yet unexplored environments to discover novel genes with biotechnological application in the food and pharmaceutical industries. PMID:26175729

  10. Strain- and plasmid-level deconvolution of a synthetic metagenome by sequencing proximity ligation products.

    Science.gov (United States)

    Beitel, Christopher W; Froenicke, Lutz; Lang, Jenna M; Korf, Ian F; Michelmore, Richard W; Eisen, Jonathan A; Darling, Aaron E

    2014-01-01

    Metagenomics is a valuable tool for the study of microbial communities but has been limited by the difficulty of "binning" the resulting sequences into groups corresponding to the individual species and strains that constitute the community. Moreover, there are presently no methods to track the flow of mobile DNA elements such as plasmids through communities or to determine which of these are co-localized within the same cell. We address these limitations by applying Hi-C, a technology originally designed for the study of three-dimensional genome structure in eukaryotes, to measure the cellular co-localization of DNA sequences. We leveraged Hi-C data generated from a simple synthetic metagenome sample to accurately cluster metagenome assembly contigs into groups that contain nearly complete genomes of each species. The Hi-C data also reliably associated plasmids with the chromosomes of their host and with each other. We further demonstrated that Hi-C data provides a long-range signal of strain-specific genotypes, indicating such data may be useful for high-resolution genotyping of microbial populations. Our work demonstrates that Hi-C sequencing data provide valuable information for metagenome analyses that are not currently obtainable by other methods. This metagenomic Hi-C method could facilitate future studies of the fine-scale population structure of microbes, as well as studies of how antibiotic resistance plasmids (or other genetic elements) mobilize in microbial communities. The method is not limited to microbiology; the genetic architecture of other heterogeneous populations of cells could also be studied with this technique. PMID:24918035

  11. Strain- and plasmid-level deconvolution of a synthetic metagenome by sequencing proximity ligation products

    Directory of Open Access Journals (Sweden)

    Christopher W. Beitel

    2014-05-01

    Full Text Available Metagenomics is a valuable tool for the study of microbial communities but has been limited by the difficulty of “binning” the resulting sequences into groups corresponding to the individual species and strains that constitute the community. Moreover, there are presently no methods to track the flow of mobile DNA elements such as plasmids through communities or to determine which of these are co-localized within the same cell. We address these limitations by applying Hi-C, a technology originally designed for the study of three-dimensional genome structure in eukaryotes, to measure the cellular co-localization of DNA sequences. We leveraged Hi-C data generated from a simple synthetic metagenome sample to accurately cluster metagenome assembly contigs into groups that contain nearly complete genomes of each species. The Hi-C data also reliably associated plasmids with the chromosomes of their host and with each other. We further demonstrated that Hi-C data provides a long-range signal of strain-specific genotypes, indicating such data may be useful for high-resolution genotyping of microbial populations. Our work demonstrates that Hi-C sequencing data provide valuable information for metagenome analyses that are not currently obtainable by other methods. This metagenomic Hi-C method could facilitate future studies of the fine-scale population structure of microbes, as well as studies of how antibiotic resistance plasmids (or other genetic elements mobilize in microbial communities. The method is not limited to microbiology; the genetic architecture of other heterogeneous populations of cells could also be studied with this technique.

  12. A case study for large-scale human microbiome analysis using JCVI's metagenomics reports (METAREP).

    Science.gov (United States)

    Goll, Johannes; Thiagarajan, Mathangi; Abubucker, Sahar; Huttenhower, Curtis; Yooseph, Shibu; Methé, Barbara A

    2012-01-01

    As metagenomic studies continue to increase in their number, sequence volume and complexity, the scalability of biological analysis frameworks has become a rate-limiting factor to meaningful data interpretation. To address this issue, we have developed JCVI Metagenomics Reports (METAREP) as an open source tool to query, browse, and compare extremely large volumes of metagenomic annotations. Here we present improvements to this software including the implementation of a dynamic weighting of taxonomic and functional annotation, support for distributed searches, advanced clustering routines, and integration of additional annotation input formats. The utility of these improvements to data interpretation are demonstrated through the application of multiple comparative analysis strategies to shotgun metagenomic data produced by the National Institutes of Health Roadmap for Biomedical Research Human Microbiome Project (HMP) (http://nihroadmap.nih.gov). Specifically, the scalability of the dynamic weighting feature is evaluated and established by its application to the analysis of over 400 million weighted gene annotations derived from 14 billion short reads as predicted by the HMP Unified Metabolic Analysis Network (HUMAnN) pipeline. Further, the capacity of METAREP to facilitate the identification and simultaneous comparison of taxonomic and functional annotations including biological pathway and individual enzyme abundances from hundreds of community samples is demonstrated by providing scenarios that describe how these data can be mined to answer biological questions related to the human microbiome. These strategies provide users with a reference of how to conduct similar large-scale metagenomic analyses using METAREP with their own sequence data, while in this study they reveal insights into the nature and extent of variation in taxonomic and functional profiles across body habitats and individuals. Over one thousand HMP WGS datasets and the latest open source code

  13. A case study for large-scale human microbiome analysis using JCVI's metagenomics reports (METAREP.

    Directory of Open Access Journals (Sweden)

    Johannes Goll

    Full Text Available As metagenomic studies continue to increase in their number, sequence volume and complexity, the scalability of biological analysis frameworks has become a rate-limiting factor to meaningful data interpretation. To address this issue, we have developed JCVI Metagenomics Reports (METAREP as an open source tool to query, browse, and compare extremely large volumes of metagenomic annotations. Here we present improvements to this software including the implementation of a dynamic weighting of taxonomic and functional annotation, support for distributed searches, advanced clustering routines, and integration of additional annotation input formats. The utility of these improvements to data interpretation are demonstrated through the application of multiple comparative analysis strategies to shotgun metagenomic data produced by the National Institutes of Health Roadmap for Biomedical Research Human Microbiome Project (HMP (http://nihroadmap.nih.gov. Specifically, the scalability of the dynamic weighting feature is evaluated and established by its application to the analysis of over 400 million weighted gene annotations derived from 14 billion short reads as predicted by the HMP Unified Metabolic Analysis Network (HUMAnN pipeline. Further, the capacity of METAREP to facilitate the identification and simultaneous comparison of taxonomic and functional annotations including biological pathway and individual enzyme abundances from hundreds of community samples is demonstrated by providing scenarios that describe how these data can be mined to answer biological questions related to the human microbiome. These strategies provide users with a reference of how to conduct similar large-scale metagenomic analyses using METAREP with their own sequence data, while in this study they reveal insights into the nature and extent of variation in taxonomic and functional profiles across body habitats and individuals. Over one thousand HMP WGS datasets and the latest

  14. Exploration of soil metagenome diversity for prospection of enzymes involved in lignocellulosic biomass conversion

    Energy Technology Data Exchange (ETDEWEB)

    Alvarez, T.M.; Squina, F.M. [Laboratorio Nacional de Luz Sincrotron (LNLS), Campinas, SP (Brazil); Paixao, D.A.A.; Franco Cairo, J.P.L.; Buchli, F.; Ruller, R. [Laboratorio Nacional de Ciencia e Tecnologia do Bioetanol (CTBE), Campinas, SP (Brazil); Prade, R. [Oklahoma State University, Sillwater, OK (United States)

    2012-07-01

    Full text: Metagenomics allows access to genetic information encoded in DNA of microorganisms recalcitrant to cultivation. They represent a reservoir of novel biocatalyst with potential application in environmental friendly techniques aiming to overcome the dependence on fossil fuels and also to diminish air and water pollution. The focus of our work is the generation of a tool kit of lignocellulolytic enzymes from soil metagenome, which could be used for second generation ethanol production. Environmental samples were collected at a sugarcane field after harvesting, where it is expected that the microbial population involved on lignocellulose degradation was enriched due to the presence of straws covering the soil. Sugarcane Bagasse-Degrading-Soil (SBDS) metagenome was massively-parallel-454-Roche-sequenced. We identified a full repertoire of genes with significant match to glycosyl hydrolases catalytic domain and carbohydrate-binding modules. Soil metagenomics libraries cloned into pUC19 were screened through functional assays. CMC-agar screening resulted in positive clones, revealing new cellulases coding genes. Through a CMC-zymogram it was possible to observe that one of these genes, nominated as E-1, corresponds to an enzyme that is secreted to the extracellular medium, suggesting that the cloned gene carried the original signal peptide. Enzymatic assays and analysis through capillary electrophoresis showed that E-1 was able to cleave internal glycosidic bonds of cellulose. New rounds of functional screenings through chromogenic substrates are being conducted aiming the generation of a library of lignocellulolytic enzymes derived from soil metagenome, which may become key component for development of second generation biofuels. (author)

  15. Exploration of soil metagenome diversity for prospection of enzymes involved in lignocellulosic biomass conversion

    International Nuclear Information System (INIS)

    Full text: Metagenomics allows access to genetic information encoded in DNA of microorganisms recalcitrant to cultivation. They represent a reservoir of novel biocatalyst with potential application in environmental friendly techniques aiming to overcome the dependence on fossil fuels and also to diminish air and water pollution. The focus of our work is the generation of a tool kit of lignocellulolytic enzymes from soil metagenome, which could be used for second generation ethanol production. Environmental samples were collected at a sugarcane field after harvesting, where it is expected that the microbial population involved on lignocellulose degradation was enriched due to the presence of straws covering the soil. Sugarcane Bagasse-Degrading-Soil (SBDS) metagenome was massively-parallel-454-Roche-sequenced. We identified a full repertoire of genes with significant match to glycosyl hydrolases catalytic domain and carbohydrate-binding modules. Soil metagenomics libraries cloned into pUC19 were screened through functional assays. CMC-agar screening resulted in positive clones, revealing new cellulases coding genes. Through a CMC-zymogram it was possible to observe that one of these genes, nominated as E-1, corresponds to an enzyme that is secreted to the extracellular medium, suggesting that the cloned gene carried the original signal peptide. Enzymatic assays and analysis through capillary electrophoresis showed that E-1 was able to cleave internal glycosidic bonds of cellulose. New rounds of functional screenings through chromogenic substrates are being conducted aiming the generation of a library of lignocellulolytic enzymes derived from soil metagenome, which may become key component for development of second generation biofuels. (author)

  16. Biotechnological applications of functional metagenomics in the food and pharmaceutical industries

    Directory of Open Access Journals (Sweden)

    Laura M Coughlan

    2015-06-01

    Full Text Available Microorganisms are found throughout nature, thriving in a vast range of environmental conditions. The majority of them are unculturable or difficult to culture by traditional methods. Metagenomics enables the study of all microorganisms, regardless of whether they can be cultured or not, through the analysis of genomic data obtained directly from an environmental sample, providing knowledge of the species present and allowing the extraction of information regarding the functionality of microbial communities in their natural habitat. Function-based screenings, following the cloning and expression of metagenomic DNA in a heterologous host, can be applied to the discovery of novel proteins of industrial interest encoded by the genes of previously inaccessible microorganisms. Functional metagenomics has considerable potential in the food and pharmaceutical industries, where it can, for instance, aid (i the identification of enzymes with desirable technological properties, capable of catalysing novel reactions or replacing existing chemically synthesized catalysts which may be difficult or expensive to produce, and able to work under a wide range of environmental conditions encountered in food and pharmaceutical processing cycles including extreme conditions of temperature, pH, osmolarity, etc; (ii the discovery of novel bioactives including antimicrobials active against microorganisms of concern both in food and medical settings; (iii the investigation of industrial and societal issues such as antibiotic resistance development. This review article summarizes the state-of-the-art functional metagenomic methods available and discusses the potential of functional metagenomic approaches to mine as yet unexplored environments to discover novel genes with biotechnological application in the food and pharmaceutical industries.

  17. Biotechnological applications of functional metagenomics in the food and pharmaceutical industries.

    Science.gov (United States)

    Coughlan, Laura M; Cotter, Paul D; Hill, Colin; Alvarez-Ordóñez, Avelino

    2015-01-01

    Microorganisms are found throughout nature, thriving in a vast range of environmental conditions. The majority of them are unculturable or difficult to culture by traditional methods. Metagenomics enables the study of all microorganisms, regardless of whether they can be cultured or not, through the analysis of genomic data obtained directly from an environmental sample, providing knowledge of the species present, and allowing the extraction of information regarding the functionality of microbial communities in their natural habitat. Function-based screenings, following the cloning and expression of metagenomic DNA in a heterologous host, can be applied to the discovery of novel proteins of industrial interest encoded by the genes of previously inaccessible microorganisms. Functional metagenomics has considerable potential in the food and pharmaceutical industries, where it can, for instance, aid (i) the identification of enzymes with desirable technological properties, capable of catalyzing novel reactions or replacing existing chemically synthesized catalysts which may be difficult or expensive to produce, and able to work under a wide range of environmental conditions encountered in food and pharmaceutical processing cycles including extreme conditions of temperature, pH, osmolarity, etc; (ii) the discovery of novel bioactives including antimicrobials active against microorganisms of concern both in food and medical settings; (iii) the investigation of industrial and societal issues such as antibiotic resistance development. This review article summarizes the state-of-the-art functional metagenomic methods available and discusses the potential of functional metagenomic approaches to mine as yet unexplored environments to discover novel genes with biotechnological application in the food and pharmaceutical industries.

