A novel salt-tolerant chitobiosidase discovered by genetic screening of a metagenomic library derived from chitin-amended agricultural soil

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Cretoiu, Mariana Silvia; Berini, Francesca; Kielak, Anna Maria; Marinelli, Flavia; van Elsas, Jan Dirk

2015-01-01

Here, we report on the construction of a metagenomic library from a chitin-amended disease-suppressive agricultural soil and its screening for genes that encode novel chitinolytic enzymes. The library, constructed in fosmids in an Escherichia coli host, comprised 145,000 clones containing inserts of

Functional Screening of Antibiotic Resistance Genes from a Representative Metagenomic Library of Food Fermenting Microbiota

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Chiara Devirgiliis

2014-01-01

Full Text Available Lactic acid bacteria (LAB represent the predominant microbiota in fermented foods. Foodborne LAB have received increasing attention as potential reservoir of antibiotic resistance (AR determinants, which may be horizontally transferred to opportunistic pathogens. We have previously reported isolation of AR LAB from the raw ingredients of a fermented cheese, while AR genes could be detected in the final, marketed product only by PCR amplification, thus pointing at the need for more sensitive microbial isolation techniques. We turned therefore to construction of a metagenomic library containing microbial DNA extracted directly from the food matrix. To maximize yield and purity and to ensure that genomic complexity of the library was representative of the original bacterial population, we defined a suitable protocol for total DNA extraction from cheese which can also be applied to other lipid-rich foods. Functional library screening on different antibiotics allowed recovery of ampicillin and kanamycin resistant clones originating from Streptococcus salivarius subsp. thermophilus and Lactobacillus helveticus genomes. We report molecular characterization of the cloned inserts, which were fully sequenced and shown to confer AR phenotype to recipient bacteria. We also show that metagenomics can be applied to food microbiota to identify underrepresented species carrying specific genes of interest.

Identification of aminoglycoside and β-lactam resistance genes from within an infant gut functional metagenomic library.

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Fiona Fouhy

Full Text Available The infant gut microbiota develops rapidly during the first 2 years of life, acquiring microorganisms from diverse sources. During this time, significant opportunities exist for the infant to acquire antibiotic resistant bacteria, which can become established and constitute the infant gut resistome. With increased antibiotic resistance limiting our ability to treat bacterial infections, investigations into resistance reservoirs are highly pertinent. This study aimed to explore the nascent resistome in antibiotically-naïve infant gut microbiomes, using a combination of metagenomic approaches. Faecal samples from 22 six-month-old infants without previous antibiotic exposure were used to construct a pooled metagenomic library, which was functionally screened for ampicillin and gentamicin resistance. Our library of ∼220Mb contained 0.45 ampicillin resistant hits/Mb and 0.059 gentamicin resistant hits/Mb. PCR-based analysis of fosmid clones and uncloned metagenomic DNA, revealed a diverse and abundant aminoglycoside and β-lactam resistance reservoir within the infant gut, with resistance determinants exhibiting homology to those found in common gut inhabitants, including Escherichia coli, Enterococcus sp., and Clostridium difficile, as well as to genes from cryptic environmental bacteria. Notably, the genes identified differed from those revealed when a sequence-driven PCR-based screen of metagenomic DNA was employed. Carriage of these antibiotic resistance determinants conferred substantial, but varied (2-512x, increases in antibiotic resistance to their bacterial host. These data provide insights into the infant gut resistome, revealing the presence of a varied aminoglycoside and β-lactam resistance reservoir even in the absence of selective pressure, confirming the infant resistome establishes early in life, perhaps even at birth.

Est16, a New Esterase Isolated from a Metagenomic Library of a Microbial Consortium Specializing in Diesel Oil Degradation.

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Pereira, Mariana Rangel; Mercaldi, Gustavo Fernando; Maester, Thaís Carvalho; Balan, Andrea; Lemos, Eliana Gertrudes de Macedo

2015-01-01

Lipolytic enzymes have attracted attention from a global market because they show enormous biotechnological potential for applications such as detergent production, leather processing, cosmetics production, and use in perfumes and biodiesel. Due to the intense demand for biocatalysts, a metagenomic approach provides methods of identifying new enzymes. In this study, an esterase designated as Est16 was selected from 4224 clones of a fosmid metagenomic library, revealing an 87% amino acid identity with an esterase/lipase (accession number ADM63076.1) from an uncultured bacterium. Phylogenetic studies showed that the enzyme belongs to family V of bacterial lipolytic enzymes and has sequence and structural similarities with an aryl-esterase from Pseudomonas fluorescens and a patented Anti-Kazlauskas lipase (patent number US20050153404). The protein was expressed and purified as a highly soluble, thermally stable enzyme that showed a preference for basic pH. Est16 exhibited activity toward a wide range of substrates and the highest catalytic efficiency against p-nitrophenyl butyrate and p-nitrophenyl valerate. Est16 also showed tolerance to the presence of organic solvents, detergents and metals. Based on molecular modeling, we showed that the large alpha-beta domain is conserved in the patented enzymes but not the substrate pocket. Here, it was demonstrated that a metagenomic approach is suitable for discovering the lipolytic enzyme diversity and that Est16 has the biotechnological potential for use in industrial processes.

Est16, a New Esterase Isolated from a Metagenomic Library of a Microbial Consortium Specializing in Diesel Oil Degradation.

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Mariana Rangel Pereira

Full Text Available Lipolytic enzymes have attracted attention from a global market because they show enormous biotechnological potential for applications such as detergent production, leather processing, cosmetics production, and use in perfumes and biodiesel. Due to the intense demand for biocatalysts, a metagenomic approach provides methods of identifying new enzymes. In this study, an esterase designated as Est16 was selected from 4224 clones of a fosmid metagenomic library, revealing an 87% amino acid identity with an esterase/lipase (accession number ADM63076.1 from an uncultured bacterium. Phylogenetic studies showed that the enzyme belongs to family V of bacterial lipolytic enzymes and has sequence and structural similarities with an aryl-esterase from Pseudomonas fluorescens and a patented Anti-Kazlauskas lipase (patent number US20050153404. The protein was expressed and purified as a highly soluble, thermally stable enzyme that showed a preference for basic pH. Est16 exhibited activity toward a wide range of substrates and the highest catalytic efficiency against p-nitrophenyl butyrate and p-nitrophenyl valerate. Est16 also showed tolerance to the presence of organic solvents, detergents and metals. Based on molecular modeling, we showed that the large alpha-beta domain is conserved in the patented enzymes but not the substrate pocket. Here, it was demonstrated that a metagenomic approach is suitable for discovering the lipolytic enzyme diversity and that Est16 has the biotechnological potential for use in industrial processes.

Bioproduction and characterization of extracellular melanin-like pigment from industrially polluted metagenomic library equipped Escherichia coli.

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Amin, Shivani; Rastogi, Rajesh P; Sonani, Ravi R; Ray, Arabinda; Sharma, Rakesh; Madamwar, Datta

2018-04-15

To explore the potential genes from the industrially polluted Amlakhadi canal, located in Ankleshwar, Gujarat, India, its community genome was extracted and cloned into E. coli EPI300™-T1 R using a fosmid vector (pCC2 FOS™) generating a library of 3,92,000 clones with average size of 40kb of DNA-insert. From this library, the clone DM1 producing brown colored melanin-like pigment was isolated and characterized. For over expression of the pigment, further sub-cloning of the clone DM1 was done. Sub-clone containing 10kb of the insert was sequenced for gene identification. The amino acids sequence of a protein 4-Hydroxyphenylpyruvate dioxygenase (HPPD), which is know to be involved in melanin biosynthesis was obtained from the gene sequence. The sequence-homology based 3D structure model of HPPD was constructed and analyzed. The physico-chemical nature of pigment was further analysed using 1 H and 13 C NMR, LC-MS, FTIR and UV-visible spectroscopy. The pigment was readily soluble in DMSO with an absorption maximum around 290nm. Based on the genetic and chemical characterization, the compound was confirmed as melanin-like pigment. The present results indicate that the metagenomic library from industrially polluted environment generated a microbial tool for the production of melanin-like pigment. Copyright © 2018 Elsevier B.V. All rights reserved.

Metagenomic approaches to exploit the biotechnological potential of the microbial consortia of marine sponges.

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Kennedy, Jonathan; Marchesi, Julian R; Dobson, Alan D W

2007-05-01

Natural products isolated from sponges are an important source of new biologically active compounds. However, the development of these compounds into drugs has been held back by the difficulties in achieving a sustainable supply of these often-complex molecules for pre-clinical and clinical development. Increasing evidence implicates microbial symbionts as the source of many of these biologically active compounds, but the vast majority of the sponge microbial community remain uncultured. Metagenomics offers a biotechnological solution to this supply problem. Metagenomes of sponge microbial communities have been shown to contain genes and gene clusters typical for the biosynthesis of biologically active natural products. Heterologous expression approaches have also led to the isolation of secondary metabolism gene clusters from uncultured microbial symbionts of marine invertebrates and from soil metagenomic libraries. Combining a metagenomic approach with heterologous expression holds much promise for the sustainable exploitation of the chemical diversity present in the sponge microbial community.

Mining the metagenome of activated biomass of an industrial wastewater treatment plant by a novel method.

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Sharma, Nandita; Tanksale, Himgouri; Kapley, Atya; Purohit, Hemant J

2012-12-01

Metagenomic libraries herald the era of magnifying the microbial world, tapping into the vast metabolic potential of uncultivated microbes, and enhancing the rate of discovery of novel genes and pathways. In this paper, we describe a method that facilitates the extraction of metagenomic DNA from activated sludge of an industrial wastewater treatment plant and its use in mining the metagenome via library construction. The efficiency of this method was demonstrated by the large representation of the bacterial genome in the constructed metagenomic libraries and by the functional clones obtained. The BAC library represented 95.6 times the bacterial genome, while, the pUC library represented 41.7 times the bacterial genome. Twelve clones in the BAC library demonstrated lipolytic activity, while four clones demonstrated dioxygenase activity. Four clones in pUC library tested positive for cellulase activity. This method, using FTA cards, not only can be used for library construction, but can also store the metagenome at room temperature.

Expanding the Repertoire of Carbapenem-Hydrolyzing Metallo-ß-Lactamases by Functional Metagenomic Analysis of Soil Microbiota

DEFF Research Database (Denmark)

Gudeta, Dereje D.; Bortolaia, Valeria; Pollini, Simona

2016-01-01

, diversity and functionality of carbapenemase-encoding genes in soil microbiota by functional metagenomics. Ten plasmid libraries were generated by cloning metagenomic DNA from agricultural (n = 6) and grassland (n = 4) soil into Escherichia coli. The libraries were cultured on amoxicillin-containing agar......% identity). RAIphy analysis indicated that six enzymes (CRD3-1, GRD23-1, DHT2-1, SPN79-1, ALG6-1, and ALG11-1) originated from Proteobacteria, two (PEDO-1 and ESP-2) from Bacteroidetes and one (GRD33-1) from Gemmatimonadetes. All MBLs detected in soil microbiota were functional when expressed in E. coli...... approaches targeted different subpopulations in soil microbiota....

Hybrid sequencing approach applied to human fecal metagenomic clone libraries revealed clones with potential biotechnological applications.

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Džunková, Mária; D'Auria, Giuseppe; Pérez-Villarroya, David; Moya, Andrés

2012-01-01

Natural environments represent an incredible source of microbial genetic diversity. Discovery of novel biomolecules involves biotechnological methods that often require the design and implementation of biochemical assays to screen clone libraries. However, when an assay is applied to thousands of clones, one may eventually end up with very few positive clones which, in most of the cases, have to be "domesticated" for downstream characterization and application, and this makes screening both laborious and expensive. The negative clones, which are not considered by the selected assay, may also have biotechnological potential; however, unfortunately they would remain unexplored. Knowledge of the clone sequences provides important clues about potential biotechnological application of the clones in the library; however, the sequencing of clones one-by-one would be very time-consuming and expensive. In this study, we characterized the first metagenomic clone library from the feces of a healthy human volunteer, using a method based on 454 pyrosequencing coupled with a clone-by-clone Sanger end-sequencing. Instead of whole individual clone sequencing, we sequenced 358 clones in a pool. The medium-large insert (7-15 kb) cloning strategy allowed us to assemble these clones correctly, and to assign the clone ends to maintain the link between the position of a living clone in the library and the annotated contig from the 454 assembly. Finally, we found several open reading frames (ORFs) with previously described potential medical application. The proposed approach allows planning ad-hoc biochemical assays for the clones of interest, and the appropriate sub-cloning strategy for gene expression in suitable vectors/hosts.