  18. Detection and identification of Trichophyton tonsurans from clinical isolates and hairbrush samples by loop-mediated isothermal amplification system.

    Science.gov (United States)

    Yo, Ayaka; Yamamoto, Mikachi; Nakayama, Takako; Ishikawa, Jun; Makimura, Koichi

    2016-09-01

    Since the 1990s, there have been reports of the spread of dermatophytosis caused by Trichophyton tonsurans among contact sports athletes in several countries, including Japan. This study was performed to develop a loop-mediated isothermal amplification (LAMP) system for rapid and accurate detection and identification of T. tonsurans from clinical isolates or hairbrush samples for diagnosis and to prevent the spread of infection. A specific primer set was prepared by comparing the whole genome sequence of T. tonsurans with those of six other closely related dermatophytes. After confirming the sensitivity and specificity of this system, LAMP assay was performed using 37 clinical samples obtained from three healthy volunteers and 24 judo athletes. A total of 155 fungal isolates (56 strains of various standard fungi, 96 identified T. tonsurans isolates, three hairbrush-cultured isolates from judo athletes) and 37 hairbrush samples (34 samples from 24 judo athletes, and three samples from three healthy volunteers) were used for culture and LAMP assay, respectively. The assay showed no cross-reactivity to standard strains other than T. tonsurans. The detection limit was 100 copies of DNA template per tube. All of the 96 T. tonsurans isolates were amplified, and all samples from healthy volunteers showed negative results. Four of the 34 hairbrush samples obtained from judo athletes showed positive results in LAMP assay, and two of the four were positive in both culture and LAMP assay. We developed a rapid LAMP system with high specificity and sensitivity for diagnosis of T. tonsurans infection. PMID:26892741

  19. Detection and identification of Trichophyton tonsurans from clinical isolates and hairbrush samples by loop-mediated isothermal amplification system.

    Science.gov (United States)

    Yo, Ayaka; Yamamoto, Mikachi; Nakayama, Takako; Ishikawa, Jun; Makimura, Koichi

    2016-09-01

    Since the 1990s, there have been reports of the spread of dermatophytosis caused by Trichophyton tonsurans among contact sports athletes in several countries, including Japan. This study was performed to develop a loop-mediated isothermal amplification (LAMP) system for rapid and accurate detection and identification of T. tonsurans from clinical isolates or hairbrush samples for diagnosis and to prevent the spread of infection. A specific primer set was prepared by comparing the whole genome sequence of T. tonsurans with those of six other closely related dermatophytes. After confirming the sensitivity and specificity of this system, LAMP assay was performed using 37 clinical samples obtained from three healthy volunteers and 24 judo athletes. A total of 155 fungal isolates (56 strains of various standard fungi, 96 identified T. tonsurans isolates, three hairbrush-cultured isolates from judo athletes) and 37 hairbrush samples (34 samples from 24 judo athletes, and three samples from three healthy volunteers) were used for culture and LAMP assay, respectively. The assay showed no cross-reactivity to standard strains other than T. tonsurans. The detection limit was 100 copies of DNA template per tube. All of the 96 T. tonsurans isolates were amplified, and all samples from healthy volunteers showed negative results. Four of the 34 hairbrush samples obtained from judo athletes showed positive results in LAMP assay, and two of the four were positive in both culture and LAMP assay. We developed a rapid LAMP system with high specificity and sensitivity for diagnosis of T. tonsurans infection.

  20. Metagenomic studies of the Red Sea.

    Science.gov (United States)

    Behzad, Hayedeh; Ibarra, Martin Augusto; Mineta, Katsuhiko; Gojobori, Takashi

    2016-02-01

    Metagenomics has significantly advanced the field of marine microbial ecology, revealing the vast diversity of previously unknown microbial life forms in different marine niches. The tremendous amount of data generated has enabled identification of a large number of microbial genes (metagenomes), their community interactions, adaptation mechanisms, and their potential applications in pharmaceutical and biotechnology-based industries. Comparative metagenomics reveals that microbial diversity is a function of the local environment, meaning that unique or unusual environments typically harbor novel microbial species with unique genes and metabolic pathways. The Red Sea has an abundance of unique characteristics; however, its microbiota is one of the least studied among marine environments. The Red Sea harbors approximately 25 hot anoxic brine pools, plus a vibrant coral reef ecosystem. Physiochemical studies describe the Red Sea as an oligotrophic environment that contains one of the warmest and saltiest waters in the world with year-round high UV radiations. These characteristics are believed to have shaped the evolution of microbial communities in the Red Sea. Over-representation of genes involved in DNA repair, high-intensity light responses, and osmoregulation were found in the Red Sea metagenomic databases suggesting acquisition of specific environmental adaptation by the Red Sea microbiota. The Red Sea brine pools harbor a diverse range of halophilic and thermophilic bacterial and archaeal communities, which are potential sources of enzymes for pharmaceutical and biotechnology-based application. Understanding the mechanisms of these adaptations and their function within the larger ecosystem could also prove useful in light of predicted global warming scenarios where global ocean temperatures are expected to rise by 1-3°C in the next few decades. In this review, we provide an overview of the published metagenomic studies that were conducted in the Red Sea, and

  1. "ISA-Lation" of Single-Stranded Positive-Sense RNA Viruses from Non-Infectious Clinical/Animal Samples.

    Directory of Open Access Journals (Sweden)

    Fabien Aubry

    Full Text Available Isolation of viral pathogens from clinical and/or animal samples has traditionally relied on either cell cultures or laboratory animal model systems. However, virus viability is notoriously susceptible to adverse conditions that may include inappropriate procedures for sample collection, storage temperature, support media and transportation. Using our recently described ISA method, we have developed a novel procedure to isolate infectious single-stranded positive-sense RNA viruses from clinical or animal samples. This approach, that we have now called "ISA-lation", exploits the capacity of viral cDNA subgenomic fragments to re-assemble and produce infectious viral RNA in susceptible cells. Here, it was successfully used to rescue enterovirus, Chikungunya and Tick-borne encephalitis viruses from a variety of inactivated animal and human samples. ISA-lation represents an effective option to rescue infectious virus from clinical and/or animal samples that may have deteriorated during the collection and storage period, but also potentially overcomes logistic and administrative difficulties generated when complying with current health and safety and biosecurity guidelines associated with shipment of infectious viral material.

  2. Metagenomics: Retrospect and Prospects in High Throughput Age

    OpenAIRE

    Satish Kumar; Kishore Kumar Krishnani; Bharat Bhushan; Manoj Pandit Brahmane

    2015-01-01

    In recent years, metagenomics has emerged as a powerful tool for mining of hidden microbial treasure in a culture independent manner. In the last two decades, metagenomics has been applied extensively to exploit concealed potential of microbial communities from almost all sorts of habitats. A brief historic progress made over the period is discussed in terms of origin of metagenomics to its current state and also the discovery of novel biological functions of commercial importance from metage...

  3. Current and future trends in metagenomics : Development of knowledge bases

    Science.gov (United States)

    Mori, Hiroshi; Yamada, Takuji; Kurokawa, Ken

    Microbes are essential for every part of life on Earth. Numerous microbes inhabit the biosphere, many of which are uncharacterized or uncultivable. They form a complex microbial community that deeply affects against surrounding environments. Metagenome analysis provides a radically new way of examining such complex microbial community without isolation or cultivation of individual bacterial community members. In this article, we present a brief discussion about a metagenomics and the development of knowledge bases, and also discuss about the future trends in metagenomics.

  4. Identification and Genome Characterization of the First Sicinivirus Isolate from Chickens in Mainland China by Using Viral Metagenomics.

    Science.gov (United States)

    Zhou, Hongzhuan; Zhu, Shanshan; Quan, Rong; Wang, Jing; Wei, Li; Yang, Bing; Xu, Fuzhou; Wang, Jinluo; Chen, Fuyong; Liu, Jue

    2015-01-01

    Unlike traditional virus isolation and sequencing approaches, sequence-independent amplification based viral metagenomics technique allows one to discover unexpected or novel viruses efficiently while bypassing culturing step. Here we report the discovery of the first Sicinivirus isolate (designated as strain JSY) of picornaviruses from commercial layer chickens in mainland China by using a viral metagenomics technique. This Sicinivirus isolate, which contains a whole genome of 9,797 nucleotides (nt) excluding the poly(A) tail, possesses one of the largest picornavirus genome so far reported, but only shares 88.83% and 82.78% of amino acid sequence identity to that of ChPV1 100C (KF979332) and Sicinivirus 1 strain UCC001 (NC_023861), respectively. The complete 939 nt 5'UTR of the isolate strain contains at least twelve stem-loop domains (A-L), representing the highest set of loops reported within Sicinivirus genus. The conserved 'barbell-like' structure was also present in the 272 nt 3'UTR of the isolate as that in the 3' UTR of Sicinivirus 1 strain UCC001. The 8,586 nt large open reading frame encodes a 2,862 amino acids polyprotein precursor. Moreover, Sicinivirus infection might be widely present in commercial chicken farms in Yancheng region of the Jiangsu Province as evidenced by all the tested stool samples from three different farms being positive (17/17) for Sicinivirus detection. This is the first report on identification of Sicinivirus in commercial layer chickens with a severe clinical disease in mainland China, however, further studies are needed to evaluate the pathogenic potential of this picornavirus in chickens.

  5. FOCUS: an alignment-free model to identify organisms in metagenomes using non-negative least squares

    Directory of Open Access Journals (Sweden)

    Genivaldo Gueiros Z. Silva

    2014-06-01

    Full Text Available One of the major goals in metagenomics is to identify the organisms present in a microbial community from unannotated shotgun sequencing reads. Taxonomic profiling has valuable applications in biological and medical research, including disease diagnostics. Most currently available approaches do not scale well with increasing data volumes, which is important because both the number and lengths of the reads provided by sequencing platforms keep increasing. Here we introduce FOCUS, an agile composition based approach using non-negative least squares (NNLS to report the organisms present in metagenomic samples and profile their abundances. FOCUS was tested with simulated and real metagenomes, and the results show that our approach accurately predicts the organisms present in microbial communities. FOCUS was implemented in Python. The source code and web-sever are freely available at http://edwards.sdsu.edu/FOCUS.

  6. Algal biogeography: metagenomics shows distribution of a picoplanktonic pelagophyte.

    Science.gov (United States)

    Raven, John A

    2012-09-11

    How can we determine the distribution of uncultured marine microorganisms? Targeted metagenomics has provided the complete chloroplast genome sequence, and the distribution, for a picoplanktonic pelagophyte alga.

  7. SmashCommunity: A metagenomic annotation and analysis tool

    DEFF Research Database (Denmark)

    Arumugam, Manimozhiyan; Harrington, Eoghan D; Foerstner, Konrad U;

    2010-01-01

    SUMMARY: SmashCommunity is a stand-alone metagenomic annotation and analysis pipeline suitable for data from Sanger and 454 sequencing technologies. It supports state-of-the-art software for essential metagenomic tasks such as assembly and gene prediction. It provides tools to estimate...... the quantitative phylogenetic and functional compositions of metagenomes, to compare compositions of multiple metagenomes and to produce intuitive visual representations of such analyses. AVAILABILITY: SmashCommunity is freely available at http://www.bork.embl.de/software/smash CONTACT: bork@embl.de....

  8. Chemistry Testing on Plasma Versus Serum Samples in Dialysis Patients: Clinical and Quality Improvement Implications.

    Science.gov (United States)

    Carey, Roger Neill; Jani, Chinu; Johnson, Curtis; Pearce, Jim; Hui-Ng, Patricia; Lacson, Eduardo

    2016-09-01

    Plasma samples collected in tubes containing separator gels have replaced serum samples for most chemistry tests in many hospital and commercial laboratories. Use of plasma samples for blood tests in the dialysis population eliminates delays in sample processing while waiting for clotting to complete, laboratory technical issues associated with fibrin formation, repeat sample collection, and patient care issues caused by delay of results because of incompletely clotted specimens. Additionally, a larger volume of plasma is produced than serum for the same amount of blood collected. Plasma samples are also acceptable for most chemical tests involved in the care of patients with ESRD. This information becomes very important when United States regulatory requirements for ESRD inadvertently limit the type of sample that can be used for government reporting, quality assessment, and value-based payment initiatives. In this narrative, we summarize the renal community experience and how the subsequent resolution of the acceptability of phosphorus levels measured from serum and plasma samples may have significant implications in the country's continued development of a value-based Medicare ESRD Quality Incentive Program.