Metagenomics as a Tool for Enzyme Discovery: Hydrolytic Enzymes from Marine-Related Metagenomes.

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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.

Evaluation of a pooled strategy for high-throughput sequencing of cosmid clones from metagenomic libraries.

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Lam, Kathy N; Hall, Michael W; Engel, Katja; Vey, Gregory; Cheng, Jiujun; Neufeld, Josh D; Charles, Trevor C

2014-01-01

High-throughput sequencing methods have been instrumental in the growing field of metagenomics, with technological improvements enabling greater throughput at decreased costs. Nonetheless, the economy of high-throughput sequencing cannot be fully leveraged in the subdiscipline of functional metagenomics. In this area of research, environmental DNA is typically cloned to generate large-insert libraries from which individual clones are isolated, based on specific activities of interest. Sequence data are required for complete characterization of such clones, but the sequencing of a large set of clones requires individual barcode-based sample preparation; this can become costly, as the cost of clone barcoding scales linearly with the number of clones processed, and thus sequencing a large number of metagenomic clones often remains cost-prohibitive. We investigated a hybrid Sanger/Illumina pooled sequencing strategy that omits barcoding altogether, and we evaluated this strategy by comparing the pooled sequencing results to reference sequence data obtained from traditional barcode-based sequencing of the same set of clones. Using identity and coverage metrics in our evaluation, we show that pooled sequencing can generate high-quality sequence data, without producing problematic chimeras. Though caveats of a pooled strategy exist and further optimization of the method is required to improve recovery of complete clone sequences and to avoid circumstances that generate unrecoverable clone sequences, our results demonstrate that pooled sequencing represents an effective and low-cost alternative for sequencing large sets of metagenomic clones.

Phylogenetic characterization of a biogas plant microbial community integrating clone library 16S-rDNA sequences and metagenome sequence data obtained by 454-pyrosequencing.

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Kröber, Magdalena; Bekel, Thomas; Diaz, Naryttza N; Goesmann, Alexander; Jaenicke, Sebastian; Krause, Lutz; Miller, Dimitri; Runte, Kai J; Viehöver, Prisca; Pühler, Alfred; Schlüter, Andreas

2009-06-01

The phylogenetic structure of the microbial community residing in a fermentation sample from a production-scale biogas plant fed with maize silage, green rye and liquid manure was analysed by an integrated approach using clone library sequences and metagenome sequence data obtained by 454-pyrosequencing. Sequencing of 109 clones from a bacterial and an archaeal 16S-rDNA amplicon library revealed that the obtained nucleotide sequences are similar but not identical to 16S-rDNA database sequences derived from different anaerobic environments including digestors and bioreactors. Most of the bacterial 16S-rDNA sequences could be assigned to the phylum Firmicutes with the most abundant class Clostridia and to the class Bacteroidetes, whereas most archaeal 16S-rDNA sequences cluster close to the methanogen Methanoculleus bourgensis. Further sequences of the archaeal library most probably represent so far non-characterised species within the genus Methanoculleus. A similar result derived from phylogenetic analysis of mcrA clone sequences. The mcrA gene product encodes the alpha-subunit of methyl-coenzyme-M reductase involved in the final step of methanogenesis. BLASTn analysis applying stringent settings resulted in assignment of 16S-rDNA metagenome sequence reads to 62 16S-rDNA amplicon sequences thus enabling frequency of abundance estimations for 16S-rDNA clone library sequences. Ribosomal Database Project (RDP) Classifier processing of metagenome 16S-rDNA reads revealed abundance of the phyla Firmicutes, Bacteroidetes and Euryarchaeota and the orders Clostridiales, Bacteroidales and Methanomicrobiales. Moreover, a large fraction of 16S-rDNA metagenome reads could not be assigned to lower taxonomic ranks, demonstrating that numerous microorganisms in the analysed fermentation sample of the biogas plant are still unclassified or unknown.

Isolation of a novel alkaline-stable lipase from a metagenomic library and its specific application for milkfat flavor production.

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Peng, Qing; Wang, Xu; Shang, Meng; Huang, Jinjin; Guan, Guohua; Li, Ying; Shi, Bo

2014-01-04

Lipolytic enzymes are commonly used to produce desired flavors in lipolyzed milkfat (LMF) manufacturing processes. However, the choice of enzyme is critical because it determines the final profile of fatty acids released and the consequent flavor of the product. We previously constructed a metagenomic library from marine sediments, to explore the novel enzymes which have unique properties useful in flavor-enhancing LMF. A novel lipase Est_p6 was isolated from a metagenomic library and was expressed highly in E.coli. Bioinformatic analysis indicated that Est_p6 belongs to lipolytic enzyme family IV, the molecular weight of purified Est_p6 was estimated at 36 kDa by SDS-PAGE. The hydrolytic activity of the enzyme was stable under alkaline condition and the optimal temperature was 50°C. It had a high specific activity (2500 U/mg) toward pNP butyrate (pNP-C4), with K(m) and V(max) values of 1.148 mM and 3497 μmol∙min⁻¹∙mg⁻¹, respectively. The enzyme activity was enhanced by DTT and was not significantly inhibited by PMSF, EDTA or SDS. This enzyme also showed high hydrolysis specificity for myristate (C14) and palmitate (C16). It seems that Est_p6 has safety for commercial LMF flavor production and food manufacturing processes. The ocean is a vast and largely unexplored resource for enzymes. According the outstanding alkaline-stability of Est_p6 and it produced myristic acid and palmitic acid more efficiently than other free fatty acids in lipolyzed milkfat. This novel lipase may be used to impart a distinctive and desirable flavor and odor in milkfat flavor production.

Hybrid sequencing approach applied to human fecal metagenomic clone libraries revealed clones with potential biotechnological applications.

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Mária Džunková

Full Text Available Natural environments represent an incredible source of microbial genetic diversity. Discovery of novel biomolecules involves biotechnological methods that often require the design and implementation of biochemical assays to screen clone libraries. However, when an assay is applied to thousands of clones, one may eventually end up with very few positive clones which, in most of the cases, have to be "domesticated" for downstream characterization and application, and this makes screening both laborious and expensive. The negative clones, which are not considered by the selected assay, may also have biotechnological potential; however, unfortunately they would remain unexplored. Knowledge of the clone sequences provides important clues about potential biotechnological application of the clones in the library; however, the sequencing of clones one-by-one would be very time-consuming and expensive. In this study, we characterized the first metagenomic clone library from the feces of a healthy human volunteer, using a method based on 454 pyrosequencing coupled with a clone-by-clone Sanger end-sequencing. Instead of whole individual clone sequencing, we sequenced 358 clones in a pool. The medium-large insert (7-15 kb cloning strategy allowed us to assemble these clones correctly, and to assign the clone ends to maintain the link between the position of a living clone in the library and the annotated contig from the 454 assembly. Finally, we found several open reading frames (ORFs with previously described potential medical application. The proposed approach allows planning ad-hoc biochemical assays for the clones of interest, and the appropriate sub-cloning strategy for gene expression in suitable vectors/hosts.

Novel polyhydroxyalkanoate copolymers produced in Pseudomonas putida by metagenomic polyhydroxyalkanoate synthases.

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Cheng, Jiujun; Charles, Trevor C

2016-09-01

Bacterially produced biodegradable polyhydroxyalkanoates (PHAs) with versatile properties can be achieved using different PHA synthases (PhaCs). This work aims to expand the diversity of known PhaCs via functional metagenomics and demonstrates the use of these novel enzymes in PHA production. Complementation of a PHA synthesis-deficient Pseudomonas putida strain with a soil metagenomic cosmid library retrieved 27 clones expressing either class I, class II, or unclassified PHA synthases, and many did not have close sequence matches to known PhaCs. The composition of PHA produced by these clones was dependent on both the supplied growth substrates and the nature of the PHA synthase, with various combinations of short-chain-length (SCL) and medium-chain-length (MCL) PHA. These data demonstrate the ability to isolate diverse genes for PHA synthesis by functional metagenomics and their use for the production of a variety of PHA polymer and copolymer mixtures.

Evaluation of ddRADseq for reduced representation metagenome sequencing

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Michael Y. Liu

2017-09-01

Full Text Available Background Profiling of microbial communities via metagenomic shotgun sequencing has enabled researches to gain unprecedented insight into microbial community structure and the functional roles of community members. This study describes a method and basic analysis for a metagenomic adaptation of the double digest restriction site associated DNA sequencing (ddRADseq protocol for reduced representation metagenome profiling. Methods This technique takes advantage of the sequence specificity of restriction endonucleases to construct an Illumina-compatible sequencing library containing DNA fragments that are between a pair of restriction sites located within close proximity. This results in a reduced sequencing library with coverage breadth that can be tuned by size selection. We assessed the performance of the metagenomic ddRADseq approach by applying the full method to human stool samples and generating sequence data. Results The ddRADseq data yields a similar estimate of community taxonomic profile as obtained from shotgun metagenome sequencing of the same human stool samples. No obvious bias with respect to genomic G + C content and the estimated relative species abundance was detected. Discussion Although ddRADseq does introduce some bias in taxonomic representation, the bias is likely to be small relative to DNA extraction bias. ddRADseq appears feasible and could have value as a tool for metagenome-wide association studies.

Structural and mechanistic analysis of a β-glycoside phosphorylase identified by screening a metagenomic library.

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Macdonald, Spencer S; Patel, Ankoor; Larmour, Veronica L C; Morgan-Lang, Connor; Hallam, Steven J; Mark, Brian L; Withers, Stephen G

2018-03-02

Glycoside phosphorylases have considerable potential as catalysts for the assembly of useful glycans for products ranging from functional foods and prebiotics to novel materials. However, the substrate diversity of currently identified phosphorylases is relatively small, limiting their practical applications. To address this limitation, we developed a high-throughput screening approach using the activated substrate 2,4-dinitrophenyl β-d-glucoside (DNPGlc) and inorganic phosphate for identifying glycoside phosphorylase activity and used it to screen a large insert metagenomic library. The initial screen, based on release of 2,4-dinitrophenyl from DNPGlc in the presence of phosphate, identified the gene bglP, encoding a retaining β-glycoside phosphorylase from the CAZy GH3 family. Kinetic and mechanistic analysis of the gene product, BglP, confirmed a double displacement ping-pong mechanism involving a covalent glycosyl-enzyme intermediate. X-ray crystallographic analysis provided insights into the phosphate-binding mode and identified a key glutamine residue in the active site important for substrate recognition. Substituting this glutamine for a serine swapped the substrate specificity from glucoside to N -acetylglucosaminide. In summary, we present a high-throughput screening approach for identifying β-glycoside phosphorylases, which was robust, simple to implement, and useful in identifying active clones within a metagenomics library. Implementation of this screen enabled discovery of a new glycoside phosphorylase class and has paved the way to devising simple ways in which enzyme specificity can be encoded and swapped, which has implications for biotechnological applications. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Bioprospecting metagenomics of decaying wood: mining for new glycoside hydrolases

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Li Luen-Luen

2011-08-01

Full Text Available Abstract Background To efficiently deconstruct recalcitrant plant biomass to fermentable sugars in industrial processes, biocatalysts of higher performance and lower cost are required. The genetic diversity found in the metagenomes of natural microbial biomass decay communities may harbor such enzymes. Our goal was to discover and characterize new glycoside hydrolases (GHases from microbial biomass decay communities, especially those from unknown or never previously cultivated microorganisms. Results From the metagenome sequences of an anaerobic microbial community actively decaying poplar biomass, we identified approximately 4,000 GHase homologs. Based on homology to GHase families/activities of interest and the quality of the sequences, candidates were selected for full-length cloning and subsequent expression. As an alternative strategy, a metagenome expression library was constructed and screened for GHase activities. These combined efforts resulted in the cloning of four novel GHases that could be successfully expressed in Escherichia coli. Further characterization showed that two enzymes showed significant activity on p-nitrophenyl-α-L-arabinofuranoside, one enzyme had significant activity against p-nitrophenyl-β-D-glucopyranoside, and one enzyme showed significant activity against p-nitrophenyl-β-D-xylopyranoside. Enzymes were also tested in the presence of ionic liquids. Conclusions Metagenomics provides a good resource for mining novel biomass degrading enzymes and for screening of cellulolytic enzyme activities. The four GHases that were cloned may have potential application for deconstruction of biomass pretreated with ionic liquids, as they remain active in the presence of up to 20% ionic liquid (except for 1-ethyl-3-methylimidazolium diethyl phosphate. Alternatively, ionic liquids might be used to immobilize or stabilize these enzymes for minimal solvent processing of biomass.