  9. WISC-III and CAS: Which Correlates Higher with Achievement for a Clinical Sample?

    Science.gov (United States)

    Naglieri, Jack A.; De Lauder, Brianna Y.; Goldstein, Sam; Schwebech, Adam

    2006-01-01

    The relationships between Wechsler Intelligence Scale for Children-Third Edition (WISC-III) and the Cognitive Assessment System (CAS) with the Woodcock-Johnson Tests of Achievement (WJ-III) were examined for a sample of 119 children (87 males and 32 females) ages 6 to 16. The sample was comprised of children who were referred to a specialty clinic…

  10. The metagenomics RAST server – a public resource for the automatic phylogenetic and functional analysis of metagenomes

    OpenAIRE

    Stevens R; Rodriguez A. (ed.); Paczian T; Kubal M; Glass EM; Olson R; D'Souza M; Paarmann D; Meyer F; Wilke A; Wilkening J; Edwards RA

    2008-01-01

    Abstract Background Random community genomes (metagenomes) are now commonly used to study microbes in different environments. Over the past few years, the major challenge associated with metagenomics shifted from generating to analyzing sequences. High-throughput, low-cost next-generation sequencing has provided access to metagenomics to a wide range of researchers. Results A high-throughput pipeline has been constructed to provide high-performance computing to all researchers interested in u...

  11. Quantifying environmental adaptation of metabolic pathways in metagenomics

    DEFF Research Database (Denmark)

    Gianoulis, Tara A; Raes, Jeroen; Patel, Prianka V;

    2009-01-01

    of particular pathways and subnetworks reflects the adaptation of microbial communities across environments and habitats-i.e., how network dynamics relates to environmental features. Previous research has treated environments as discrete, somewhat simplified classes (e.g., terrestrial vs. marine), and searched......Recently, approaches have been developed to sample the genetic content of heterogeneous environments (metagenomics). However, by what means these sequences link distinct environmental conditions with specific biological processes is not well understood. Thus, a major challenge is how the usage...... of weighted pathways that maximally covaries with a combination of environmental variables (many-to-many), which we term a metabolic footprint. Applied to available aquatic datasets, we identified footprints predictive of their environment that can potentially be used as biosensors. For example, we show...

  12. Metagenomic approach for discovering new pathogens in infection disease outbreaks

    Directory of Open Access Journals (Sweden)

    Emanuela Giombini

    2011-09-01

    Full Text Available Viruses represent the most abundant biological components on earth.They can be found in every environment, from deep layers of oceans to animal bodies.Although several viruses have been isolated and sequenced, in each environment there are millions of different types of viruses that have not been identified yet.The advent of nextgeneration sequencing technologies with their high throughput capabilities make possible to study in a single experiment all the community of microorganisms present in a particular sample “microbioma”.They made more feasible the application of the metagenomic approach, by which it is also possible to discover and identify new pathogens, that may pose a threat to public health.This paper summarizes the most recent applications of nextgeneration sequencing to discover new viral pathogens during the occurrence of infection disease outbreaks.

  13. Genomics and metagenomics in medical microbiology.

    Science.gov (United States)

    Padmanabhan, Roshan; Mishra, Ajay Kumar; Raoult, Didier; Fournier, Pierre-Edouard

    2013-12-01

    Over the last two decades, sequencing tools have evolved from laborious time-consuming methodologies to real-time detection and deciphering of genomic DNA. Genome sequencing, especially using next generation sequencing (NGS) has revolutionized the landscape of microbiology and infectious disease. This deluge of sequencing data has not only enabled advances in fundamental biology but also helped improve diagnosis, typing of pathogen, virulence and antibiotic resistance detection, and development of new vaccines and culture media. In addition, NGS also enabled efficient analysis of complex human micro-floras, both commensal, and pathological, through metagenomic methods, thus helping the comprehension and management of human diseases such as obesity. This review summarizes technological advances in genomics and metagenomics relevant to the field of medical microbiology.

  14. Evaluation of inhibitor-resistant real-time PCR methods for diagnostics in clinical and environmental samples.

    Science.gov (United States)

    Trombley Hall, Adrienne; McKay Zovanyi, Ashley; Christensen, Deanna Rose; Koehler, Jeffrey William; Devins Minogue, Timothy

    2013-01-01

    Polymerase chain reaction (PCR) is commonly used for pathogen detection in clinical and environmental samples. These sample matrices often contain inhibitors of PCR, which is a primary reason for sample processing; however, the purification process is highly inefficient, becoming unacceptable at lower signature concentrations. One potential solution is direct PCR assessment without sample processing. Here, we evaluated nine inhibitor-resistant PCR reagents for direct detection of Francisella tularensis in seven different clinical and environmental samples using an established real-time PCR assay to assess ability to overcome PCR inhibition. While several of these reagents were designed for standard PCR, the described inhibitor resistant properties (ex. Omni Klentaq can amplify target DNA samples of up to 20% whole blood or soil) led to our evaluation with real-time PCR. A preliminary limit of detection (LOD) was determined for each chemistry in whole blood and buffer, and LODs (20 replicates) were determined for the top five chemistries in each matrix (buffer, whole blood, sputum, stool, swab, soil, and sand). Not surprisingly, no single chemistry performed the best across all of the different matrices evaluated. For instance, Phusion Blood Direct PCR Kit, Phire Hot Start DNA polymerase, and Phire Hot Start DNA polymerase with STR Boost performed best for direct detection in whole blood while Phire Hot Start DNA polymerase with STR Boost were the only reagents to yield an LOD in the femtogram range for soil. Although not the best performer across all matrices, KAPA Blood PCR kit produced the most consistent results among the various conditions assessed. Overall, while these inhibitor resistant reagents show promise for direct amplification of complex samples by real-time PCR, the amount of template required for detection would not be in a clinically relevant range for most matrices. PMID:24040090

  15. Evaluation of inhibitor-resistant real-time PCR methods for diagnostics in clinical and environmental samples.

    Directory of Open Access Journals (Sweden)

    Adrienne Trombley Hall

    Full Text Available Polymerase chain reaction (PCR is commonly used for pathogen detection in clinical and environmental samples. These sample matrices often contain inhibitors of PCR, which is a primary reason for sample processing; however, the purification process is highly inefficient, becoming unacceptable at lower signature concentrations. One potential solution is direct PCR assessment without sample processing. Here, we evaluated nine inhibitor-resistant PCR reagents for direct detection of Francisella tularensis in seven different clinical and environmental samples using an established real-time PCR assay to assess ability to overcome PCR inhibition. While several of these reagents were designed for standard PCR, the described inhibitor resistant properties (ex. Omni Klentaq can amplify target DNA samples of up to 20% whole blood or soil led to our evaluation with real-time PCR. A preliminary limit of detection (LOD was determined for each chemistry in whole blood and buffer, and LODs (20 replicates were determined for the top five chemistries in each matrix (buffer, whole blood, sputum, stool, swab, soil, and sand. Not surprisingly, no single chemistry performed the best across all of the different matrices evaluated. For instance, Phusion Blood Direct PCR Kit, Phire Hot Start DNA polymerase, and Phire Hot Start DNA polymerase with STR Boost performed best for direct detection in whole blood while Phire Hot Start DNA polymerase with STR Boost were the only reagents to yield an LOD in the femtogram range for soil. Although not the best performer across all matrices, KAPA Blood PCR kit produced the most consistent results among the various conditions assessed. Overall, while these inhibitor resistant reagents show promise for direct amplification of complex samples by real-time PCR, the amount of template required for detection would not be in a clinically relevant range for most matrices.

  16. Draft Genome Sequence of Uncultured SAR324 Bacterium lautmerah10, Binned from a Red Sea Metagenome

    KAUST Repository

    Haroon, Mohamed F.

    2016-02-11

    A draft genome of SAR324 bacterium lautmerah10 was assembled from a metagenome of a surface water sample from the Red Sea, Saudi Arabia. The genome is more complete and has a higher G+C content than that of previously sequenced SAR324 representatives. Its genomic information shows a versatile metabolism that confers an advantage to SAR324, which is reflected in its distribution throughout different depths of the marine water column.

  17. Biotechnological applications of functional metagenomics in the food and pharmaceutical industries

    OpenAIRE

    Coughlan, Laura M.; Paul David Cotter; Colin eHill; Avelino eAlvarez-Ordóñez

    2015-01-01

    Microorganisms are found throughout nature, thriving in a vast range of environmental conditions. The majority of them are unculturable or difficult to culture by traditional methods. Metagenomics enables the study of all microorganisms, regardless of whether they can be cultured or not, through the analysis of genomic data obtained directly from an environmental sample, providing knowledge of the species present and allowing the extraction of information regarding the functionality of microb...

  18. SUPER-FOCUS: a tool for agile functional analysis of shotgun metagenomic data

    OpenAIRE

    Genivaldo Gueiros Z. Silva; Green, Kevin T.; Dutilh, Bas E.; Robert A Edwards

    2015-01-01

    Summary: Analyzing the functional profile of a microbial community from unannotated shotgun sequencing reads is one of the important goals in metagenomics. Functional profiling has valuable applications in biological research because it identifies the abundances of the functional genes of the organisms present in the original sample, answering the question what they can do. Currently, available tools do not scale well with increasing data volumes, which is important because both the number an...

  19. Biotechnological applications of functional metagenomics in the food and pharmaceutical industries

    OpenAIRE

    Coughlan, Laura M.; Paul D Cotter; Hill, Colin; Alvarez-Ordóñez, Avelino

    2015-01-01

    Microorganisms are found throughout nature, thriving in a vast range of environmental conditions. The majority of them are unculturable or difficult to culture by traditional methods. Metagenomics enables the study of all microorganisms, regardless of whether they can be cultured or not, through the analysis of genomic data obtained directly from an environmental sample, providing knowledge of the species present, and allowing the extraction of information regarding the functionality of micro...

  20. Fecal metagenomics for the simultaneous assessment of diet, parasites, and population genetics of an understudied primate

    OpenAIRE

    Srivathsan, Amrita; Ang, Andie; Vogler, Alfried P; Meier, Rudolf

    2016-01-01

    Background Rapid habitat loss and degradation are responsible for population decline in a growing number of species. Understanding the natural history of these species is important for designing conservation strategies, such as habitat enhancements or ex-situ conservation. The acquisition of observational data may be difficult for rare and declining species, but metagenomics and metabarcoding can provide novel kinds of information. Here we use these methods for analysing fecal samples from an...

  1. ISOLATION AND SPECIATION OF CANDIDA FROM CLINICAL SAMPLES IN A TERTIARY CARE HOSPITAL AT KURNOOL, ANDHRAPRADESH, INDIA

    Directory of Open Access Journals (Sweden)

    Dasari

    2014-12-01

    Full Text Available : Candida is one of the most frequently encountered opportunistic fungi that cause infection in humans. The pathogenesis of Candida is complex and probably varies with each infection. This study was conducted to understand the prevalence of Candida from various clinical specimens of patients and to show the emergence of Non albicans Candida in clinical samples. This study also focused on the antifungal susceptibility which guides the clinicians to treat the infection effectively. METHODS: Clinical samples were collected from outpatients and inpatients of Government General Hospital, Kurnool over a period of one year from March2008 to June2009. Isolation, culture, speciation of Candida was done by using standard methods. Antifungal susceptibility testing was done by disc diffusion technique against amphotericin B, nystatin, fluconazole and clotrimazole. RESULTS: Candida manifests in various sites depending on the predisposing factors and immune status of the person. In this study we found the association of Candida with various predisposing factors (Pregnancy, Oral contraceptive pills’s, Immune suppression, Diabetes. This study observed the dominance of non-albicans Candida (51% in the clinical samples over Candida albicans (49%. The maximum antifungal susceptibility was observed against amphotericin B in both the albicans and non-albicans Candia, but non-albicans Candida showed maximum resistance to azoles. CONCLUSION: Candida albicans was the most predominant species (49% isolated in various clinical samples. There was an increase in the prevalence of non albicans Candida in this study. Among the nonalbicans Candida (51% Candid tropicalis was the commonest species isolated. Candida albicans showed maximum susceptibility to amphotericin B and maximum resistance to azoles was seen in nonalbicans Candida.

  2. The GAAS metagenomic tool and its estimations of viral and microbial average genome size in four major biomes.

    Directory of Open Access Journals (Sweden)

    Florent E Angly

    2009-12-01

    Full Text Available Metagenomic studies characterize both the composition and diversity of uncultured viral and microbial communities. BLAST-based comparisons have typically been used for such analyses; however, sampling biases, high percentages of unknown sequences, and the use of arbitrary thresholds to find significant similarities can decrease the accuracy and validity of estimates. Here, we present Genome relative Abundance and Average Size (GAAS, a complete software package that provides improved estimates of community composition and average genome length for metagenomes in both textual and graphical formats. GAAS implements a novel methodology to control for sampling bias via length normalization, to adjust for multiple BLAST similarities by similarity weighting, and to select significant similarities using relative alignment lengths. In benchmark tests, the GAAS method was robust to both high percentages of unknown sequences and to variations in metagenomic sequence read lengths. Re-analysis of the Sargasso Sea virome using GAAS indicated that standard methodologies for metagenomic analysis may dramatically underestimate the abundance and importance of organisms with small genomes in environmental systems. Using GAAS, we conducted a meta-analysis of microbial and viral average genome lengths in over 150 metagenomes from four biomes to determine whether genome lengths vary consistently between and within biomes, and between microbial and viral communities from the same environment. Significant differences between biomes and within aquatic sub-biomes (oceans, hypersaline systems, freshwater, and microbialites suggested that average genome length is a fundamental property of environments driven by factors at the sub-biome level. The behavior of paired viral and microbial metagenomes from the same environment indicated that microbial and viral average genome sizes are independent of each other, but indicative of community responses to stressors and

  3. [Clinical characteristics and social determinants in a sample of non-homebound elderly].

    Science.gov (United States)

    Morsch, Patricia; Pereira, Gustavo Nunes; Navarro, Joel Hirtz do Nascimento; Trevisan, Margarete Diprat; Lopes, Diene Gomes Colvara; Bós, Ângelo José Gonçalves

    2015-05-01

    This study aimed to assess social and clinical factors associated with the fact that older adults (≥ 60 years) go out of their homes. The study interviewed 5,898 older adults identified through home visits, randomly selected in 59 cities in the State of Rio Grande do Sul, Brazil. Multivariate logistic regression was used to assess the association between the outcome and independent variables. Factors associated with going out were being men, younger and married, presence of arthrosis, ease in performing specific activities, and good self-rated health. Heart disease was a negative factor for going out. Given the importance of social activity for quality of life and the World Health Organization policy for active aging, it is extremely important to consider clinical conditions that allow the older adults to remain active in the community. Studies like this can help to adjust public policies for the elderly, especially acting on modifiable clinical and functional conditions. PMID:26083177

  4. CAMERA: A Community Resource for Metagenomics

    OpenAIRE

    Rekha Seshadri; Kravitz, Saul A; Larry Smarr; Paul Gilna; Marvin Frazier

    2007-01-01

    Microbes are responsible for most of the chemical transformations that are crucial to sustaining life on Earth. Their ability to inhabit almost any environmental niche suggests that they possess an incredible diversity of physiological capabilities. However, we have little to no information on a majority of the millions of microbial species that are predicted to exist, mainly because of our inability to culture them in the laboratory. A growing discipline called metagenomics allows u...

  5. Metagenomic scaffolds enable combinatorial lignin transformation

    OpenAIRE

    Strachan, Cameron R.; Singh, Rahul; VanInsberghe, David; Ievdokymenko, Kateryna; Budwill, Karen; Mohn, William W.; Eltis, Lindsay D.; Steven J Hallam

    2014-01-01

    Plant biomass conversion into biofuels and chemicals can reduce human reliance on petroleum and promote sustainable biorefining processes. The structural polymer lignin can comprise up to 40% of plant biomass, but resists decomposition into valuable monoaromatic compounds. In this study, we devised a previously unidentified biosensor responsive to lignin transformation products. We used this biosensor in a functional screen to recover metagenomic scaffolds sourced from coal bed bacterial comm...

  6. Generating viral metagenomes from the coral holobiont.

    Science.gov (United States)

    Weynberg, Karen D; Wood-Charlson, Elisha M; Suttle, Curtis A; van Oppen, Madeleine J H

    2014-01-01

    Reef-building corals comprise multipartite symbioses where the cnidarian animal is host to an array of eukaryotic and prokaryotic organisms, and the viruses that infect them. These viruses are critical elements of the coral holobiont, serving not only as agents of mortality, but also as potential vectors for lateral gene flow, and as elements encoding a variety of auxiliary metabolic functions. Consequently, understanding the functioning and health of the coral holobiont requires detailed knowledge of the associated viral assemblage and its function. Currently, the most tractable way of uncovering viral diversity and function is through metagenomic approaches, which is inherently difficult in corals because of the complex holobiont community, an extracellular mucus layer that all corals secrete, and the variety of sizes and structures of nucleic acids found in viruses. Here we present the first protocol for isolating, purifying and amplifying viral nucleic acids from corals based on mechanical disruption of cells. This method produces at least 50% higher yields of viral nucleic acids, has very low levels of cellular sequence contamination and captures wider viral diversity than previously used chemical-based extraction methods. We demonstrate that our mechanical-based method profiles a greater diversity of DNA and RNA genomes, including virus groups such as Retro-transcribing and ssRNA viruses, which are absent from metagenomes generated via chemical-based methods. In addition, we briefly present (and make publically available) the first paired DNA and RNA viral metagenomes from the coral Acropora tenuis. PMID:24847321

  7. New Extremophilic Lipases and Esterases from Metagenomics

    Science.gov (United States)

    López-López, Olalla; Cerdán, Maria E; González Siso, Maria I

    2014-01-01

    Lipolytic enzymes catalyze the hydrolysis of ester bonds in the presence of water. In media with low water content or in organic solvents, they can catalyze synthetic reactions such as esterification and transesterification. Lipases and esterases, in particular those from extremophilic origin, are robust enzymes, functional under the harsh conditions of industrial processes owing to their inherent thermostability and resistance towards organic solvents, which combined with their high chemo-, regio- and enantioselectivity make them very attractive biocatalysts for a variety of industrial applications. Likewise, enzymes from extremophile sources can provide additional features such as activity at extreme temperatures, extreme pH values or high salinity levels, which could be interesting for certain purposes. New lipases and esterases have traditionally been discovered by the isolation of microbial strains producing lipolytic activity. The Genome Projects Era allowed genome mining, exploiting homology with known lipases and esterases, to be used in the search for new enzymes. The Metagenomic Era meant a step forward in this field with the study of the metagenome, the pool of genomes in an environmental microbial community. Current molecular biology techniques make it possible to construct total environmental DNA libraries, including the genomes of unculturable organisms, opening a new window to a vast field of unknown enzymes with new and unique properties. Here, we review the latest advances and findings from research into new extremophilic lipases and esterases, using metagenomic approaches, and their potential industrial and biotechnological applications. PMID:24588890

  8. Generating viral metagenomes from the coral holobiont

    Directory of Open Access Journals (Sweden)

    Karen Dawn Weynberg

    2014-05-01

    Full Text Available Reef-building corals comprise multipartite symbioses where the cnidarian animal is host to an array of eukaryotic and prokaryotic organisms, and the viruses that infect them. These viruses are critical elements of the coral holobiont, serving not only as agents of mortality, but also as potential vectors for lateral gene flow, and as elements encoding a variety of auxiliary metabolic functions. Consequently, understanding the functioning and health of the coral holobiont requires detailed knowledge of the associated viral assemblage and its function. Currently, the most tractable way of uncovering viral diversity and function is through metagenomic approaches, which is inherently difficult in corals because of the complex holobiont community, an extracellular mucus layer that all corals secrete, and the variety of sizes and structures of nucleic acids found in viruses. Here we present the first protocol for isolating, purifying and amplifying viral nucleic acids from corals based on mechanical disruption of cells. This method produces at least 50% higher yields of viral nucleic acids, has very low levels of cellular sequence contamination and captures wider viral diversity than previously used chemical-based extraction methods. We demonstrate that our mechanical-based method profiles a greater diversity of DNA and RNA genomes, including virus groups such as Retro-transcribing and ssRNA viruses, which are absent from metagenomes generated via chemical-based methods. In addition, we briefly present (and make publically available the first paired DNA and RNA viral metagenomes from the coral Acropora tenuis.

  9. Preservation of nucleic acids and tissue morphology in paraffin-embedded clinical samples: comparison of five molecular fixatives.

    Science.gov (United States)

    Staff, Synnöve; Kujala, Paula; Karhu, Ritva; Rökman, Annika; Ilvesaro, Joanna; Kares, Saara; Isola, Jorma

    2013-09-01

    Formalin fixation preserves tissue morphology at the expense of macromolecule integrity. Freshly frozen samples are the golden standard for DNA and RNA analyses but require laborious deep-freezing and frozen sectioning for morphological studies. Alternative tissue stabilisation methods are therefore needed. We analysed the preservation of nucleic acids, immunohistochemical staining properties and tissue morphology in paraffin-embedded clinical tissue samples fixed with Z7, RCL2, PAXgene, Allprotect and RNAlater. Formalin-fixed and deep-frozen samples were used as controls. Immunohistochemical analyses showed good preservation of antigenicity in all except Allprotect and RNAlater-fixed samples. RNA quality, based on RNA integrity number value by Bioanalyzer, was comparable with freshly frozen samples only in PAXgene-fixed samples. According to quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses, RNA from PAXgene samples yielded results similar to freshly frozen samples. No difference between fixatives was seen in DNA analyses (PCR and real-time PCR). In conclusion, PAXgene seems to be superior to other molecular fixatives and formaldehyde.

  10. Amplicon-based metagenomics identified candidate organisms in soils that caused yield decline in strawberry.

    Science.gov (United States)

    Xu, Xiangming; Passey, Thomas; Wei, Feng; Saville, Robert; Harrison, Richard J

    2015-01-01

    A phenomenon of yield decline due to weak plant growth in strawberry was recently observed in non-chemo-fumigated soils, which was not associated with the soil fungal pathogen Verticillium dahliae, the main target of fumigation. Amplicon-based metagenomics was used to profile soil microbiota in order to identify microbial organisms that may have caused the yield decline. A total of 36 soil samples were obtained in 2013 and 2014 from four sites for metagenomic studies; two of the four sites had a yield-decline problem, the other two did not. More than 2000 fungal or bacterial operational taxonomy units (OTUs) were found in these samples. Relative abundance of individual OTUs was statistically compared for differences between samples from sites with or without yield decline. A total of 721 individual comparisons were statistically significant - involving 366 unique bacterial and 44 unique fungal OTUs. Based on further selection criteria, we focused on 34 bacterial and 17 fungal OTUs and found that yield decline resulted probably from one or more of the following four factors: (1) low abundance of Bacillus and Pseudomonas populations, which are well known for their ability of supressing pathogen development and/or promoting plant growth; (2) lack of the nematophagous fungus (Paecilomyces species); (3) a high level of two non-specific fungal root rot pathogens; and (4) wet soil conditions. This study demonstrated the usefulness of an amplicon-based metagenomics approach to profile soil microbiota and to detect differential abundance in microbes. PMID:26504572

  11. Comparative Metagenomics of Toxic Freshwater Cyanobacteria Bloom Communities on Two Continents

    Energy Technology Data Exchange (ETDEWEB)

    Steffen, Morgan M [ORNL; Li, Zhou [ORNL; Effler, Chad [Department of Microbiology, University of Tennessee; Hauser, Loren John [ORNL; Boyer, Gergory [College of Environmental Science and Forestry, State University of New York, Syracuse; Wilhelm, Steven W [ORNL

    2012-01-01

    Toxic cyanobacterial blooms have persisted in freshwater systems around the world for centuries and appear to be globally increasing in frequency and severity. Toxins produced by bloom-associated cyanobacteria can have drastic impacts on the ecosystem and surrounding communities, and bloom biomass can disrupt aquatic food webs and act as a driver for hypoxia. Little is currently known regarding the genomic content of the Microcystis strains that form blooms or the companion heterotrophic community associated with bloom events. To address these issues, we examined the bloomassociated microbial communities in single samples from Lake Erie (North America), Lake Tai (Taihu, China), and Grand Lakes St. Marys (OH, USA) using comparative metagenomics. Together the Cyanobacteria and Proteobacteria comprised .90% of each bloom bacterial community sample, although the dominant phylum varied between systems. Relative to the existing Microcystis aeruginosa NIES 843 genome, sequences from Lake Erie and Taihu revealed a number of metagenomic islands that were absent in the environmental samples. Moreover, despite variation in the phylogenetic assignments of bloomassociated organisms, the functional potential of bloom members remained relatively constant between systems. This pattern was particularly noticeable in the genomic contribution of nitrogen assimilation genes. In Taihu, the genetic elements associated with the assimilation and metabolism of nitrogen were predominantly associated with Proteobacteria, while these functions in the North American lakes were primarily contributed to by the Cyanobacteria. Our observations build on an emerging body of metagenomic surveys describing the functional potential of microbial communities as more highly conserved than that of their phylogenetic makeup within natural systems.

  12. Microbial Diversity and Biochemical Potential Encoded by Thermal Spring Metagenomes Derived from the Kamchatka Peninsula

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    Bernd Wemheuer

    2013-01-01

    Full Text Available Volcanic regions contain a variety of environments suitable for extremophiles. This study was focused on assessing and exploiting the prokaryotic diversity of two microbial communities derived from different Kamchatkian thermal springs by metagenomic approaches. Samples were taken from a thermoacidophilic spring near the Mutnovsky Volcano and from a thermophilic spring in the Uzon Caldera. Environmental DNA for metagenomic analysis was isolated from collected sediment samples by direct cell lysis. The prokaryotic community composition was examined by analysis of archaeal and bacterial 16S rRNA genes. A total number of 1235 16S rRNA gene sequences were obtained and used for taxonomic classification. Most abundant in the samples were members of Thaumarchaeota, Thermotogae, and Proteobacteria. The Mutnovsky hot spring was dominated by the Terrestrial Hot Spring Group, Kosmotoga, and Acidithiobacillus. The Uzon Caldera was dominated by uncultured members of the Miscellaneous Crenarchaeotic Group and Enterobacteriaceae. The remaining 16S rRNA gene sequences belonged to the Aquificae, Dictyoglomi, Euryarchaeota, Korarchaeota, Thermodesulfobacteria, Firmicutes, and some potential new phyla. In addition, the recovered DNA was used for generation of metagenomic libraries, which were subsequently mined for genes encoding lipolytic and proteolytic enzymes. Three novel genes conferring lipolytic and one gene conferring proteolytic activity were identified.