Ruminal metagenomic libraries as a source of relevant hemicellulolytic enzymes for biofuel production.

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Duque, Estrella; Daddaoua, Abdelali; Cordero, Baldo F; Udaondo, Zulema; Molina-Santiago, Carlos; Roca, Amalia; Solano, Jennifer; Molina-Alcaide, Eduarda; Segura, Ana; Ramos, Juan-Luis

2018-04-17

The success of second-generation (2G) ethanol technology relies on the efficient transformation of hemicellulose into monosaccharides and, particularly, on the full conversion of xylans into xylose for over 18% of fermentable sugars. We sought new hemicellulases using ruminal liquid, after enrichment of microbes with industrial lignocellulosic substrates and preparation of metagenomic libraries. Among 150 000 fosmid clones tested, we identified 22 clones with endoxylanase activity and 125 with β-xylosidase activity. These positive clones were sequenced en masse, and the analysis revealed open reading frames with a low degree of similarity with known glycosyl hydrolases families. Among them, we searched for enzymes that were thermostable (activity at > 50°C) and that operate at high rate at pH around 5. Upon a wide series of assays, the clones exhibiting the highest endoxylanase and β-xylosidase activities were identified. The fosmids were sequenced, and the corresponding genes cloned, expressed and proteins purified. We found that the activity of the most active β-xylosidase was at least 10-fold higher than that in commercial enzymatic fungal cocktails. Endoxylanase activity was in the range of fungal enzymes. Fungal enzymatic cocktails supplemented with the bacterial hemicellulases exhibited enhanced release of sugars from pretreated sugar cane straw, a relevant agricultural residue. © 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Synthesis and evaluation of a series of 6-chloro-4-methylumbelliferyl glycosides as fluorogenic reagents for screening metagenomic libraries for glycosidase activity.

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Chen, Hong-Ming; Armstrong, Zachary; Hallam, Steven J; Withers, Stephen G

2016-02-08

Screening of large enzyme libraries such as those derived from metagenomic sources requires sensitive substrates. Fluorogenic glycosides typically offer the best sensitivity but typically must be used in a stopped format to generate good signal. Use of fluorescent phenols of pKa libraries yielded a "hit rate" of 1 in 60. Hits were then readily deconvoluted with the individual substrates in a single plate to identify specific activities within each clone. The use of such a collection of substrates greatly accelerates the screening process. Copyright © 2015 Elsevier Ltd. All rights reserved.

Combined whole-cell high-throughput functional screening for identification of new nicotinamidases/pyrazinamidases in metagenomic/polygenomic libraries

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Rubén Zapata-Pérez

2016-11-01

Full Text Available Nicotinamidases catalyze the hydrolysis of the amide bond in nicotinamide to produce ammonia and nicotinic acid. These enzymes are an essential component of the NAD+ salvage pathway and are implicated in the viability of several pathogenic organisms. Its absence in humans makes them a promising drug target. In addition, although they are key analytical biocatalysts for screening modulators in relevant biomedical enzymes, such as sirtuins and poly-ADP-ribosyltransferases, no commercial sources are available. Surprisingly, the finding of an affordable source of nicotinamidase from metagenomic libraries is hindered by the absence of a suitable and fast screening method. In this manuscript, we describe the development of two new whole-cell methods using the chemical property of one of the products formed in the enzymatic reaction (pyrazinoic or nicotinic acid to form colored complexes with stable iron salts, such as ammonium ferrous sulfate or sodium nitroprusside. After optimization of the assay conditions, a fosmid polygenomic expression library obtained from deep-sea mesophilic bacteria was screened, discovering several positive clones with the ammonium ferrous sulfate method. Their quantitative rescreening with the sodium nitroprusside method allowed the finding of the first nicotinamidase with balanced catalytic efficiency towards nicotinamide (nicotinamidase activity and pyrazinamide (pyrazinamidase activity. Its biochemical characterization has also made possible the development of the first high-throughput whole-cell method for prescreening of new nicotinamidase inhibitors by the naked eye, saving time and costs in the design of future antimicrobial and antiparasitic agents.

Combined Whole-Cell High-Throughput Functional Screening for Identification of New Nicotinamidases/Pyrazinamidases in Metagenomic/Polygenomic Libraries.

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Zapata-Pérez, Rubén; García-Saura, Antonio G; Jebbar, Mohamed; Golyshin, Peter N; Sánchez-Ferrer, Álvaro

2016-01-01

Nicotinamidases catalyze the hydrolysis of the amide bond in nicotinamide (NAM) to produce ammonia and nicotinic acid (NA). These enzymes are an essential component of the NAD + salvage pathway and are implicated in the viability of several pathogenic organisms. Its absence in humans makes them a promising drug target. In addition, although they are key analytical biocatalysts for screening modulators in relevant biomedical enzymes, such as sirtuins and poly-ADP-ribosyltransferases, no commercial sources are available. Surprisingly, the finding of an affordable source of nicotinamidase from metagenomic libraries is hindered by the absence of a suitable and fast screening method. In this manuscript, we describe the development of two new whole-cell methods using the chemical property of one of the products formed in the enzymatic reaction (pyrazinoic or NA) to form colored complexes with stable iron salts, such as ammonium ferrous sulfate or sodium nitroprusside (SNP). After optimization of the assay conditions, a fosmid polygenomic expression library obtained from deep-sea mesophilic bacteria was screened, discovering several positive clones with the ammonium ferrous sulfate method. Their quantitative rescreening with the SNP method allowed the finding of the first nicotinamidase with balanced catalytic efficiency toward NAM (nicotinamidase activity) and pyrazinamide (pyrazinamidase activity). Its biochemical characterization has also made possible the development of the first high-throughput whole-cell method for prescreening of new nicotinamidase inhibitors by the naked eye, saving time and costs in the design of future antimicrobial and antiparasitic agents.

Discovery and characterizaton of a novel lipase with transesterification activity from hot spring metagenomic library

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Wei Yan

2017-03-01

Full Text Available A new gene encoding a lipase (designated as Lip-1 was identified from a metagenomic bacterial artificial chromosome(BAC library prepared from a concentrated water sample collected from a hot spring field in Niujie, Eryuan of Yunnan province in China. The open reading frame of this gene encoded 622 amino acid residues. It was cloned, fused with the oleosin gene and over expressed in Escherichia coli to prepare immobilized lipase artificial oil body AOB-sole-lip-1. The monomeric Sole-lip-1 fusion protein presented a molecular mass of 102.4 kDa. Enzyme assays using olive oil and methanol as the substrates in petroleum ether confirmed its transesterification activity. Hexadecanoic acid methyl ester, 8,11-Octadecadienoic acid methyl ester, 8-Octadecenoic acid methyl ester, and Octadecanoic acid methyl ester were detected. It showed favorable transesterification activity with optimal temperature 45 °C. Besides, the maximal biodiesel yield was obtained when the petroleum ether system as the organic solvent and the substrate methanol in 350 mmol/L (at a molar ratio of methanol of 10.5:1 and the water content was 1%. In light of these advantages, this lipase presents a promising resource for biodiesel production.

Rapid and efficient method to extract metagenomic DNA from estuarine sediments.

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Shamim, Kashif; Sharma, Jaya; Dubey, Santosh Kumar

2017-07-01

Metagenomic DNA from sediments of selective estuaries of Goa, India was extracted using a simple, fast, efficient and environment friendly method. The recovery of pure metagenomic DNA from our method was significantly high as compared to other well-known methods since the concentration of recovered metagenomic DNA ranged from 1185.1 to 4579.7 µg/g of sediment. The purity of metagenomic DNA was also considerably high as the ratio of absorbance at 260 and 280 nm ranged from 1.88 to 1.94. Therefore, the recovered metagenomic DNA was directly used to perform various molecular biology experiments viz. restriction digestion, PCR amplification, cloning and metagenomic library construction. This clearly proved that our protocol for metagenomic DNA extraction using silica gel efficiently removed the contaminants and prevented shearing of the metagenomic DNA. Thus, this modified method can be used to recover pure metagenomic DNA from various estuarine sediments in a rapid, efficient and eco-friendly manner.

Identification of eukaryotic open reading frames in metagenomic cDNA libraries made from environmental samples.

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Grant, Susan; Grant, William D; Cowan, Don A; Jones, Brian E; Ma, Yanhe; Ventosa, Antonio; Heaphy, Shaun

2006-01-01

Here we describe the application of metagenomic technologies to construct cDNA libraries from RNA isolated from environmental samples. RNAlater (Ambion) was shown to stabilize RNA in environmental samples for periods of at least 3 months at -20 degrees C. Protocols for library construction were established on total RNA extracted from Acanthamoeba polyphaga trophozoites. The methodology was then used on algal mats from geothermal hot springs in Tengchong county, Yunnan Province, People's Republic of China, and activated sludge from a sewage treatment plant in Leicestershire, United Kingdom. The Tenchong libraries were dominated by RNA from prokaryotes, reflecting the mainly prokaryote microbial composition. The majority of these clones resulted from rRNA; only a few appeared to be derived from mRNA. In contrast, many clones from the activated sludge library had significant similarity to eukaryote mRNA-encoded protein sequences. A library was also made using polyadenylated RNA isolated from total RNA from activated sludge; many more clones in this library were related to eukaryotic mRNA sequences and proteins. Open reading frames (ORFs) up to 378 amino acids in size could be identified. Some resembled known proteins over their full length, e.g., 36% match to cystatin, 49% match to ribosomal protein L32, 63% match to ribosomal protein S16, 70% to CPC2 protein. The methodology described here permits the polyadenylated transcriptome to be isolated from environmental samples with no knowledge of the identity of the microorganisms in the sample or the necessity to culture them. It has many uses, including the identification of novel eukaryotic ORFs encoding proteins and enzymes.

A Bioinformatician's Guide to Metagenomics

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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.

Tapping uncultured microorganisms through metagenomics for drug ...

African Journals Online (AJOL)

bdelnasser

reached the market using this new technology. For these reasons and others, the interest in natural products has ..... Functional metagenomic library screening strategy ..... Bertrand H, Poly F, Van VT, Lombard N, Nalin R, Vogel TM, Simonet P.

Development of high-throughput phenotyping of metagenomic clones from the human gut microbiome for modulation of eukaryotic cell growth.

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Gloux, Karine; Leclerc, Marion; Iliozer, Harout; L'Haridon, René; Manichanh, Chaysavanh; Corthier, Gérard; Nalin, Renaud; Blottière, Hervé M; Doré, Joël

2007-06-01

Metagenomic libraries derived from human intestinal microbiota (20,725 clones) were screened for epithelial cell growth modulation. Modulatory clones belonging to the four phyla represented among the metagenomic libraries were identified (hit rate, 0.04 to 8.7% depending on the screening cutoff). Several candidate loci were identified by transposon mutagenesis and subcloning.

Heterologous Expression of the Oxytetracycline Biosynthetic Pathway in Myxococcus xanthus▿

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Stevens, D. Cole; Henry, Michael R.; Murphy, Kimberly A.; Boddy, Christopher N.

2010-01-01

New natural products for drug discovery may be accessed by heterologous expression of bacterial biosynthetic pathways in metagenomic DNA libraries. However, a “universal” host is needed for this experiment. Herein, we show that Myxococcus xanthus is a potential “universal” host for heterologous expression of polyketide biosynthetic gene clusters. PMID:20208031

New Hydrocarbon Degradation Pathways in the Microbial Metagenome from Brazilian Petroleum Reservoirs

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Sierra-García, Isabel Natalia; Correa Alvarez, Javier; Pantaroto de Vasconcellos, Suzan; Pereira de Souza, Anete; dos Santos Neto, Eugenio Vaz; de Oliveira, Valéria Maia

2014-01-01

Current knowledge of the microbial diversity and metabolic pathways involved in hydrocarbon degradation in petroleum reservoirs is still limited, mostly due to the difficulty in recovering the complex community from such an extreme environment. Metagenomics is a valuable tool to investigate the genetic and functional diversity of previously uncultured microorganisms in natural environments. Using a function-driven metagenomic approach, we investigated the metabolic abilities of microbial communities in oil reservoirs. Here, we describe novel functional metabolic pathways involved in the biodegradation of aromatic compounds in a metagenomic library obtained from an oil reservoir. Although many of the deduced proteins shared homology with known enzymes of different well-described aerobic and anaerobic catabolic pathways, the metagenomic fragments did not contain the complete clusters known to be involved in hydrocarbon degradation. Instead, the metagenomic fragments comprised genes belonging to different pathways, showing novel gene arrangements. These results reinforce the potential of the metagenomic approach for the identification and elucidation of new genes and pathways in poorly studied environments and contribute to a broader perspective on the hydrocarbon degradation processes in petroleum reservoirs. PMID:24587220

Expanding the Repertoire of Carbapenem-Hydrolyzing Metallo-ß-Lactamases by Functional Metagenomic Analysis of Soil Microbiota.

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Gudeta, Dereje D; Bortolaia, Valeria; Pollini, Simona; Docquier, Jean-Denis; Rossolini, Gian M; Amos, Gregory C A; Wellington, Elizabeth M H; Guardabassi, Luca

2016-01-01

Carbapenemases are bacterial enzymes that hydrolyze carbapenems, a group of last-resort β-lactam antibiotics used for treatment of severe bacterial infections. They belong to three β-lactamase classes based amino acid sequence (A, B, and D). The aim of this study was to elucidate occurrence, diversity and functionality of carbapenemase-encoding genes in soil microbiota by functional metagenomics. Ten plasmid libraries were generated by cloning metagenomic DNA from agricultural ( n = 6) and grassland ( n = 4) soil into Escherichia coli . The libraries were cultured on amoxicillin-containing agar and up to 100 colonies per library were screened for carbapenemase production by CarbaNP test. Presumptive carbapenemases were characterized with regard to DNA sequence, minimum inhibitory concentration (MIC) of β-lactams, and imipenem hydrolysis. Nine distinct class B carbapenemases, also known as metallo-beta-lactamases (MBLs), were identified in six soil samples, including two subclass B1 (GRD23-1 and SPN79-1) and seven subclass B3 (CRD3-1, PEDO-1, GRD33-1, ESP-2, ALG6-1, ALG11-1, and DHT2-1). Except PEDO-1 and ESP-2, these enzymes were distantly related to any previously described MBLs (33 to 59% identity). RAIphy analysis indicated that six enzymes (CRD3-1, GRD23-1, DHT2-1, SPN79-1, ALG6-1, and ALG11-1) originated from Proteobacteria , two (PEDO-1 and ESP-2) from Bacteroidetes and one (GRD33-1) from Gemmatimonadetes . All MBLs detected in soil microbiota were functional when expressed in E. coli , resulting in detectable imipenem-hydrolyzing activity and significantly increased MICs of clinically relevant ß-lactams. Interestingly, the MBLs yielded by functional metagenomics generally differed from those detected in the same soil samples by antibiotic selective culture, showing that the two approaches targeted different subpopulations in soil microbiota.

A deep gold mine metagenome as a source of novel esterases

African Journals Online (AJOL)

Jane

2011-07-04

Jul 4, 2011 ... small metagenome library from the deep mine biofilm provided two esterolytic clones, ...... tuberosum) tubers, and its occurrence as genotype effect: processing .... diversity in freshwater sediment of a shallow eutrophic lake by.

Discovery of novel enzymes with industrial potential from a cold and alkaline environment by a combination of functional metagenomics and culturing.

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Vester, Jan Kjølhede; Glaring, Mikkel Andreas; Stougaard, Peter

2014-05-20

The use of cold-active enzymes has many advantages, including reduced energy consumption and easy inactivation. The ikaite columns of SW Greenland are permanently cold (4-6°C) and alkaline (above pH 10), and the microorganisms living there and their enzymes are adapted to these conditions. Since only a small fraction of the total microbial diversity can be cultured in the laboratory, a combined approach involving functional screening of a strain collection and a metagenomic library was undertaken for discovery of novel enzymes from the ikaite columns. A strain collection with 322 cultured isolates was screened for enzymatic activities identifying a large number of enzyme producers, with a high re-discovery rate to previously characterized strains. A functional expression library established in Escherichia coli identified a number of novel cold-active enzymes. Both α-amylases and β-galactosidases were characterized in more detail with respect to temperature and pH profiles and one of the β-galactosidases, BGalI17E2, was able to hydrolyze lactose at 5°C. A metagenome sequence of the expression library indicated that the majority of enzymatic activities were not detected by functional expression. Phylogenetic analysis showed that different bacterial communities were targeted with the culture dependent and independent approaches and revealed the bias of multiple displacement amplification (MDA) of DNA isolated from complex microbial communities. Many cold- and/or alkaline-active enzymes of industrial relevance were identified in the culture based approach and the majority of the enzyme-producing isolates were closely related to previously characterized strains. The function-based metagenomic approach, on the other hand, identified several enzymes (β-galactosidases, α-amylases and a phosphatase) with low homology to known sequences that were easily expressed in the production host E. coli. The β-galactosidase BGalI17E2 was able to hydrolyze lactose at low

A new tetracycline efflux gene, tet(40), is located in tandem with tet(O/32/O) in a human gut firmicute bacterium and in metagenomic library clones.

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Kazimierczak, Katarzyna A; Rincon, Marco T; Patterson, Andrea J; Martin, Jennifer C; Young, Pauline; Flint, Harry J; Scott, Karen P

2008-11-01

The bacterium Clostridium saccharolyticum K10, isolated from a fecal sample obtained from a healthy donor who had received long-term tetracycline therapy, was found to carry three tetracycline resistance genes: tet(W) and the mosaic tet(O/32/O), both conferring ribosome protection-type resistance, and a novel, closely linked efflux-type resistance gene designated tet(40). tet(40) encodes a predicted membrane-associated protein with 42% amino acid identity to tetA(P). Tetracycline did not accumulate in Escherichia coli cells expressing the Tet(40) efflux protein, and resistance to tetracycline was reduced when cells were incubated with an efflux pump inhibitor. E. coli cells carrying tet(40) had a 50% inhibitory concentration of tetracycline of 60 microg/ml. Analysis of a transconjugant from a mating between donor strain C. saccharolyticum K10 and the recipient human gut commensal bacterium Roseburia inulinivorans suggested that tet(O/32/O) and tet(40) were cotransferred on a mobile element. Sequence analysis of a 37-kb insert identified on the basis of tetracycline resistance from a metagenomic fosmid library again revealed a tandem arrangement of tet(O/32/O) and tet(40), flanked by regions with homology to parts of the VanG operon previously identified in Enterococcus faecalis. At least 10 of the metagenomic inserts that carried tet(O/32/O) also carried tet(40), suggesting that tet(40), although previously undetected, may be an abundant efflux gene.

Laboratory procedures to generate viral metagenomes.

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Thurber, Rebecca V; Haynes, Matthew; Breitbart, Mya; Wegley, Linda; Rohwer, Forest

2009-01-01

This collection of laboratory protocols describes the steps to collect viruses from various samples with the specific aim of generating viral metagenome sequence libraries (viromes). Viral metagenomics, the study of uncultured viral nucleic acid sequences from different biomes, relies on several concentration, purification, extraction, sequencing and heuristic bioinformatic methods. No single technique can provide an all-inclusive approach, and therefore the protocols presented here will be discussed in terms of hypothetical projects. However, care must be taken to individualize each step depending on the source and type of viral-particles. This protocol is a description of the processes we have successfully used to: (i) concentrate viral particles from various types of samples, (ii) eliminate contaminating cells and free nucleic acids and (iii) extract, amplify and purify viral nucleic acids. Overall, a sample can be processed to isolate viral nucleic acids suitable for high-throughput sequencing in approximately 1 week.

Mining for hemicellulases in the fungus-growing termite Pseudacanthotermes militaris using functional metagenomics.

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Bastien, Géraldine; Arnal, Grégory; Bozonnet, Sophie; Laguerre, Sandrine; Ferreira, Fernando; Fauré, Régis; Henrissat, Bernard; Lefèvre, Fabrice; Robe, Patrick; Bouchez, Olivier; Noirot, Céline; Dumon, Claire; O'Donohue, Michael

2013-05-14

The metagenomic analysis of gut microbiomes has emerged as a powerful strategy for the identification of biomass-degrading enzymes, which will be no doubt useful for the development of advanced biorefining processes. In the present study, we have performed a functional metagenomic analysis on comb and gut microbiomes associated with the fungus-growing termite, Pseudacanthotermes militaris. Using whole termite abdomens and fungal-comb material respectively, two fosmid-based metagenomic libraries were created and screened for the presence of xylan-degrading enzymes. This revealed 101 positive clones, corresponding to an extremely high global hit rate of 0.49%. Many clones displayed either β-d-xylosidase (EC 3.2.1.37) or α-l-arabinofuranosidase (EC 3.2.1.55) activity, while others displayed the ability to degrade AZCL-xylan or AZCL-β-(1,3)-β-(1,4)-glucan. Using secondary screening it was possible to pinpoint clones of interest that were used to prepare fosmid DNA. Sequencing of fosmid DNA generated 1.46 Mbp of sequence data, and bioinformatics analysis revealed 63 sequences encoding putative carbohydrate-active enzymes, with many of these forming parts of sequence clusters, probably having carbohydrate degradation and metabolic functions. Taxonomic assignment of the different sequences revealed that Firmicutes and Bacteroidetes were predominant phyla in the gut sample, while microbial diversity in the comb sample resembled that of typical soil samples. Cloning and expression in E. coli of six enzyme candidates identified in the libraries provided access to individual enzyme activities, which all proved to be coherent with the primary and secondary functional screens. This study shows that the gut microbiome of P. militaris possesses the potential to degrade biomass components, such as arabinoxylans and arabinans. Moreover, the data presented suggests that prokaryotic microorganisms present in the comb could also play a part in the degradation of biomass within the

Salt resistance genes revealed by functional metagenomics from brines and moderate-salinity rhizosphere within a hypersaline environment

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Salvador eMirete

2015-10-01

Full Text Available Hypersaline environments are considered one of the most extreme habitats on earth and microorganisms have developed diverse molecular mechanisms of adaptation to withstand these conditions. The present study was aimed at identifying novel genes involved in salt resistance from the microbial communities of brines and the rhizosphere from the Es Trenc saltern (Mallorca, Spain. The microbial diversity assessed by pyrosequencing of 16S rRNA gene libraries revealed the presence of communities that are typical in such environments. Metagenomic libraries from brine and rhizosphere samples, were transferred to the osmosensitive strain Escherichia coli MKH13, and screened for salt resistance. As a result, eleven genes that conferred salt resistance were identified, some encoding for well known proteins previously related to osmoadaptation as a glycerol and a proton pump, whereas others encoded for proteins not previously related to this function in microorganisms as DNA/RNA helicases, an endonuclease III (Nth and hypothetical proteins of unknown function. Furthermore, four of the retrieved genes were cloned and expressed in Bacillus subtilis and they also exhibited salt resistance in this bacterium, broadening the spectrum of bacterial species where these genes can operate. This is the first report of salt resistance genes recovered from metagenomes of a hypersaline environment.