  13. Total Protein Profile and Drug Resistance in Candida albicans Isolated from Clinical Samples

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    Kamal Uddin Zaidi

    2016-01-01

    Full Text Available This study was done to assess the antifungal susceptibility of clinical isolates of Candida albicans and to evaluate its total protein profile based on morphological difference on drug resistance. Hundred and twenty clinical isolates of C. albicans from various clinical specimens were tested for susceptibility against four antifungal agents, namely, fluconazole, itraconazole, amphotericin B, and ketoconazole. A significant increase of drug resistance in clinical isolates of C. albicans was observed. The study showed 50% fluconazole and itraconazole resistance at 32 μg mL−1 with a MIC50 and MIC90 values at 34 and 47 and 36 and 49 μg mL−1, respectively. All isolates were sensitive to amphotericin B and ketoconazole. The SDS-PAGE protein profile showed a prevalent band of ~52.5 kDa, indicating overexpression of gene in 72% strains with fluconazole resistance. Since the opportunistic infections of Candida spp. are increasing along with drug resistance, the total protein profile will help in understanding the evolutionary changes in drug resistance and also to characterize them.

  14. Social and Emotional Outcomes of Child Sexual Abuse: A Clinical Sample in Turkey

    Science.gov (United States)

    Ozbaran, Burcu; Erermis, Serpil; Bukusoglu, Nagehan; Bildik, Tezan; Tamar, Muge; Ercan, Eyyup Sabri; Aydin, Cahide; Cetin, Saniye Korkmaz

    2009-01-01

    Childhood sexual abuse is a traumatic life event that may cause psychiatric disorders such as posttraumatic stress disorder and depression. During 2003-2004, 20 sexually abused children were referred to the Child and Adolescent Psychiatry Clinic of Ege University in Izmir, Turkey. Two years later, the psychological adjustment of these children (M…

  15. Clinical and Laboratory Data in a Sample of Greek Children with Autism Spectrum Disorders

    Science.gov (United States)

    Ververi, Athina; Vargiami, Efthymia; Papadopoulou, Vassiliki; Tryfonas, Dimitrios; Zafeiriou, Dimitrios I.

    2012-01-01

    The purpose of this study is to describe clinical and laboratory data, as well as comorbid disorders in Greek children with autism spectrum disorders (ASD). Data were retrospectively collected for 222 children aged 1.5-9 years. The mean age at diagnosis was 43.7 [plus or minus] 17.6 months. Significantly earlier diagnoses were noted in children…

  16. Metagenomic and geochemical characterization of pockmarked sediments overlaying the Troll petroleum reservoir in the North Sea

    Directory of Open Access Journals (Sweden)

    Håvelsrud Othilde

    2012-09-01

    Full Text Available Abstract Background Pockmarks (depressions in the seabed have been discovered throughout the world’s oceans and are often related to hydrocarbon seepage. Although high concentrations of pockmarks are present in the seabed overlaying the Troll oil and gas reservoir in the northern North Sea, geological surveys have not detected hydrocarbon seepage in this area at the present time. In this study we have used metagenomics to characterize the prokaryotic communities inhabiting the surface sediments in the Troll area in relation to geochemical parameters, particularly related to hydrocarbon presence. We also investigated the possibility of increased potential for methane oxidation related to the pockmarks. Five metagenomes from pockmarks and plain seabed sediments were sequenced by pyrosequencing (Roche/454 technology. In addition, two metagenomes from seabed sediments geologically unlikely to be influenced by hydrocarbon seepage (the Oslofjord were included. The taxonomic distribution and metabolic potential of the metagenomes were analyzed by multivariate analysis and statistical comparisons to reveal variation within and between the two sampling areas. Results The main difference identified between the two sampling areas was an overabundance of predominantly autotrophic nitrifiers, especially Nitrosopumilus, and oligotrophic marine Gammaproteobacteria in the Troll metagenomes compared to the Oslofjord. Increased potential for degradation of hydrocarbons, especially aromatic hydrocarbons, was detected in two of the Troll samples: one pockmark sample and one from the plain seabed. Although presence of methanotrophic organisms was indicated in all samples, no overabundance in pockmark samples compared to the Oslofjord samples supports no, or only low level, methane seepage in the Troll pockmarks at the present time. Conclusions Given the relatively low content of total organic carbon and great depths of hydrocarbon containing sediments in the Troll

  17. Evaluation of constitutive and inducible resistance to clindamycin in clinical samples of Staphylococcus aureus from a tertiary hospital

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    Angelita Bottega

    2014-10-01

    Full Text Available Introduction Infections caused by methicillin-resistant Staphylococcus aureus (MRSA have become common in hospitals and the community environment, and this wide resistance has limited patient treatment. Clindamycin (CL represents an important alternative therapy for infections caused by S. aureus. Antimicrobial susceptibility testing using standard methods may not detect inducible CL resistance. This study was performed to detect the phenotypes of resistance to macrolides-lincosamides-streptogramin B (MLSB antibiotics, including CL, in clinical samples of S. aureus from patients at a tertiary hospital in Santa Maria, State of Rio Grande do Sul, Brazil. Methods One hundred and forty clinical isolates were submitted to the disk diffusion induction test (D-test with an erythromycin (ER disk positioned at a distance of 20mm from a CL disk. The results were interpreted according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI. Results In this study, 29 (20.7% of the 140 S. aureus samples were resistant to methicillin (MRSA, and 111 (79.3% were susceptible to methicillin (MSSA. The constitutive resistance phenotype (cMLSB was observed in 20 (14.3% MRSA samples and in 5 (3.6% MSSA samples, whereas the inducible resistance phenotype (iMLSB was observed in 3 (2.1% MRSA samples and in 8 (5.8% MSSA samples. Conclusions The D-test is essential for detecting the iMLSB phenotype because the early identification of this phenotype allows clinicians to choose an appropriate treatment for patients. Furthermore, this test is simple, easy to perform and inexpensive.

  18. Multiple stage MS in analysis of plasma, serum, urine and in vitro samples relevant to clinical and forensic toxicology.

    Science.gov (United States)

    Meyer, Golo M; Maurer, Hans H; Meyer, Markus R

    2016-01-01

    This paper reviews MS approaches applied to metabolism studies, structure elucidation and qualitative or quantitative screening of drugs (of abuse) and/or their metabolites. Applications in clinical and forensic toxicology were included using blood plasma or serum, urine, in vitro samples, liquids, solids or plant material. Techniques covered are liquid chromatography coupled to low-resolution and high-resolution multiple stage mass analyzers. Only PubMed listed studies published in English between January 2008 and January 2015 were considered. Approaches are discussed focusing on sample preparation and mass spectral settings. Comments on advantages and limitations of these techniques complete the review.

  19. Development of standardized methodology for identifying toxins in clinical samples and fish species associated with tetrodotoxin-borne poisoning incidents

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    Tai-Yuan Chen

    2016-01-01

    Full Text Available Tetrodotoxin (TTX is a naturally occurring toxin in food, especially in puffer fish. TTX poisoning is observed frequently in South East Asian regions. In TTX-derived food poisoning outbreaks, the amount of TTX recovered from suspicious fish samples or leftovers, and residual levels from biological fluids of victims are typically trace. However, liquid chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry methods have been demonstrated to qualitatively and quantitatively determine TTX in clinical samples from victims. Identification and validation of the TTX-originating seafood species responsible for a food poisoning incident is needed. A polymerase chain reaction-based method on mitochondrial DNA analysis is useful for identification of fish species. This review aims to collect pertinent information available on TTX-borne food poisoning incidents with a special emphasis on the analytical methods employed for TTX detection in clinical laboratories as well as for the identification of TTX-bearing species.

  20. Matrix Metalloproteinase-9/Neutrophil Gelatinase-Associated Lipocalin Complex Activity in Human Glioma Samples Predicts Tumor Presence and Clinical Prognosis

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    Ming-Fa Liu

    2015-01-01

    Full Text Available Matrix metalloproteinase-9/neutrophil gelatinase-associated lipocalin (MMP-9/NGAL complex activity is elevated in brain tumors and may serve as a molecular marker for brain tumors. However, the relationship between MMP-9/NGAL activity in brain tumors and patient prognosis and treatment response remains unclear. Here, we compared the clinical characteristics of glioma patients with the MMP-9/NGAL activity measured in their respective tumor and urine samples. Using gelatin zymography assays, we found that MMP-9/NGAL activity was significantly increased in tumor tissues (TT and preoperative urine samples (Preop-1d urine. Activity was reduced by seven days after surgery (Postop-1w urine and elevated again in cases of tumor recurrence. The MMP-9/NGAL status correlated well with MRI-based tumor assessments. These findings suggest that MMP-9/NGAL activity could be a novel marker to detect gliomas and predict the clinical outcome of patients.

  1. Clinically significant fatigue: prevalence and associated factors in an international sample of adults with multiple sclerosis recruited via the internet.

    Directory of Open Access Journals (Sweden)

    Tracey J Weiland

    Full Text Available Fatigue contributes a significant burden of disease for people with multiple sclerosis (PwMS. Modifiable lifestyle factors have been recognized as having a role in a range of morbidity outcomes in PwMS. There is significant potential to prevent and treat fatigue in PwMS by addressing modifiable risk factors.To explore the associations between clinically significant fatigue and demographic factors, clinical factors (health-related quality of life, disability and relapse rate and modifiable lifestyle, disease-modifying drugs (DMD and supplement use in a large international sample of PwMS.PwMS were recruited to the study via Web 2.0 platforms and completed a comprehensive survey measuring demographic, lifestyle and clinical characteristics, including health-related quality of life, disability, and relapse rate.Of 2469 participants with confirmed MS, 2138 (86.6% completed a validated measure of clinically significant fatigue, the Fatigue Severity Scale. Participants were predominantly female from English speaking countries, with relatively high levels of education, and due to recruitment methods may have been highly pro-active about engaging in lifestyle management and self-help. Approximately two thirds of our sample (1402/2138; 65.6% (95% CI 63.7-67.7 screened positive for clinically significant fatigue. Bivariate associations were present between clinically significant fatigue and several demographic, clinical, lifestyle, and medication variables. After controlling for level of disability and a range of stable socio-demographic variables, we found increased odds of fatigue associated with obesity, DMD use, poor diet, and reduced odds of fatigue with exercise, fish consumption, moderate alcohol use, and supplementation with vitamin D and flaxseed oil.This study supports strong and significant associations between clinically significant fatigue and modifiable lifestyle factors. Longitudinal follow-up of this sample may help clarify the contribution

  2. Antimicrobial resistance trends among canine Escherichia coli isolates obtained from clinical samples in the northeastern USA, 2004–2011

    OpenAIRE

    Cummings, Kevin J.; Aprea, Victor A.; Altier, Craig

    2015-01-01

    Our objectives were to describe the antimicrobial susceptibility of Escherichia coli isolates from dogs in the northeastern USA and to identify temporal trends in resistance to selected antimicrobial agents. Data were collected retrospectively for all canine E. coli isolates from clinical samples submitted to Cornell University’s Animal Health Diagnostic Center between January 1, 2004 and December 31, 2011. Antimicrobial susceptibility testing was performed on 3519 canine E. coli isolates; fr...

  3. Rapid Detection/pathotyping of Newcastle disease virus isolates in clinical samples using real time polymerase chain reaction assay

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Abdul Wajid, Muhammad Wasim, Tahir Yaqub, Shafqat F Rehmani, Tasra Bibi, Nadia Mukhtar, Javed Muhammad, Umar Bacha, Suliman Qadir Afridi, Muhammad Nauman Zahid, Zia u ddin, Muhammad Zubair Shabbir, Kamran Abbas & Muneer Ahmad ### Abstract In the present protocol we describe the real time reverse transcription polymerase chain reaction (rRT-PCR) assay for the rapid detection/pathotyping of Newcastle disease virus (NDV) isoaltes in clinical samples. Fusion gene and matrix ...

  4. Birth Order and Sibling Gender Ratio of a Clinical Sample of Children and Adolescents Diagnosed with Attention Deficit Hyperactivity Disorder

    OpenAIRE

    Ahmad Ghanizadeh; Marzie Abotorabi-Zarchi; Mohammad Reza Mohammadi; Ali Firoozabadi

    2012-01-01

    Objective: It is not clear whether sibling’s gender ratio is associated with attention deficit hyperactivity disorder (ADHD). This study examines whether inattentiveness severity and hyperactivity/impulsivity severity are associated with birth order of children with ADHD.Method: Participants are a clinical sample of 173 children and adolescents with ADHD and 43 ones without ADHD. Diagnoses were made using Diagnostic and Statistical Manual of Mental Disorders forth edition-Text Revision (DSM-I...