Isolation and characterization of a novel endoglucanase from a Bursaphelenchus xylophilus metagenomic library.

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Lin Zhang

Full Text Available A novel gene (designated as cen219 encoding endoglucanase was isolated from a Bursaphelenchus xylophilus metagenomic library by functional screening. Sequence analysis revealed that cen219 encoded a protein of 367 amino acids. SDS-PAGE analysis of purified endoglucanase suggested that Cen219 was a monomeric enzyme with a molecular mass of 40 kDa. The optimum temperature and pH for endoglucanase activity of Cen219 was separately 50 °C and 6.0. It was stable from 30 to 50 °C, and from pH 4.0 to 7.0. The activity was significantly enhanced by Mn(2+ and dramatically reduced by detergent SDS and metals Fe(3+, Cu(2+ or Hg(2+. The enzyme hydrolyzed a wide range of β-1, 3-, and β-1, 4-linked polysaccharides, with varying activities. Activities towards microcrystalline cellulose and filter paper were relatively high, while the highest activity was towards oat gum. The Km and Vmax of Cen219 towards CMC was 17.37 mg/ml and 333.33 U/mg, respectively. The findings have an insight into understanding the molecular basis of host-parasite interactions in B. xylophilus species. The properties also make Cen219 an interesting enzyme for biotechnological application.

A response regulator from a soil metagenome enhances resistance to the β-lactam antibiotic carbenicillin in Escherichia coli.

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Heather K Allen

Full Text Available Functional metagenomic analysis of soil metagenomes is a method for uncovering as-yet unidentified mechanisms for antibiotic resistance. Here we report an unconventional mode by which a response regulator derived from a soil metagenome confers resistance to the β-lactam antibiotic carbenicillin in Escherichia coli. A recombinant clone (βlr16 harboring a 5,169 bp DNA insert was selected from a metagenomic library previously constructed from a remote Alaskan soil. The βlr16 clone conferred specific resistance to carbenicillin, with limited increases in resistance to other tested antibiotics, including other β-lactams (penicillins and cephalosporins, rifampin, ciprofloxacin, erythromycin, chloramphenicol, nalidixic acid, fusidic acid, and gentamicin. Resistance was more pronounced at 24°C than at 37°C. Zone-of-inhibition assays suggested that the mechanism of carbenicillin resistance was not due to antibiotic inactivation. The DNA insert did not encode any genes known to confer antibiotic resistance, but did have two putative open reading frames (ORFs that were annotated as a metallopeptidase and a two-component response regulator. Transposon mutagenesis and subcloning of the two ORFs followed by phenotypic assays showed that the response regulator gene was necessary and sufficient to confer the resistance phenotype. Quantitative reverse transcriptase PCR showed that the response regulator suppressed expression of the ompF porin gene, independently of the small RNA regulator micF, and enhanced expression of the acrD, mdtA, and mdtB efflux pump genes. This work demonstrates that antibiotic resistance can be achieved by the modulation of gene regulation by heterologous DNA. Functional analyses such as these can be important for making discoveries in antibiotic resistance gene biology and ecology.

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)

MinION™ nanopore sequencing of environmental metagenomes: a synthetic approach.

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Brown, Bonnie L; Watson, Mick; Minot, Samuel S; Rivera, Maria C; Franklin, Rima B

2017-03-01

Environmental metagenomic analysis is typically accomplished by assigning taxonomy and/or function from whole genome sequencing or 16S amplicon sequences. Both of these approaches are limited, however, by read length, among other technical and biological factors. A nanopore-based sequencing platform, MinION™, produces reads that are ≥1 × 104 bp in length, potentially providing for more precise assignment, thereby alleviating some of the limitations inherent in determining metagenome composition from short reads. We tested the ability of sequence data produced by MinION (R7.3 flow cells) to correctly assign taxonomy in single bacterial species runs and in three types of low-complexity synthetic communities: a mixture of DNA using equal mass from four species, a community with one relatively rare (1%) and three abundant (33% each) components, and a mixture of genomic DNA from 20 bacterial strains of staggered representation. Taxonomic composition of the low-complexity communities was assessed by analyzing the MinION sequence data with three different bioinformatic approaches: Kraken, MG-RAST, and One Codex. Results: Long read sequences generated from libraries prepared from single strains using the version 5 kit and chemistry, run on the original MinION device, yielded as few as 224 to as many as 3497 bidirectional high-quality (2D) reads with an average overall study length of 6000 bp. For the single-strain analyses, assignment of reads to the correct genus by different methods ranged from 53.1% to 99.5%, assignment to the correct species ranged from 23.9% to 99.5%, and the majority of misassigned reads were to closely related organisms. A synthetic metagenome sequenced with the same setup yielded 714 high quality 2D reads of approximately 5500 bp that were up to 98% correctly assigned to the species level. Synthetic metagenome MinION libraries generated using version 6 kit and chemistry yielded from 899 to 3497 2D reads with lengths averaging 5700 bp with up

A function-based screen for seeking RubisCO active clones from metagenomes: novel enzymes influencing RubisCO activity.

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Böhnke, Stefanie; Perner, Mirjam

2015-03-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a key enzyme of the Calvin cycle, which is responsible for most of Earth's primary production. Although research on RubisCO genes and enzymes in plants, cyanobacteria and bacteria has been ongoing for years, still little is understood about its regulation and activation in bacteria. Even more so, hardly any information exists about the function of metagenomic RubisCOs and the role of the enzymes encoded on the flanking DNA owing to the lack of available function-based screens for seeking active RubisCOs from the environment. Here we present the first solely activity-based approach for identifying RubisCO active fosmid clones from a metagenomic library. We constructed a metagenomic library from hydrothermal vent fluids and screened 1056 fosmid clones. Twelve clones exhibited RubisCO activity and the metagenomic fragments resembled genes from Thiomicrospira crunogena. One of these clones was further analyzed. It contained a 35.2 kb metagenomic insert carrying the RubisCO gene cluster and flanking DNA regions. Knockouts of twelve genes and two intergenic regions on this metagenomic fragment demonstrated that the RubisCO activity was significantly impaired and was attributed to deletions in genes encoding putative transcriptional regulators and those believed to be vital for RubisCO activation. Our new technique revealed a novel link between a poorly characterized gene and RubisCO activity. This screen opens the door to directly investigating RubisCO genes and respective enzymes from environmental samples.

Metagenome Analyses of Corroded Concrete Wastewater Pipe Biofilms Reveals a Complex Microbial System

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Analysis of whole-metagenome pyrosequencing data and 16S rRNA gene clone libraries was used to determine microbial composition and functional genes associated with biomass harvested from crown (top) and invert (bottom) sections of a corroded wastewater pipe. Taxonomic and functio...

Automated and Accurate Estimation of Gene Family Abundance from Shotgun Metagenomes.

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Stephen Nayfach

2015-11-01

Full Text Available Shotgun metagenomic DNA sequencing is a widely applicable tool for characterizing the functions that are encoded by microbial communities. Several bioinformatic tools can be used to functionally annotate metagenomes, allowing researchers to draw inferences about the functional potential of the community and to identify putative functional biomarkers. However, little is known about how decisions made during annotation affect the reliability of the results. Here, we use statistical simulations to rigorously assess how to optimize annotation accuracy and speed, given parameters of the input data like read length and library size. We identify best practices in metagenome annotation and use them to guide the development of the Shotgun Metagenome Annotation Pipeline (ShotMAP. ShotMAP is an analytically flexible, end-to-end annotation pipeline that can be implemented either on a local computer or a cloud compute cluster. We use ShotMAP to assess how different annotation databases impact the interpretation of how marine metagenome and metatranscriptome functional capacity changes across seasons. We also apply ShotMAP to data obtained from a clinical microbiome investigation of inflammatory bowel disease. This analysis finds that gut microbiota collected from Crohn's disease patients are functionally distinct from gut microbiota collected from either ulcerative colitis patients or healthy controls, with differential abundance of metabolic pathways related to host-microbiome interactions that may serve as putative biomarkers of disease.

A novel esterase gene cloned from a metagenomic library from neritic sediments of the South China Sea

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2011-01-01

Background Marine microbes are a large and diverse group, which are exposed to a wide variety of pressure, temperature, salinity, nutrient availability and other environmental conditions. They provide a huge potential source of novel enzymes with unique properties that may be useful in industry and biotechnology. To explore the lipolytic genetic resources in the South China Sea, 23 sediment samples were collected in the depth South China Sea sediments assemblage in plasmid vector containing about 194 Mb of community DNA was prepared. Screening of a part of the unamplified library resulted in isolation of 15 unique lipolytic clones with the ability to hydrolyze tributyrin. A positive recombinant clone (pNLE1), containing a novel esterase (Est_p1), was successfully expressed in E. coli and purified. In a series of assays, Est_p1 displayed maximal activity at pH 8.57, 40°C, with ρ-Nitrophenyl butyrate (C4) as substrate. Compared to other metagenomic esterases, Est_p1 played a notable role in specificity for substrate C4 (kcat/Km value 11,500 S-1m M-1) and showed no inhibited by phenylmethylsulfonyl fluoride, suggested that the substrate binding pocket was suitable for substrate C4 and the serine active-site residue was buried at the bottom of substrate binding pocket which sheltered by a lid structure. Conclusions Esterase, which specificity towards short chain fatty acids, especially butanoic acid, is commercially available as potent flavoring tools. According the outstanding activity and specificity for substrate C4, Est_p1 has potential application in flavor industries requiring hydrolysis of short chain esters. PMID:22067554

Identification of nitrogen-fixing genes and gene clusters from metagenomic library of acid mine drainage.

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Zhimin Dai

Full Text Available Biological nitrogen fixation is an essential function of acid mine drainage (AMD microbial communities. However, most acidophiles in AMD environments are uncultured microorganisms and little is known about the diversity of nitrogen-fixing genes and structure of nif gene cluster in AMD microbial communities. In this study, we used metagenomic sequencing to isolate nif genes in the AMD microbial community from Dexing Copper Mine, China. Meanwhile, a metagenome microarray containing 7,776 large-insertion fosmids was constructed to screen novel nif gene clusters. Metagenomic analyses revealed that 742 sequences were identified as nif genes including structural subunit genes nifH, nifD, nifK and various additional genes. The AMD community is massively dominated by the genus Acidithiobacillus. However, the phylogenetic diversity of nitrogen-fixing microorganisms is much higher than previously thought in the AMD community. Furthermore, a 32.5-kb genomic sequence harboring nif, fix and associated genes was screened by metagenome microarray. Comparative genome analysis indicated that most nif genes in this cluster are most similar to those of Herbaspirillum seropedicae, but the organization of the nif gene cluster had significant differences from H. seropedicae. Sequence analysis and reverse transcription PCR also suggested that distinct transcription units of nif genes exist in this gene cluster. nifQ gene falls into the same transcription unit with fixABCX genes, which have not been reported in other diazotrophs before. All of these results indicated that more novel diazotrophs survive in the AMD community.

Identification of nitrogen-fixing genes and gene clusters from metagenomic library of acid mine drainage.

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Dai, Zhimin; Guo, Xue; Yin, Huaqun; Liang, Yili; Cong, Jing; Liu, Xueduan

2014-01-01

Biological nitrogen fixation is an essential function of acid mine drainage (AMD) microbial communities. However, most acidophiles in AMD environments are uncultured microorganisms and little is known about the diversity of nitrogen-fixing genes and structure of nif gene cluster in AMD microbial communities. In this study, we used metagenomic sequencing to isolate nif genes in the AMD microbial community from Dexing Copper Mine, China. Meanwhile, a metagenome microarray containing 7,776 large-insertion fosmids was constructed to screen novel nif gene clusters. Metagenomic analyses revealed that 742 sequences were identified as nif genes including structural subunit genes nifH, nifD, nifK and various additional genes. The AMD community is massively dominated by the genus Acidithiobacillus. However, the phylogenetic diversity of nitrogen-fixing microorganisms is much higher than previously thought in the AMD community. Furthermore, a 32.5-kb genomic sequence harboring nif, fix and associated genes was screened by metagenome microarray. Comparative genome analysis indicated that most nif genes in this cluster are most similar to those of Herbaspirillum seropedicae, but the organization of the nif gene cluster had significant differences from H. seropedicae. Sequence analysis and reverse transcription PCR also suggested that distinct transcription units of nif genes exist in this gene cluster. nifQ gene falls into the same transcription unit with fixABCX genes, which have not been reported in other diazotrophs before. All of these results indicated that more novel diazotrophs survive in the AMD community.

Identification of Nitrogen-Fixing Genes and Gene Clusters from Metagenomic Library of Acid Mine Drainage

Science.gov (United States)

Yin, Huaqun; Liang, Yili; Cong, Jing; Liu, Xueduan

2014-01-01

Biological nitrogen fixation is an essential function of acid mine drainage (AMD) microbial communities. However, most acidophiles in AMD environments are uncultured microorganisms and little is known about the diversity of nitrogen-fixing genes and structure of nif gene cluster in AMD microbial communities. In this study, we used metagenomic sequencing to isolate nif genes in the AMD microbial community from Dexing Copper Mine, China. Meanwhile, a metagenome microarray containing 7,776 large-insertion fosmids was constructed to screen novel nif gene clusters. Metagenomic analyses revealed that 742 sequences were identified as nif genes including structural subunit genes nifH, nifD, nifK and various additional genes. The AMD community is massively dominated by the genus Acidithiobacillus. However, the phylogenetic diversity of nitrogen-fixing microorganisms is much higher than previously thought in the AMD community. Furthermore, a 32.5-kb genomic sequence harboring nif, fix and associated genes was screened by metagenome microarray. Comparative genome analysis indicated that most nif genes in this cluster are most similar to those of Herbaspirillum seropedicae, but the organization of the nif gene cluster had significant differences from H. seropedicae. Sequence analysis and reverse transcription PCR also suggested that distinct transcription units of nif genes exist in this gene cluster. nifQ gene falls into the same transcription unit with fixABCX genes, which have not been reported in other diazotrophs before. All of these results indicated that more novel diazotrophs survive in the AMD community. PMID:24498417

A broad pH range and processive chitinase from a metagenome library

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S.S. Thimoteo

Full Text Available Chitinases are hydrolases that degrade chitin, a polymer of N-acetylglucosamine linked β(1-4 present in the exoskeleton of crustaceans, insects, nematodes and fungal cell walls. A metagenome fosmid library from a wastewater-contaminated soil was functionally screened for chitinase activity leading to the isolation and identification of a chitinase gene named metachi18A. The metachi18A gene was subcloned and overexpressed in Escherichia coli BL21 and the MetaChi18A chitinase was purified by affinity chromatography as a 6xHis-tagged fusion protein. The MetaChi18A enzyme is a 92-kDa protein with a conserved active site domain of glycosyl hydrolases family 18. It hydrolyses colloidal chitin with an optimum pH of 5 and temperature of 50°C. Moreover, the enzyme retained at least 80% of its activity in the pH range from 4 to 9 and 98% at 600 mM NaCl. Thin layer chromatography analyses identified chitobiose as the main product of MetaChi18A on chitin polymers as substrate. Kinetic analysis showed inhibition of MetaChi18A activity at high concentrations of colloidal chitin and 4-methylumbelliferyl N,N′-diacetylchitobiose and sigmoid kinetics at low concentrations of colloidal chitin, indicating a possible conformational change to lead the chitin chain from the chitin-binding to the catalytic domain. The observed stability and activity of MetaChi18A over a wide range of conditions suggest that this chitinase, now characterized, may be suitable for application in the industrial processing of chitin.

Microbiota composition, gene pool and its expression in Gir cattle (Bos indicus) rumen under different forage diets using metagenomic and metatranscriptomic approaches.

Science.gov (United States)

Pandit, Ramesh J; Hinsu, Ankit T; Patel, Shriram H; Jakhesara, Subhash J; Koringa, Prakash G; Bruno, Fosso; Psifidi, Androniki; Shah, S V; Joshi, Chaitanya G

2018-03-09

Zebu (Bos indicus) is a domestic cattle species originating from the Indian subcontinent and now widely domesticated on several continents. In this study, we were particularly interested in understanding the functionally active rumen microbiota of an important Zebu breed, the Gir, under different dietary regimes. Metagenomic and metatranscriptomic data were compared at various taxonomic levels to elucidate the differential microbial population and its functional dynamics in Gir cattle rumen under different roughage dietary regimes. Different proportions of roughage rather than the type of roughage (dry or green) modulated microbiome composition and the expression of its gene pool. Fibre degrading bacteria (i.e. Clostridium, Ruminococcus, Eubacterium, Butyrivibrio, Bacillus and Roseburia) were higher in the solid fraction of rumen (Pcomparison of metagenomic shotgun and metatranscriptomic sequencing appeared to be a much richer source of information compared to conventional metagenomic analysis. Copyright © 2018 Elsevier GmbH. All rights reserved.

Metagenomic Taxonomy-Guided Database-Searching Strategy for Improving Metaproteomic Analysis.

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Xiao, Jinqiu; Tanca, Alessandro; Jia, Ben; Yang, Runqing; Wang, Bo; Zhang, Yu; Li, Jing

2018-04-06

Metaproteomics provides a direct measure of the functional information by investigating all proteins expressed by a microbiota. However, due to the complexity and heterogeneity of microbial communities, it is very hard to construct a sequence database suitable for a metaproteomic study. Using a public database, researchers might not be able to identify proteins from poorly characterized microbial species, while a sequencing-based metagenomic database may not provide adequate coverage for all potentially expressed protein sequences. To address this challenge, we propose a metagenomic taxonomy-guided database-search strategy (MT), in which a merged database is employed, consisting of both taxonomy-guided reference protein sequences from public databases and proteins from metagenome assembly. By applying our MT strategy to a mock microbial mixture, about two times as many peptides were detected as with the metagenomic database only. According to the evaluation of the reliability of taxonomic attribution, the rate of misassignments was comparable to that obtained using an a priori matched database. We also evaluated the MT strategy with a human gut microbial sample, and we found 1.7 times as many peptides as using a standard metagenomic database. In conclusion, our MT strategy allows the construction of databases able to provide high sensitivity and precision in peptide identification in metaproteomic studies, enabling the detection of proteins from poorly characterized species within the microbiota.

Caught in the middle with multiple displacement amplification: the myth of pooling for avoiding multiple displacement amplification bias in a metagenome.

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Marine, Rachel; McCarren, Coleen; Vorrasane, Vansay; Nasko, Dan; Crowgey, Erin; Polson, Shawn W; Wommack, K Eric

2014-01-30

Shotgun metagenomics has become an important tool for investigating the ecology of microorganisms. Underlying these investigations is the assumption that metagenome sequence data accurately estimates the census of microbial populations. Multiple displacement amplification (MDA) of microbial community DNA is often used in cases where it is difficult to obtain enough DNA for sequencing; however, MDA can result in amplification biases that may impact subsequent estimates of population census from metagenome data. Some have posited that pooling replicate MDA reactions negates these biases and restores the accuracy of population analyses. This assumption has not been empirically tested. Using mock viral communities, we examined the influence of pooling on population-scale analyses. In pooled and single reaction MDA treatments, sequence coverage of viral populations was highly variable and coverage patterns across viral genomes were nearly identical, indicating that initial priming biases were reproducible and that pooling did not alleviate biases. In contrast, control unamplified sequence libraries showed relatively even coverage across phage genomes. MDA should be avoided for metagenomic investigations that require quantitative estimates of microbial taxa and gene functional groups. While MDA is an indispensable technique in applications such as single-cell genomics, amplification biases cannot be overcome by combining replicate MDA reactions. Alternative library preparation techniques should be utilized for quantitative microbial ecology studies utilizing metagenomic sequencing approaches.

Metagenomic discovery of novel enzymes and biosurfactants in a slaughterhouse biofilm microbial community

Science.gov (United States)

Thies, Stephan; Rausch, Sonja Christina; Kovacic, Filip; Schmidt-Thaler, Alexandra; Wilhelm, Susanne; Rosenau, Frank; Daniel, Rolf; Streit, Wolfgang; Pietruszka, Jörg; Jaeger, Karl-Erich

2016-01-01

DNA derived from environmental samples is a rich source of novel bioactive molecules. The choice of the habitat to be sampled predefines the properties of the biomolecules to be discovered due to the physiological adaptation of the microbial community to the prevailing environmental conditions. We have constructed a metagenomic library in Escherichia coli DH10b with environmental DNA (eDNA) isolated from the microbial community of a slaughterhouse drain biofilm consisting mainly of species from the family Flavobacteriaceae. By functional screening of this library we have identified several lipases, proteases and two clones (SA343 and SA354) with biosurfactant and hemolytic activities. Sequence analysis of the respective eDNA fragments and subsequent structure homology modelling identified genes encoding putative N-acyl amino acid synthases with a unique two-domain organisation. The produced biosurfactants were identified by NMR spectroscopy as N-acyltyrosines with N-myristoyltyrosine as the predominant species. Critical micelle concentration and reduction of surface tension were similar to those of chemically synthesised N-myristoyltyrosine. Furthermore, we showed that the newly isolated N-acyltyrosines exhibit antibiotic activity against various bacteria. This is the first report describing the successful application of functional high-throughput screening assays for the identification of biosurfactant producing clones within a metagenomic library. PMID:27271534

Isolation and characterization of novel lipases/esterases from a bovine rumen metagenome.

Science.gov (United States)

Privé, Florence; Newbold, C Jamie; Kaderbhai, Naheed N; Girdwood, Susan G; Golyshina, Olga V; Golyshin, Peter N; Scollan, Nigel D; Huws, Sharon A

2015-07-01

Improving the health beneficial fatty acid content of meat and milk is a major challenge requiring an increased understanding of rumen lipid metabolism. In this study, we isolated and characterized rumen bacterial lipases/esterases using functional metagenomics. Metagenomic libraries were constructed from DNA extracted from strained rumen fluid (SRF), solid-attached bacteria (SAB) and liquid-associated rumen bacteria (LAB), ligated into a fosmid vector and subsequently transformed into an Escherichia coli host. Fosmid libraries consisted of 7,744; 8,448; and 7,680 clones with an average insert size of 30 to 35 kbp for SRF, SAB and LAB, respectively. Transformants were screened on spirit blue agar plates containing tributyrin for lipase/esterase activity. Five SAB and four LAB clones exhibited lipolytic activity, and no positive clones were found in the SRF library. Fosmids from positive clones were pyrosequenced and twelve putative lipase/esterase genes and two phospholipase genes retrieved. Although the derived proteins clustered into diverse esterase and lipase families, a degree of novelty was seen, with homology ranging from 40 to 78% following BlastP searches. Isolated lipases/esterases exhibited activity against mostly short- to medium-chain substrates across a range of temperatures and pH. The function of these novel enzymes recovered in ruminal metabolism needs further investigation, alongside their potential industrial uses.

Analyses of an expressed sequence tag library from Taenia solium, Cysticerca.