  5. Anxiety and depression in parents of a Brazilian non-clinical sample of attention-deficit/ hyperactivity disorder (ADHD) students

    OpenAIRE

    D. Segenreich; D. Fortes; G. Coutinho; G. Pastura; Mattos, P

    2009-01-01

    Higher prevalence rates of anxiety and depression have been reported in parents of children with attention-deficit/hyperactivity disorder (ADHD). The interaction between the burden of ADHD in offspring, a higher prevalence rate of this highly inherited disorder in parents, and comorbidities may explain this finding. Our objective was to investigate levels of ADHD, anxious and depressive symptomatology, and their relationship in parents of ADHD children from a non-clinical sample using a dimen...

  6. Adult ADHD Symptoms and Five Factor Model Traits in a Clinical Sample: A Structural Equation Modeling Approach

    OpenAIRE

    Knouse, Laura E.; Traeger, Lara; O’Cleirigh, Conall; Safren, Steven A.

    2013-01-01

    Relationships among Attention-Deficit/Hyperactivity Disorder (ADHD) symptoms and adult personality traits have not been examined in larger clinically diagnosed samples. We collected multi-source ADHD symptom and self-report NEO Five-Factor Inventory (Costa & McCrae, 1992a) data from 117 adults with ADHD and tested symptom-trait associations using structural equation modeling. The final model fit the data. Inattention was positively associated with Neuroticism and negatively associated with Co...

  7. Functionalized graphene oxide for clinical glucose biosensing in urine and serum samples

    OpenAIRE

    Veerapandian M; Seo YT; Shin H; Yun K; Lee MH

    2012-01-01

    Murugan Veerapandian,1 Yeong-Tai Seo,2 Hyunkyung Shin,3 Kyusik Yun,1 Min-Ho Lee41Department of Bionanotechnology, Gachon University, Gyeonggi-Do, 2School of Electrical Engineering and Computer Science, Seoul National University, Seoul, 3Department of Mathematics and Information, Gachon University, Gyeonggi-Do, 4Korea Electronics Technology Institute, Medical IT Technology, Gyeonggi-Do, Republic of KoreaAbstract: A novel clinical glucose biosensor fabricated using functionalized metalloid-poly...

  8. Simultaneous Detection of Four Human Pathogenic Microsporidian Species from Clinical Samples by Oligonucleotide Microarray

    OpenAIRE

    Wang, Zheng; Orlandi, Palmer A.; David A Stenger

    2005-01-01

    Microsporidian species have been rapidly emerging as human enteric pathogens in immunocompromised and immunocompetent individuals in recent years. Routine diagnostic techniques for microsporidia in clinical laboratories are laborious and insensitive and tend to underestimate their presence. In most instances, they are unable to differentiate species of spores due to their small sizes and similar morphologies. In this study, we report the development of another protozoan oligonucleotide microa...

  9. Evolution and Diversity of Listeria monocytogenes from Clinical and Food Samples in Shanghai, China.

    Science.gov (United States)

    Zhang, Jianmin; Cao, Guojie; Xu, Xuebin; Allard, Marc; Li, Peng; Brown, Eric; Yang, Xiaowei; Pan, Haijian; Meng, Jianghong

    2016-01-01

    Listeria monocytogenes is a significant foodborne pathogen causing severe systemic infections in humans with high mortality rates. The objectives of this work were to establish a phylogenetic framework of L. monocytogenes from China and to investigate sequence diversity among different serotypes. We selected 17 L. monocytogenes strains recovered from patients and foods in China representing serotypes 1/2a, 1/2b, and 1/2c. Draft genome sequences were determined using Illumina MiSeq technique and associated protocols. Open reading frames were assigned using prokaryotic genome annotation pipeline by NCBI. Twenty-four published genomes were included for comparative genomic and phylogenetic analysis. More than 154,000 single nucleotide polymorphisms (SNPs) were identified from multiple genome alignment and used to reconstruct maximum likelihood phylogenetic tree. The 41 genomes were differentiated into lineages I and II, which consisted of 4 and 11 subgroups, respectively. A clinical strain from China (SHL009) contained significant SNP differences compared to the rest genomes, whereas clinical strain SHL001 shared most recent common ancestor with strain SHL017 from food. Moreover, clinical strains SHL004 and SHL015 clustered together with two strains (08-5578 and 08-5923) recovered from an outbreak in Canada. Partial sequences of a plasmid found in the Canadian strain were also present in SHL004. We investigated the presence of various genes and gene clusters associated with virulence and subgroup-specific genes, including internalins, L. monocytogenes pathogenicity islands (LIPIs), L. monocytogenes genomic islands (LGIs), stress survival islet 1 (SSI-1), and clustered regularly interspaced short palindromic repeats (CRISPR)/cas system. A novel genomic island, denoted as LGI-2 was identified. Comparative sequence analysis revealed differences among the L. monocytogenes strains related to virulence, survival abilities, and attributes against foreign genetic elements. L

  10. Clinical Diagnosis by Whole-Genome Sequencing of a Prenatal Sample

    OpenAIRE

    Talkowski, Michael E.; Ordulu, Zehra; Pillalamarri, Vamsee; Benson, Carol B.; Blumenthal, Ian; Connolly, Susan; Hanscom, Carrie; Hussain, Naveed; Pereira, Shahrin; Picker, Jonathan; Rosenfeld, Jill A.; Shaffer, Lisa G.; Wilkins-Haug, Louise E.; Gusella, James F.; Morton, Cynthia C.

    2012-01-01

    Conventional cytogenetic testing offers low-resolution detection of balanced karyotypic abnormalities but cannot provide the precise, gene-level knowledge required to predict outcomes. The use of high-resolution whole-genome deep sequencing is currently impractical for the purpose of routine clinical care. We show here that whole-genome “jumping libraries” can offer an immediately applicable, nucleotide-level complement to conventional genetic diagnostics within a time frame that allows for c...

  11. Tales from the crypt and coral reef: the successes and challenges of identifying new herpesviruses using metagenomics

    Directory of Open Access Journals (Sweden)

    Charlotte Jane Houldcroft

    2015-03-01

    Full Text Available Herpesviruses are ubiquitous double-stranded DNA viruses infecting many animals, with the capacity to cause disease in both immunocompetent and immunocompromised hosts. Different herpesviruses have different cell tropisms, and have been detected in a diverse range of tissues and sample types.Metagenomics – encompassing viromics – analyses the nucleic acid of a tissue or other sample in an unbiased manner, making few or no prior assumptions about which viruses may be present in a sample. This approach has successfully discovered a number of novel herpesviruses. Furthermore, metagenomic analysis can identify herpesviruses with high degrees of sequence divergence from known herpesviruses and does not rely upon culturing large quantities of viral material.Metagenomics has had success in two areas of herpesvirus sequencing: firstly, the discovery of novel exogenous and endogenous herpesviruses in primates, bats and cnidarians; and secondly, in characterising large areas of the genomes of herpesviruses previously only known from small fragments, revealing unexpected diversity. This review will discuss the successes and challenges of using metagenomics to identify novel herpesviruses, and future directions within the field.

  12. Genetic diversity of Cryptosporidium identified in clinical samples from cities in Brazil and Argentina

    Science.gov (United States)

    Peralta, Regina Helena Saramago; Velásquez, Jorge Néstor; Cunha, Flavia de Souza; Pantano, María Laura; Sodré, Fernando Campos; da Silva, Sidnei; Astudillo, Osvaldo Germán; Peralta, José Mauro; Carnevale, Silvana

    2016-01-01

    The identification and characterisation of Cryptosporidiumgenotypes and subtypes are fundamental to the study of cryptosporidiosis epidemiology, aiding in prevention and control strategies. The objective was to determine the genetic diversity ofCryptosporidium in samples obtained from hospitals of Rio de Janeiro, Brazil, and Buenos Aires, Argentina. Samples were analysed by microscopy and TaqMan polymerase chain reaction (PCR) assays forCryptosporidium detection, genotyped by nested-PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene and subtyped by DNA sequencing of the gp60 gene. Among the 89 samples from Rio de Janeiro, Cryptosporidium spp were detected in 26 by microscopy/TaqMan PCR. In samples from Buenos Aires,Cryptosporidium was diagnosed in 15 patients of the 132 studied. The TaqMan PCR and the nested-PCR-RFLP detected Cryptosporidium parvum, Cryptosporidium hominis, and co-infections of both species. In Brazilian samples, the subtypes IbA10G2 and IIcA5G3 were observed. The subtypes found in Argentinean samples were IbA10G2, IaA10G1R4, IaA11G1R4, and IeA11G3T3, and mixed subtypes of Ia and IIa families were detected in the co-infections. C. hominis was the species more frequently detected, and subtype family Ib was reported in both countries. Subtype diversity was higher in Buenos Aires than in Rio de Janeiro and two new subtypes were described for the first time. PMID:26814641

  13. Conceptual Model of Clinical Governance Information System for Statistical Indicators by Using UML in Two Sample Hospitals

    Science.gov (United States)

    Jeddi, Fatemeh Rangraz; Farzandipoor, Mehrdad; Arabfard, Masoud; Hosseini, Azam Haj Mohammad

    2016-01-01

    Objective: The purpose of this study was investigating situation and presenting a conceptual model for clinical governance information system by using UML in two sample hospitals. Background: However, use of information is one of the fundamental components of clinical governance; but unfortunately, it does not pay much attention to information management. Material and Methods: A cross sectional study was conducted in October 2012- May 2013. Data were gathered through questionnaires and interviews in two sample hospitals. Face and content validity of the questionnaire has been confirmed by experts. Data were collected from a pilot hospital and reforms were carried out and Final questionnaire was prepared. Data were analyzed by descriptive statistics and SPSS 16 software. Results: With the scenario derived from questionnaires, UML diagrams are presented by using Rational Rose 7 software. The results showed that 32.14 percent Indicators of the hospitals were calculated. Database was not designed and 100 percent of the hospital’s clinical governance was required to create a database. Conclusion: Clinical governance unit of hospitals to perform its mission, do not have access to all the needed indicators. Defining of Processes and drawing of models and creating of database are essential for designing of information systems. PMID:27147804

  14. Detection of Rare Drug Resistance Mutations by Digital PCR in a Human Influenza A Virus Model System and Clinical Samples.

    Science.gov (United States)

    Whale, Alexandra S; Bushell, Claire A; Grant, Paul R; Cowen, Simon; Gutierrez-Aguirre, Ion; O'Sullivan, Denise M; Žel, Jana; Milavec, Mojca; Foy, Carole A; Nastouli, Eleni; Garson, Jeremy A; Huggett, Jim F

    2016-02-01

    Digital PCR (dPCR) is being increasingly used for the quantification of sequence variations, including single nucleotide polymorphisms (SNPs), due to its high accuracy and precision in comparison with techniques such as quantitative PCR (qPCR) and melt curve analysis. To develop and evaluate dPCR for SNP detection using DNA, RNA, and clinical samples, an influenza virus model of resistance to oseltamivir (Tamiflu) was used. First, this study was able to recognize and reduce off-target amplification in dPCR quantification, thereby enabling technical sensitivities down to 0.1% SNP abundance at a range of template concentrations, a 50-fold improvement on the qPCR assay used routinely in the clinic. Second, a method was developed for determining the false-positive rate (background) signal. Finally, comparison of dPCR with qPCR results on clinical samples demonstrated the potential impact dPCR could have on clinical research and patient management by earlier (trace) detection of rare drug-resistant sequence variants. Ultimately this could reduce the quantity of ineffective drugs taken and facilitate early switching to alternative medication when available. In the short term such methods could advance our understanding of microbial dynamics and therapeutic responses in a range of infectious diseases such as HIV, viral hepatitis, and tuberculosis. Furthermore, the findings presented here are directly relevant to other diagnostic areas, such as the detection of rare SNPs in malignancy, monitoring of graft rejection, and fetal screening. PMID:26659206

  15. Survey and visual detection of Zaire ebolavirus in clinical samples targeting the nucleoprotein gene in Sierra Leone

    Directory of Open Access Journals (Sweden)

    Jing eYuan

    2015-12-01

    Full Text Available Ebola virus (EBOV can lead to severe hemorrhagic fever with a high risk of death in humans and other primates. To guide treatment and prevent spread of the viral infection, a rapid and sensitive detection method is required for clinical samples. Here, we described and evaluated a reverse transcription loop-mediated isothermal amplification (RT-LAMP method to detect Zaire ebolavirus using the nucleoprotein gene (NP as a target sequence. Two different techniques were used, a calcein/Mn2+ complex chromogenic method and real-time turbidity monitoring. The RT-LAMP assay detected the NP target sequence with a limit of 4.56 copies/μL within 45 min under 61°C, a similar even or increase in sensitivity than that of real-time reverse transcription-polymerase chain reaction (RT-PCR. Additionally, all pseudoviral particles or non- Zaire EBOV genomes were negative for LAMP detection, indicating that the assay was highly specific for EBOV. To appraise the availability of the RT-LAMP method for use in clinical diagnosis of EBOV, of 417 blood or swab samples collected from patients with clinically suspected infections in Sierra Leone, 307 were identified for RT-LAMP-based surveillance of EBOV. Therefore, the highly specific and sensitive RT-LAMP method allows the rapid detection of EBOV, and is a suitable tool for clinical screening, diagnosis, and primary quarantine purposes.

  16. Inflammatory cytokine concentrations in uterine flush and serum samples from dairy cows with clinical or subclinical endometritis.

    Science.gov (United States)

    Kim, Ill-Hwa; Kang, Hyun-Gu; Jeong, Jae-Kwan; Hur, Tai-Young; Jung, Young-Hun

    2014-08-01

    The objective of this study was to compare the concentrations of inflammatory cytokines in uterine flush and serum from healthy postpartum dairy cows and cows with clinical or subclinical endometritis. Clinical endometritis was diagnosed by observation of vaginal discharges (>50% pus) and subclinical endometritis was diagnosed by evaluation of uterine cytology (neutrophils >18%) at 4 weeks postpartum. Uterine flush was obtained from 48 cows at 4, 6, and 8 weeks postpartum for evaluation of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, IL-8, and IL-10 concentrations. Serum samples were obtained from 34 cows just after calving and at 1, 2, 4, 6, and 8 weeks postpartum for evaluation of TNF-α, IL-1β, and IL-6 concentrations. Concentrations of TNF-α, IL-6, and IL-10 were greater (P < 0.05) in cows with clinical endometritis than in cows with subclinical endometritis and healthy controls, whereas concentrations of IL-8 in both cows with clinical and subclinical endometritis were greater (P < 0.005) than in controls. Overall, IL-6 and IL-10 concentrations decreased during the postpartum period. IL-1β concentrations in cows with clinical endometritis decreased (P < 0.0005) during the postpartum, whereas concentrations in cows with subclinical endometritis and controls did not change significantly with time; at 4 weeks postpartum, concentrations were greater (P < 0.0001) in cows with clinical endometritis. There were no significant effects of group, sampling time, or interaction on serum cytokine concentrations. In conclusion, cows with endometritis have greater inflammatory cytokine concentrations in uterine flush than healthy cows, but no differences were observed in serum. PMID:24933095

  17. Comorbidity of psychiatric disorders with Internet addiction in a clinical sample: the effect of personality, defense style and psychopathology.

    Science.gov (United States)

    Floros, Georgios; Siomos, Konstantinos; Stogiannidou, Ariadni; Giouzepas, Ioannis; Garyfallos, Georgios

    2014-12-01

    This study aims to contribute to the understanding of underlying causes for the development of Internet Addiction Disorder (IAD) and assess comorbidity with other mental disorders through the analysis of data from a clinical sample of college students who presented for treatment of IAD. The clinical sample of our study has demonstrated a high percentage of comorbidity with Axis I and II disorders, while the temporal precedence of the establishment of those disorders cannot lead to specific conclusions. Half of the sample (25/50) presented with comorbidity of another Axis I disorder and 38% (19/50) with a concurrent Axis II personality disorder. The majority of Axis I disorders (51.85%) were reported before the onset of IAD, 33.3% after the onset while it was unclear in 14.81% of cases. The examination of a path model demonstrated that important contributions to the understanding of this disorder can be made through concepts from the neurobiological, trait personality paradigm, as well as from the psychodynamic defense style paradigm. Comorbid psychopathology can further exacerbate the presentation of IAD through a direct link, regardless of the underlying personality structure. The clinician treating IAD patients should complete a clinical evaluation for comorbid Axis I and II diagnoses since their presence may signify a more serious presentation.

  18. Molecular method for the characterization of Coxiella burnetii from clinical and environmental samples: variability of genotypes in Spain

    Directory of Open Access Journals (Sweden)

    Jado Isabel

    2012-06-01

    Full Text Available Abstract Background Coxiella burnetii is a highly clonal microorganism which is difficult to culture, requiring BSL3 conditions for its propagation. This leads to a scarce availability of isolates worldwide. On the other hand, published methods of characterization have delineated up to 8 different genomic groups and 36 genotypes. However, all these methodologies, with the exception of one that exhibited limited discriminatory power (3 genotypes, rely on performing between 10 and 20 PCR amplifications or sequencing long fragments of DNA, which make their direct application to clinical samples impracticable and leads to a scarce accessibility of data on the circulation of C. burnetii genotypes. Results To assess the variability of this organism in Spain, we have developed a novel method that consists of a multiplex (8 targets PCR and hybridization with specific probes that reproduce the previous classification of this organism into 8 genomic groups, and up to 16 genotypes. It allows for a direct characterization from clinical and environmental samples in a single run, which will help in the study of the different genotypes circulating in wild and domestic cycles as well as from sporadic human cases and outbreaks. The method has been validated with reference isolates. A high variability of C. burnetii has been found in Spain among 90 samples tested, detecting 10 different genotypes, being those adaA negative associated with acute Q fever cases presenting as fever of intermediate duration with liver involvement and with chronic cases. Genotypes infecting humans are also found in sheep, goats, rats, wild boar and ticks, and the only genotype found in cattle has never been found among our clinical samples. Conclusions This newly developed methodology has permitted to demonstrate that C. burnetii is highly variable in Spain. With the data presented here, cattle seem not to participate in the transmission of C. burnetii to humans in the samples studied

  19. Cross-cutting activities: Soil quality and soil metagenomics

    OpenAIRE

    Motavalli, Peter P.; Garrett, Karen A.

    2008-01-01

    This presentation reports on the work of the SANREM CRSP cross-cutting activities "Assessing and Managing Soil Quality for Sustainable Agricultural Systems" and "Soil Metagenomics to Construct Indicators of Soil Degradation." The introduction gives an overview of the extensiveness of soil degradation globally and defines soil quality. The objectives of the soil quality cross cutting activity are: CCRA-4 (Soil Metagenomics)

  20. Online semi-supervised learning: algorithm and application in metagenomics

    NARCIS (Netherlands)

    S. Imangaliyev; B. Keijser; W. Crielaard; E. Tsivtsivadze

    2013-01-01

    As the amount of metagenomic data grows rapidly, online statistical learning algorithms are poised to play key role in metagenome analysis tasks. Frequently, data are only partially labeled, namely dataset contains partial information about the problem of interest. This work presents an algorithm an

  1. Online Semi-Supervised Learning: Algorithm and Application in Metagenomics

    NARCIS (Netherlands)

    Imangaliyev, S.; Keijser, B.J.F.; Crielaard, W.; Tsivtsivadze, E.

    2013-01-01

    As the amount of metagenomic data grows rapidly, online statistical learning algorithms are poised to play key rolein metagenome analysis tasks. Frequently, data are only partially labeled, namely dataset contains partial information about the problem of interest. This work presents an algorithm and

  2. Recovering full-length viral genomes from metagenomes

    NARCIS (Netherlands)

    S.L. Smits (Saskia); R. Bodewes (Rogier); A. Ruiz-Gonzalez (Aritz); V. Baumgärtner (Volkmar); M.P.G. Koopmans D.V.M. (Marion); A.D.M.E. Osterhaus (Albert); A. Schürch (Anita)

    2015-01-01

    textabstractInfectious disease metagenomics is driven by the question: "what is causing the disease?" in contrast to classical metagenome studies which are guided by "what is out there?" In case of a novel virus, a first step to eventually establishing etiology can be to recover a full-length viral

  3. Glycemic Control in a Clinic-Based Sample of Diabetics in M'Bour Senegal

    Science.gov (United States)

    BeLue, Rhonda; Ndiaye, Khadidiatou; NDao, Fatou; Ba, Fatou Niass Niang; Diaw, Mor

    2016-01-01

    Background: Sub-Saharan Africa (SSA) including Senegal is faced with a significant and increasing burden of type 2 diabetes. However, little information is available about diabetes management among Senegalese diabetics. Purpose: The current study aims to describe the level of glycemic control among a convenience sample of diabetics who receive…

  4. A prospective survey of Aspergillus spp. in respiratory tract samples: prevalence, clinical impact and antifungal susceptibility

    DEFF Research Database (Denmark)

    Mortensen, K L; Johansen, H K; Fuursted, K;

    2011-01-01

    ) was evaluated using modified EORTC/MSG criteria. A total of 11,368 airway samples were received. Growth of Aspergillus spp. was found in 129 and 151 patients using routine and extended incubation, respectively. Three patients had proven IA (2%), 11 probable (7%), four had allergic bronchopulmonary aspergillosis...

  5. Usefulness of chorionic villus sampling for prenatal diagnosis of thalassaemia: a clinical study in eastern India

    Directory of Open Access Journals (Sweden)

    Sujoy Dasgupta

    2015-06-01

    Conclusions: Thalassaemia is a prevalent condition in Eastern India. Chorionic villus sampling is an effective and safe method for early diagnosis of fetal thalassaemia that helps to prevent birth of thalassaemic babies. [Int J Reprod Contracept Obstet Gynecol 2015; 4(3.000: 790-794

  6. Reliability and Validity of the Spanish Adaptation of EOSS, Comparing Normal and Clinical Samples

    Science.gov (United States)

    Valero-Aguayo, Luis; Ferro-Garcia, Rafael; Lopez-Bermudez, Miguel Angel; de Huralde, Ma. Angeles Selva-Lopez

    2012-01-01

    The Experiencing of Self Scale (EOSS) was created for the evaluation of Functional Analytic Psychotherapy (Kohlenberg & Tsai, 1991, 2001, 2008) in relation to the concept of the experience of personal self as socially and verbally constructed. This paper presents a reliability and validity study of the EOSS with a Spanish sample (582 participants,…

  7. Genotypic characterization, invasion index and antimicrobial resistance pattern in Listeria monocytogenes strains isolated from clinical samples

    Directory of Open Access Journals (Sweden)

    Behrooz Sadeghi Kalani

    2015-06-01

    Conclusions: Since MLVA is a high-throughput screening method that is fairly inexpensive, easy to accomplish, rapid, and trustworthy, it is well suited to interlaboratory comparisons during epidemiological investigations. Also further studies of larger samples from a variety of sources such as food and animal specimens recommended comparing by MLVA method.

  8. Moleculo long-read sequencing facilitates assembly and resolves functionally active genomic bins from complex soil metagenomes

    Energy Technology Data Exchange (ETDEWEB)

    White, Richard A.; Bottos, Eric M.; Roy Chowdhury, Taniya; Zucker, Jeremy D.; Brislawn, Colin J.; Nicora, Carrie D.; Fansler, Sarah J.; Glaesemann, Kurt R.; Glass, Kevin A.; Jansson, Janet K.

    2016-07-28

    Soil metagenomics has been touted as the "grand challenge" for metagenomes, as the high microbial diversity, sample complexity, and spatial heterogeneity of soils makes them unamenable to current sequencing and assembly platforms. Here we aimed to improve soil metagenomic sequence assembly by applying a synthetic long read sequencing technology (i.e. Moleculo) from three locations within Konza native prairie station in Kansas. In total, we obtained 520 GB of raw sequence data; 239 GB of short read data from the Joint Genome Institute (JGI), an additional 97 GB from Moleculo sequencing, plus 184 GB of rapid mode sequence data. The Moleculo data alone yielded over 5,600 reads greater than 10 kbp in length, mapping over 95% of the total sequence data. Hybrid assembly of all data resulted in more than 10,000 contigs over 10 kbp in length. The Moleculo sub-assemblies captured much of the functional potential of the soil community, in that 92% of the functional enzyme commission numbers (EC) predicted from the metagenome were also detected in metatranscriptome data. The Moleculo sub-assembly enabled binning of more than 100 novel soil microbial genomic bins. Candidatus Pseudomonas janssonensis strain KNPRW21, was the first genome obtained from a native soil metagenome by direct binning. By mapping RNA-Seq (i.e. metatranscriptomic) sequence reads back to the bins, we found that several low abundance Acidobacteria bins were highly transcriptionally active, whereas the highly abundant Verruomicrobia bins were not. Using Moleculo long reads alone or combined with conventional short read metagenomic data is therefore a useful tool for resolving complex soil microbial communities.

  9. Metagenomics-Guided Mining of Commercially Useful Biocatalysts from Marine Microorganisms.

    Science.gov (United States)

    Uria, A R; Zilda, D S

    2016-01-01

    Marine microorganisms are a rich reservoir of highly diverse and unique biocatalysts that offer potential applications in food, pharmaceutical, fuel, and cosmetic industries. The fact that only less than 1% of microbes in any marine habitats can be cultured under standard laboratory conditions has hampered access to their extraordinary biocatalytic potential. Metagenomics has recently emerged as a powerful and well-established tool to investigate the vast majority of hidden uncultured microbial diversity for the discovery of novel industrially relevant enzymes from different types of environmental samples, such as seawater, marine sediment, and symbiotic microbial consortia. We discuss here in this review about approaches and methods in metagenomics that have been used and can potentially be used to mine commercially useful biocatalysts from uncultured marine microbes. PMID:27452163

  10. The role of metagenomics in understanding the human microbiome in health and disease.

    Science.gov (United States)

    Martín, Rebeca; Miquel, Sylvie; Langella, Philippe; Bermúdez-Humarán, Luis G

    2014-04-01

    The term microbiome refers to the genetic material of the catalog of microbial taxa associated with humans. As in all ecosystems, the microbiota reaches a dynamic equilibrium in the human body, which can be altered by environmental factors and external stimuli. Metagenomics is a relatively new field of study of microbial genomes within diverse environmental samples, which is of increasing importance in microbiology. The introduction of this ecological perception of microbiology is the key to achieving real knowledge about the influence of the microbiota in human health and disease. The aim of this review is to summarize the link between the human microbiota (focusing on the intestinal, vaginal, skin, and airway body sites) and health from this ecological point of view, highlighting the contribution of metagenomics in the advance of this field.