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Jonas Lundström

Full Text Available BACKGROUND: Neurocysticercosis is a disease caused by the oral ingestion of eggs from the human parasitic worm Taenia solium. Although drugs are available they are controversial because of the side effects and poor efficiency. An expressed sequence tag (EST library is a method used to describe the gene expression profile and sequence of mRNA from a specific organism and stage. Such information can be used in order to find new targets for the development of drugs and to get a better understanding of the parasite biology. METHODS AND FINDINGS: Here an EST library consisting of 5760 sequences from the pig cysticerca stage has been constructed. In the library 1650 unique sequences were found and of these, 845 sequences (52% were novel to T. solium and not identified within other EST libraries. Furthermore, 918 sequences (55% were of unknown function. Amongst the 25 most frequently expressed sequences 6 had no relevant similarity to other sequences found in the Genbank NR DNA database. A prediction of putative signal peptides was also performed and 4 among the 25 were found to be predicted with a signal peptide. Proposed vaccine and diagnostic targets T24, Tsol18/HP6 and Tso31d could also be identified among the 25 most frequently expressed. CONCLUSIONS: An EST library has been produced from pig cysticerca and analyzed. More than half of the different ESTs sequenced contained a sequence with no suggested function and 845 novel EST sequences have been identified. The library increases the knowledge about what genes are expressed and to what level. It can also be used to study different areas of research such as drug and diagnostic development together with parasite fitness via e.g. immune modulation.

Diverse circovirus-like genome architectures revealed by environmental metagenomics.

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Rosario, Karyna; Duffy, Siobain; Breitbart, Mya

2009-10-01

Single-stranded DNA (ssDNA) viruses with circular genomes are the smallest viruses known to infect eukaryotes. The present study identified 10 novel genomes similar to ssDNA circoviruses through data-mining of public viral metagenomes. The metagenomic libraries included samples from reclaimed water and three different marine environments (Chesapeake Bay, British Columbia coastal waters and Sargasso Sea). All the genomes have similarities to the replication (Rep) protein of circoviruses; however, only half have genomic features consistent with known circoviruses. Some of the genomes exhibit a mixture of genomic features associated with different families of ssDNA viruses (i.e. circoviruses, geminiviruses and parvoviruses). Unique genome architectures and phylogenetic analysis of the Rep protein suggest that these viruses belong to novel genera and/or families. Investigating the complex community of ssDNA viruses in the environment can lead to the discovery of divergent species and help elucidate evolutionary links between ssDNA viruses.

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.

Marine metagenomics: strategies for the discovery of novel enzymes with biotechnological applications from marine environments

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Dobson Alan DW

2008-08-01

Full Text Available Abstract Metagenomic based strategies have previously been successfully employed as powerful tools to isolate and identify enzymes with novel biocatalytic activities from the unculturable component of microbial communities from various terrestrial environmental niches. Both sequence based and function based screening approaches have been employed to identify genes encoding novel biocatalytic activities and metabolic pathways from metagenomic libraries. While much of the focus to date has centred on terrestrial based microbial ecosystems, it is clear that the marine environment has enormous microbial biodiversity that remains largely unstudied. Marine microbes are both extremely abundant and diverse; the environments they occupy likewise consist of very diverse niches. As culture-dependent methods have thus far resulted in the isolation of only a tiny percentage of the marine microbiota the application of metagenomic strategies holds great potential to study and exploit the enormous microbial biodiversity which is present within these marine environments.

Culture-independent discovery of natural products from soil metagenomes.

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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.

Potential and pitfalls of eukaryotic metagenome skimming: a test case for lichens.

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Greshake, Bastian; Zehr, Simonida; Dal Grande, Francesco; Meiser, Anjuli; Schmitt, Imke; Ebersberger, Ingo

2016-03-01

Whole-genome shotgun sequencing of multispecies communities using only a single library layout is commonly used to assess taxonomic and functional diversity of microbial assemblages. Here, we investigate to what extent such metagenome skimming approaches are applicable for in-depth genomic characterizations of eukaryotic communities, for example lichens. We address how to best assemble a particular eukaryotic metagenome skimming data, what pitfalls can occur, and what genome quality can be expected from these data. To facilitate a project-specific benchmarking, we introduce the concept of twin sets, simulated data resembling the outcome of a particular metagenome sequencing study. We show that the quality of genome reconstructions depends essentially on assembler choice. Individual tools, including the metagenome assemblers Omega and MetaVelvet, are surprisingly sensitive to low and uneven coverages. In combination with the routine of assembly parameter choice to optimize the assembly N50 size, these tools can preclude an entire genome from the assembly. In contrast, MIRA, an all-purpose overlap assembler, and SPAdes, a multisized de Bruijn graph assembler, facilitate a comprehensive view on the individual genomes across a wide range of coverage ratios. Testing assemblers on a real-world metagenome skimming data from the lichen Lasallia pustulata demonstrates the applicability of twin sets for guiding method selection. Furthermore, it reveals that the assembly outcome for the photobiont Trebouxia sp. falls behind the a priori expectation given the simulations. Although the underlying reasons remain still unclear, this highlights that further studies on this organism require special attention during sequence data generation and downstream analysis. © 2015 John Wiley & Sons Ltd.

An exceptionally cold-adapted alpha-amylase from a metagenomic library of a cold and alkaline environment

DEFF Research Database (Denmark)

Vester, Jan Kjølhede; Glaring, Mikkel Andreas; Stougaard, Peter

2015-01-01

A cold-active α-amylase, AmyI3C6, identified by a functional metagenomics approach was expressed in Escherichia coli and purified to homogeneity. Sequence analysis showed that the AmyI3C6 amylase was similar to α-amylases from the class Clostridia and revealed classical characteristics of cold......-adapted enzymes, as did comparison of the kinetic parameters Km and kcat to a mesophilic α-amylase. AmyI3C6 was shown to be heat-labile. Temperature optimum was at 10-15 °C, and more than 70 % of the relative activity was retained at 1 °C. The pH optimum of AmyI3C6 was at pH 8-9, and the enzyme displayed activity...... in two commercial detergents tested, suggesting that the AmyI3C6 α-amylase may be useful as a detergent enzyme in environmentally friendly, low-temperature laundry processes....

Generation and evaluation of mammalian secreted and membrane protein expression libraries for high-throughput target discovery.

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Panavas, Tadas; Lu, Jin; Liu, Xuesong; Winkis, Ann-Marie; Powers, Gordon; Naso, Michael F; Amegadzie, Bernard

2011-09-01

Expressed protein libraries are becoming a critical tool for new target discovery in the pharmaceutical industry. In order to get the most meaningful and comprehensive results from protein library screens, it is essential to have library proteins in their native conformation with proper post-translation modifications. This goal is achieved by expressing untagged human proteins in a human cell background. We optimized the transfection and cell culture conditions to maximize protein expression in a 96-well format so that the expression levels were comparable with the levels observed in shake flasks. For detection purposes, we engineered a 'tag after stop codon' system. Depending on the expression conditions, it was possible to express either native or tagged proteins from the same expression vector set. We created a human secretion protein library of 1432 candidates and a small plasma membrane protein set of about 500 candidates. Utilizing the optimized expression conditions, we expressed and analyzed both libraries by SDS-PAGE gel electrophoresis and Western blotting. Two thirds of secreted proteins could be detected by Western-blot analyses; almost half of them were visible on Coomassie stained gels. In this paper, we describe protein expression libraries that can be easily produced in mammalian expression systems in a 96-well format, with one protein expressed per well. The libraries and methods described allow for the development of robust, high-throughput functional screens designed to assay for protein specific functions associated with a relevant disease-specific activity. Copyright © 2011 Elsevier Inc. All rights reserved.

Applying Shannon's information theory to bacterial and phage genomes and metagenomes

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Akhter, Sajia; Bailey, Barbara A.; Salamon, Peter; Aziz, Ramy K.; Edwards, Robert A.

2013-01-01

All sequence data contain inherent information that can be measured by Shannon's uncertainty theory. Such measurement is valuable in evaluating large data sets, such as metagenomic libraries, to prioritize their analysis and annotation, thus saving computational resources. Here, Shannon's index of complete phage and bacterial genomes was examined. The information content of a genome was found to be highly dependent on the genome length, GC content, and sequence word size. In metagenomic sequences, the amount of information correlated with the number of matches found by comparison to sequence databases. A sequence with more information (higher uncertainty) has a higher probability of being significantly similar to other sequences in the database. Measuring uncertainty may be used for rapid screening for sequences with matches in available database, prioritizing computational resources, and indicating which sequences with no known similarities are likely to be important for more detailed analysis.

Bioinformatics tools for quantitative and functional metagenome and metatranscriptome data analysis in microbes.

Science.gov (United States)

Niu, Sheng-Yong; Yang, Jinyu; McDermaid, Adam; Zhao, Jing; Kang, Yu; Ma, Qin

2017-05-08

Metagenomic and metatranscriptomic sequencing approaches are more frequently being used to link microbiota to important diseases and ecological changes. Many analyses have been used to compare the taxonomic and functional profiles of microbiota across habitats or individuals. While a large portion of metagenomic analyses focus on species-level profiling, some studies use strain-level metagenomic analyses to investigate the relationship between specific strains and certain circumstances. Metatranscriptomic analysis provides another important insight into activities of genes by examining gene expression levels of microbiota. Hence, combining metagenomic and metatranscriptomic analyses will help understand the activity or enrichment of a given gene set, such as drug-resistant genes among microbiome samples. Here, we summarize existing bioinformatics tools of metagenomic and metatranscriptomic data analysis, the purpose of which is to assist researchers in deciding the appropriate tools for their microbiome studies. Additionally, we propose an Integrated Meta-Function mapping pipeline to incorporate various reference databases and accelerate functional gene mapping procedures for both metagenomic and metatranscriptomic analyses. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Exploration of noncoding sequences in metagenomes.

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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.

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)

Metagenome Assembly at the DOE JGI (Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

Energy Technology Data Exchange (ETDEWEB)

Chain, Patrick

2011-10-13

Patrick Chain of DOE JGI at LANL, Co-Chair of the Metagenome-specific Assembly session, on Metagenome Assembly at the DOE JGIat the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

Functional cloning using pFB retroviral cDNA expression libraries.

Science.gov (United States)

Felts, Katherine A; Chen, Keith; Zaharee, Kim; Sundar, Latha; Limjoco, Jamie; Miller, Anna; Vaillancourt, Peter

2002-09-01

Retroviral cDNA expression libraries allow the efficient introduction of complex cDNA libraries into virtually any mitotic cell type for screening based on gene function. The cDNA copy number per cell can be easily controlled by adjusting the multiplicity of infection, thus cell populations may be generated in which >90% of infected cells contain one to three cDNAs. We describe the isolation of two known oncogenes and one cell-surface receptor from a human Burkitt's lymphoma (Daudi) cDNA library inserted into the high-titer retroviral vector pFB.

Exploring neighborhoods in the metagenome universe.

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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.

Microbiological profile of chicken carcasses: A comparative analysis using shotgun metagenomic sequencing

Directory of Open Access Journals (Sweden)

Alessandra De Cesare

2018-04-01

Full Text Available In the last few years metagenomic and 16S rRNA sequencing have completly changed the microbiological investigations of food products. In this preliminary study, the microbiological profile of chicken carcasses collected from animals fed with different diets were tested by using shotgun metagenomic sequencing. A total of 15 carcasses have been collected at the slaughetrhouse at the end of the refrigeration tunnel from chickens reared for 35 days and fed with a control diet (n=5, a diet supplemented with 1500 FTU/kg of commercial phytase (n=5 and a diet supplemented with 1500 FTU/kg of commercial phytase and 3g/kg of inositol (n=5. Ten grams of neck and breast skin were obtained from each carcass and submited to total DNA extraction by using the DNeasy Blood & Tissue Kit (Qiagen. Sequencing libraries have been prepared by using the Nextera XT DNA Library Preparation Kit (Illumina and sequenced in a HiScanSQ (Illumina at 100 bp in paired ends. A number of sequences ranging between 5 and 9 million was obtained for each sample. Sequence analysis showed that Proteobacteria and Firmicutes represented more than 98% of whole bacterial populations associated to carcass skin in all groups but their abundances were different between groups. Moraxellaceae and other degradative bacteria showed a significantly higher abundance in the control compared to the treated groups. Furthermore, Clostridium perfringens showed a relative frequency of abundance significantly higher in the group fed with phytase and Salmonella enterica in the group fed with phytase plus inositol. The results of this preliminary study showed that metagenome sequencing is suitable to investigate and monitor carcass microbiota in order to detect specific pathogenic and/or degradative populations.