  11. Use of metagenomic approaches to isolate lipolytic genes from activated sludge.

    Science.gov (United States)

    Liaw, Ren-Bao; Cheng, Mei-Ping; Wu, Ming-Che; Lee, Chia-Yin

    2010-11-01

    The aims of this study were to access the bacterial diversity and isolate lipolytic genes using the metagenomic approach in activated sludge of a swine wastewater treatment facility. On the basis of BLASTN analysis of 16S rRNA gene clones, most of these communities (90%) were of uncultivated bacteria. The metagenomic library was constructed using a plasmid vector and DNA extracted directly from activated sludge samples. The average insert size was approximately 5.1 kb. A total of 12 unique and lipolytic clones were obtained using the tributyrin plate assay. The rate of discovering a lipolytic clone in this study was as high as 0.31%. Molecular analysis revealed that most of the 16 putative lipolytic enzymes showed 28-55% identity with non-redundant protein sequences in the database. Briefly, this study demonstrates that activated sludge is an ideal bioresource for isolating new lipolytic enzymes. PMID:20639117

  12. Evaluating the mobility potential of antibiotic resistance genes in environmental resistomes without metagenomics

    Science.gov (United States)

    Pärnänen, Katariina; Karkman, Antti; Tamminen, Manu; Lyra, Christina; Hultman, Jenni; Paulin, Lars; Virta, Marko

    2016-01-01

    Antibiotic resistance genes are ubiquitous in the environment. However, only a fraction of them are mobile and able to spread to pathogenic bacteria. Until now, studying the mobility of antibiotic resistance genes in environmental resistomes has been challenging due to inadequate sensitivity and difficulties in contig assembly of metagenome based methods. We developed a new cost and labor efficient method based on Inverse PCR and long read sequencing for studying mobility potential of environmental resistance genes. We applied Inverse PCR on sediment samples and identified 79 different MGE clusters associated with the studied resistance genes, including novel mobile genetic elements, co-selected resistance genes and a new putative antibiotic resistance gene. The results show that the method can be used in antibiotic resistance early warning systems. In comparison to metagenomics, Inverse PCR was markedly more sensitive and provided more data on resistance gene mobility and co-selected resistances. PMID:27767072

  13. Metagenomics-Guided Mining of Commercially Useful Biocatalysts from Marine Microorganisms.

    Science.gov (United States)

    Uria, A R; Zilda, D S

    2016-01-01

    Marine microorganisms are a rich reservoir of highly diverse and unique biocatalysts that offer potential applications in food, pharmaceutical, fuel, and cosmetic industries. The fact that only less than 1% of microbes in any marine habitats can be cultured under standard laboratory conditions has hampered access to their extraordinary biocatalytic potential. Metagenomics has recently emerged as a powerful and well-established tool to investigate the vast majority of hidden uncultured microbial diversity for the discovery of novel industrially relevant enzymes from different types of environmental samples, such as seawater, marine sediment, and symbiotic microbial consortia. We discuss here in this review about approaches and methods in metagenomics that have been used and can potentially be used to mine commercially useful biocatalysts from uncultured marine microbes.

  14. Survey and rapid detection of Klebsiella pneumoniae in clinical samples targeting the rcsA gene in Beijing, China

    Directory of Open Access Journals (Sweden)

    Derong eDong

    2015-05-01

    Full Text Available Klebsiella pneumoniae is a wide-spread nosocomial pathogen. A rapid and sensitive molecular method for the detection of K. pneumoniae in clinical samples is needed to guide therapeutic treatment. In this study, we first described a loop-mediated isothermal amplification (LAMP method for the rapid detection of capsular polysaccharide synthesis regulating gene rcsA from K. pneumoniae in clinical samples by using two methods including real-time turbidity monitoring and fluorescence detection to assess the reaction. Then dissemination of K. pneumoniae strains was investigated from ICU patients in three top hospitals in Beijing, China. The results showed that the detection limit of the LAMP method was 0.115 pg/µl DNA within 60 min under isothermal conditions (61°C, a 100-fold increase in sensitivity compared with conventional PCR. All 30 non- K. pneumoniae strains tested were negative for LAMP detection, indicating the high specificity of the LAMP reaction. To evaluate the application of the LAMP assay to clinical diagnosis, of 110 clinical sputum samples collected from ICU patients with clinically suspected multi-resistant infections in China, a total of 32 K. pneumoniae isolates were identified for LAMP-based surveillance of rcsA. All isolates belonged to nine different K. pneumoniae multilocus sequence typing (MLST groups. Strikingly, of the 32 K. pneumoniae strains, 18 contained the Klebsiella pneumoniae Carbapenemase (KPC-encoding gene blaKPC-2 and had high resistance to β-lactam antibiotics. Moreover, K. pneumoniae WJ-64 was discovered to contain blaKPC-2 and blaNDM-1 genes simultaneously in the isolate. Our data showed the high prevalence of blaKPC-2 among K. pneumoniae and co-occurrence of many resistant genes in the clinical strains signal a rapid and continuing evolution of K. pneumoniae. In conclusion, we have developed a rapid and sensitive visual K. pneumoniae detection LAMP assay, which could be a useful tool for clinical screening

  15. Survey and rapid detection of Klebsiella pneumoniae in clinical samples targeting the rcsA gene in Beijing, China.

    Science.gov (United States)

    Dong, Derong; Liu, Wei; Li, Huan; Wang, Yufei; Li, Xinran; Zou, Dayang; Yang, Zhan; Huang, Simo; Zhou, Dongsheng; Huang, Liuyu; Yuan, Jing

    2015-01-01

    Klebsiella pneumoniae is a wide-spread nosocomial pathogen. A rapid and sensitive molecular method for the detection of K. pneumoniae in clinical samples is needed to guide therapeutic treatment. In this study, we first described a loop-mediated isothermal amplification (LAMP) method for the rapid detection of capsular polysaccharide synthesis regulating gene rcsA from K. pneumoniaein clinical samples by using two methods including real-time turbidity monitoring and fluorescence detection to assess the reaction. Then dissemination of K. pneumoniae strains was investigated from ICU patients in three top hospitals in Beijing, China. The results showed that the detection limit of the LAMP method was 0.115 pg/μl DNA within 60 min under isothermal conditions (61°C), a 100-fold increase in sensitivity compared with conventional PCR. All 30 non- K. pneumoniae strains tested were negative for LAMP detection, indicating the high specificity of the LAMP reaction. To evaluate the application of the LAMP assay to clinical diagnosis, of 110 clinical sputum samples collected from ICU patients with clinically suspected multi-resistant infections in China, a total of 32 K. pneumoniae isolates were identified for LAMP-based surveillance of rcsA. All isolates belonged to nine different K. pneumoniae multilocus sequence typing (MLST) groups. Strikingly, of the 32 K. pneumoniae strains, 18 contained the Klebsiella pneumoniae Carbapenemase (KPC)-encoding gene bla KPC-2 and had high resistance to β-lactam antibiotics. Moreover, K. pneumoniae WJ-64 was discovered to contain bla KPC-2 and bla NDM-1genes simultaneously in the isolate. Our data showed the high prevalence of bla KPC-2 among K. pneumoniae and co-occurrence of many resistant genes in the clinical strains signal a rapid and continuing evolution of K. pneumoniae. In conclusion, we have developed a rapid and sensitive visual K. pneumoniae detection LAMP assay, which could be a useful tool for clinical screening, on

  16. The Characterization of Novel Tissue Microbiota Using an Optimized 16S Metagenomic Sequencing Pipeline.

    Directory of Open Access Journals (Sweden)

    Jérôme Lluch

    Full Text Available Substantial progress in high-throughput metagenomic sequencing methodologies has enabled the characterisation of bacteria from various origins (for example gut and skin. However, the recently-discovered bacterial microbiota present within animal internal tissues has remained unexplored due to technical difficulties associated with these challenging samples.We have optimized a specific 16S rDNA-targeted metagenomics sequencing (16S metabarcoding pipeline based on the Illumina MiSeq technology for the analysis of bacterial DNA in human and animal tissues. This was successfully achieved in various mouse tissues despite the high abundance of eukaryotic DNA and PCR inhibitors in these samples. We extensively tested this pipeline on mock communities, negative controls, positive controls and tissues and demonstrated the presence of novel tissue specific bacterial DNA profiles in a variety of organs (including brain, muscle, adipose tissue, liver and heart.The high throughput and excellent reproducibility of the method ensured exhaustive and precise coverage of the 16S rDNA bacterial variants present in mouse tissues. This optimized 16S metagenomic sequencing pipeline will allow the scientific community to catalogue the bacterial DNA profiles of different tissues and will provide a database to analyse host/bacterial interactions in relation to homeostasis and disease.

  17. Metagenomics and Bioinformatics in Microbial Ecology: Current Status and Beyond.

    Science.gov (United States)

    Hiraoka, Satoshi; Yang, Ching-Chia; Iwasaki, Wataru

    2016-09-29

    Metagenomic approaches are now commonly used in microbial ecology to study microbial communities in more detail, including many strains that cannot be cultivated in the laboratory. Bioinformatic analyses make it possible to mine huge metagenomic datasets and discover general patterns that govern microbial ecosystems. However, the findings of typical metagenomic and bioinformatic analyses still do not completely describe the ecology and evolution of microbes in their environments. Most analyses still depend on straightforward sequence similarity searches against reference databases. We herein review the current state of metagenomics and bioinformatics in microbial ecology and discuss future directions for the field. New techniques will allow us to go beyond routine analyses and broaden our knowledge of microbial ecosystems. We need to enrich reference databases, promote platforms that enable meta- or comprehensive analyses of diverse metagenomic datasets, devise methods that utilize long-read sequence information, and develop more powerful bioinformatic methods to analyze data from diverse perspectives. PMID:27383682

  18. Metagenomics and Bioinformatics in Microbial Ecology: Current Status and Beyond

    Science.gov (United States)

    Hiraoka, Satoshi; Yang, Ching-chia; Iwasaki, Wataru

    2016-01-01

    Metagenomic approaches are now commonly used in microbial ecology to study microbial communities in more detail, including many strains that cannot be cultivated in the laboratory. Bioinformatic analyses make it possible to mine huge metagenomic datasets and discover general patterns that govern microbial ecosystems. However, the findings of typical metagenomic and bioinformatic analyses still do not completely describe the ecology and evolution of microbes in their environments. Most analyses still depend on straightforward sequence similarity searches against reference databases. We herein review the current state of metagenomics and bioinformatics in microbial ecology and discuss future directions for the field. New techniques will allow us to go beyond routine analyses and broaden our knowledge of microbial ecosystems. We need to enrich reference databases, promote platforms that enable meta- or comprehensive analyses of diverse metagenomic datasets, devise methods that utilize long-read sequence information, and develop more powerful bioinformatic methods to analyze data from diverse perspectives. PMID:27383682

  19. Assessment of metagenomic assembly using simulated next generation sequencing data

    DEFF Research Database (Denmark)

    Mende, Daniel R; Waller, Alison S; Sunagawa, Shinichi;

    2012-01-01

    Due to the complexity of the protocols and a limited knowledge of the nature of microbial communities, simulating metagenomic sequences plays an important role in testing the performance of existing tools and data analysis methods with metagenomic data. We developed metagenomic read simulators...... with platform-specific (Sanger, pyrosequencing, Illumina) base-error models, and simulated metagenomes of differing community complexities. We first evaluated the effect of rigorous quality control on Illumina data. Although quality filtering removed a large proportion of the data, it greatly improved....... Although the increase in contig length was accompanied by increased chimericity, it resulted in more complete genes and a better characterization of the functional repertoire. The metagenomic simulators developed for this research are freely available....

  20. Metagenomics: Retrospect and Prospects in High Throughput Age

    Directory of Open Access Journals (Sweden)

    Satish Kumar

    2015-01-01

    Full Text Available In recent years, metagenomics has emerged as a powerful tool for mining of hidden microbial treasure in a culture independent manner. In the last two decades, metagenomics has been applied extensively to exploit concealed potential of microbial communities from almost all sorts of habitats. A brief historic progress made over the period is discussed in terms of origin of metagenomics to its current state and also the discovery of novel biological functions of commercial importance from metagenomes of diverse habitats. The present review also highlights the paradigm shift of metagenomics from basic study of community composition to insight into the microbial community dynamics for harnessing the full potential of uncultured microbes with more emphasis on the implication of breakthrough developments, namely, Next Generation Sequencing, advanced bioinformatics tools, and systems biology.