Evaluation of a transposase protocol for rapid generation of shotgun high-throughput sequencing libraries from nanogram quantities of DNA.

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Marine, Rachel; Polson, Shawn W; Ravel, Jacques; Hatfull, Graham; Russell, Daniel; Sullivan, Matthew; Syed, Fraz; Dumas, Michael; Wommack, K Eric

2011-11-01

Construction of DNA fragment libraries for next-generation sequencing can prove challenging, especially for samples with low DNA yield. Protocols devised to circumvent the problems associated with low starting quantities of DNA can result in amplification biases that skew the distribution of genomes in metagenomic data. Moreover, sample throughput can be slow, as current library construction techniques are time-consuming. This study evaluated Nextera, a new transposon-based method that is designed for quick production of DNA fragment libraries from a small quantity of DNA. The sequence read distribution across nine phage genomes in a mock viral assemblage met predictions for six of the least-abundant phages; however, the rank order of the most abundant phages differed slightly from predictions. De novo genome assemblies from Nextera libraries provided long contigs spanning over half of the phage genome; in four cases where full-length genome sequences were available for comparison, consensus sequences were found to match over 99% of the genome with near-perfect identity. Analysis of areas of low and high sequence coverage within phage genomes indicated that GC content may influence coverage of sequences from Nextera libraries. Comparisons of phage genomes prepared using both Nextera and a standard 454 FLX Titanium library preparation protocol suggested that the coverage biases according to GC content observed within the Nextera libraries were largely attributable to bias in the Nextera protocol rather than to the 454 sequencing technology. Nevertheless, given suitable sequence coverage, the Nextera protocol produced high-quality data for genomic studies. For metagenomics analyses, effects of GC amplification bias would need to be considered; however, the library preparation standardization that Nextera provides should benefit comparative metagenomic analyses.

Identification of immune protective genes of Eimeria maxima through cDNA expression library screening.

Science.gov (United States)

Yang, XinChao; Li, MengHui; Liu, JianHua; Ji, YiHong; Li, XiangRui; Xu, LiXin; Yan, RuoFeng; Song, XiaoKai

2017-02-16

Eimeria maxima is one of the most prevalent Eimeria species causing avian coccidiosis, and results in huge economic loss to the global poultry industry. Current control strategies, such as anti-coccidial medication and live vaccines have been limited because of their drawbacks. The third generation anticoccidial vaccines including the recombinant vaccines as well as DNA vaccines have been suggested as a promising alternative strategy. To date, only a few protective antigens of E. maxima have been reported. Hence, there is an urgent need to identify novel protective antigens of E. maxima for the development of neotype anticoccidial vaccines. With the aim of identifying novel protective genes of E. maxima, a cDNA expression library of E. maxima sporozoites was constructed using Gateway technology. Subsequently, the cDNA expression library was divided into 15 sub-libraries for cDNA expression library immunization (cDELI) using parasite challenged model in chickens. Protective sub-libraries were selected for the next round of screening until individual protective clones were obtained, which were further sequenced and analyzed. Adopting the Gateway technology, a high-quality entry library was constructed, containing 9.2 × 10 6 clones with an average inserted fragments length of 1.63 kb. The expression library capacity was 2.32 × 10 7 colony-forming units (cfu) with an average inserted fragments length of 1.64 Kb. The expression library was screened using parasite challenged model in chickens. The screening yielded 6 immune protective genes including four novel protective genes of EmJS-1, EmRP, EmHP-1 and EmHP-2, and two known protective genes of EmSAG and EmCKRS. EmJS-1 is the selR domain-containing protein of E. maxima whose function is unknown. EmHP-1 and EmHP-2 are the hypothetical proteins of E. maxima. EmRP and EmSAG are rhomboid-like protein and surface antigen glycoproteins of E. maxima respectively, and involved in invasion of the parasite. Our

In vitro and in silico characterization of metagenomic soil-derived cellulases capable of hydrolyzing oil palm empty fruit bunch

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Laura Marcela Palma Medina

2017-09-01

Full Text Available Diversification of raw material for biofuel production is of interest to both academia and industry. One attractive substrate is a renewable lignocellulosic material such as oil palm (Elaeis guineensis Jacq. empty fruit bunch (OPEFB, which is a byproduct of the palm oil industry. This study aimed to characterize cellulases active against this substrate. Cellulases with activity against OPEFB were identified from a metagenomic library obtained from DNA extracted from a high-Andean forest ecosystem. Our findings show that the highest cellulolytic activities were obtained at pH and temperature ranges of 4–10 and 30 °C–60 °C, respectively. Due to the heterogeneous character of the system, degradation profiles were fitted to a fractal-like kinetic model, evidencing transport mass transfer limitations. The sequence analysis of the metagenomic library inserts revealed three glycosyl hydrolase families. Finally, molecular docking simulations of the cellulases were carried out corroborating possible exoglucanase and β-glucosidase activity.

EST Express: PHP/MySQL based automated annotation of ESTs from expression libraries.

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Smith, Robin P; Buchser, William J; Lemmon, Marcus B; Pardinas, Jose R; Bixby, John L; Lemmon, Vance P

2008-04-10

Several biological techniques result in the acquisition of functional sets of cDNAs that must be sequenced and analyzed. The emergence of redundant databases such as UniGene and centralized annotation engines such as Entrez Gene has allowed the development of software that can analyze a great number of sequences in a matter of seconds. We have developed "EST Express", a suite of analytical tools that identify and annotate ESTs originating from specific mRNA populations. The software consists of a user-friendly GUI powered by PHP and MySQL that allows for online collaboration between researchers and continuity with UniGene, Entrez Gene and RefSeq. Two key features of the software include a novel, simplified Entrez Gene parser and tools to manage cDNA library sequencing projects. We have tested the software on a large data set (2,016 samples) produced by subtractive hybridization. EST Express is an open-source, cross-platform web server application that imports sequences from cDNA libraries, such as those generated through subtractive hybridization or yeast two-hybrid screens. It then provides several layers of annotation based on Entrez Gene and RefSeq to allow the user to highlight useful genes and manage cDNA library projects.

EST Express: PHP/MySQL based automated annotation of ESTs from expression libraries

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Pardinas Jose R

2008-04-01

Full Text Available Abstract Background Several biological techniques result in the acquisition of functional sets of cDNAs that must be sequenced and analyzed. The emergence of redundant databases such as UniGene and centralized annotation engines such as Entrez Gene has allowed the development of software that can analyze a great number of sequences in a matter of seconds. Results We have developed "EST Express", a suite of analytical tools that identify and annotate ESTs originating from specific mRNA populations. The software consists of a user-friendly GUI powered by PHP and MySQL that allows for online collaboration between researchers and continuity with UniGene, Entrez Gene and RefSeq. Two key features of the software include a novel, simplified Entrez Gene parser and tools to manage cDNA library sequencing projects. We have tested the software on a large data set (2,016 samples produced by subtractive hybridization. Conclusion EST Express is an open-source, cross-platform web server application that imports sequences from cDNA libraries, such as those generated through subtractive hybridization or yeast two-hybrid screens. It then provides several layers of annotation based on Entrez Gene and RefSeq to allow the user to highlight useful genes and manage cDNA library projects.

A primer on metagenomics.

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John C Wooley

2010-02-01

Full Text Available 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.

Critical Assessment of Metagenome Interpretation – a benchmark of computational metagenomics software

Science.gov (United States)

Sczyrba, Alexander; Hofmann, Peter; Belmann, Peter; Koslicki, David; Janssen, Stefan; Dröge, Johannes; Gregor, Ivan; Majda, Stephan; Fiedler, Jessika; Dahms, Eik; Bremges, Andreas; Fritz, Adrian; Garrido-Oter, Ruben; Jørgensen, Tue Sparholt; Shapiro, Nicole; Blood, Philip D.; Gurevich, Alexey; Bai, Yang; Turaev, Dmitrij; DeMaere, Matthew Z.; Chikhi, Rayan; Nagarajan, Niranjan; Quince, Christopher; Meyer, Fernando; Balvočiūtė, Monika; Hansen, Lars Hestbjerg; Sørensen, Søren J.; Chia, Burton K. H.; Denis, Bertrand; Froula, Jeff L.; Wang, Zhong; Egan, Robert; Kang, Dongwan Don; Cook, Jeffrey J.; Deltel, Charles; Beckstette, Michael; Lemaitre, Claire; Peterlongo, Pierre; Rizk, Guillaume; Lavenier, Dominique; Wu, Yu-Wei; Singer, Steven W.; Jain, Chirag; Strous, Marc; Klingenberg, Heiner; Meinicke, Peter; Barton, Michael; Lingner, Thomas; Lin, Hsin-Hung; Liao, Yu-Chieh; Silva, Genivaldo Gueiros Z.; Cuevas, Daniel A.; Edwards, Robert A.; Saha, Surya; Piro, Vitor C.; Renard, Bernhard Y.; Pop, Mihai; Klenk, Hans-Peter; Göker, Markus; Kyrpides, Nikos C.; Woyke, Tanja; Vorholt, Julia A.; Schulze-Lefert, Paul; Rubin, Edward M.; Darling, Aaron E.; Rattei, Thomas; McHardy, Alice C.

2018-01-01

In metagenome analysis, computational methods for assembly, taxonomic profiling and binning are key components facilitating downstream biological data interpretation. However, a lack of consensus about benchmarking datasets and evaluation metrics complicates proper performance assessment. The Critical Assessment of Metagenome Interpretation (CAMI) challenge has engaged the global developer community to benchmark their programs on datasets of unprecedented complexity and realism. Benchmark metagenomes were generated from ~700 newly sequenced microorganisms and ~600 novel viruses and plasmids, including genomes with varying degrees of relatedness to each other and to publicly available ones and representing common experimental setups. Across all datasets, assembly and genome binning programs performed well for species represented by individual genomes, while performance was substantially affected by the presence of related strains. Taxonomic profiling and binning programs were proficient at high taxonomic ranks, with a notable performance decrease below the family level. Parameter settings substantially impacted performances, underscoring the importance of program reproducibility. While highlighting current challenges in computational metagenomics, the CAMI results provide a roadmap for software selection to answer specific research questions. PMID:28967888

A Study on the Interaction of Rhodamine B with Methylthioadenosine Phosphorylase Protein Sourced from an Antarctic Soil Metagenomic Library

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Bujacz, Anna; Wierzbicka-Woś, Anna; Kur, Józef

2013-01-01

The presented study examines the phenomenon of the fluorescence under UV light excitation (312 nm) of E. coli cells expressing a novel metagenomic-derived putative methylthioadenosine phosphorylase gene, called rsfp, grown on LB agar supplemented with a fluorescent dye rhodamine B. For this purpose, an rsfp gene was cloned and expressed in an LMG194 E. coli strain using an arabinose promoter. The resulting RSFP protein was purified and its UV-VIS absorbance spectrum and emission spectrum were assayed. Simultaneously, the same spectroscopic studies were carried out for rhodamine B in the absence or presence of RSFP protein or native E. coli proteins, respectively. The results of the spectroscopic studies suggested that the fluorescence of E. coli cells expressing rsfp gene under UV illumination is due to the interaction of rhodamine B molecules with the RSFP protein. Finally, this interaction was proved by a crystallographic study and then by site-directed mutagenesis of rsfp gene sequence. The crystal structures of RSFP apo form (1.98 Å) and complex RSFP/RB (1.90 Å) show a trimer of RSFP molecules located on the crystallographic six fold screw axis. The RSFP complex with rhodamine B revealed the binding site for RB, in the pocket located on the interface between symmetry related monomers. PMID:23383268

Web Resources for Metagenomics Studies

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Pravin Dudhagara

2015-10-01

Full Text Available 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.

Assembling large, complex environmental metagenomes

Energy Technology Data Exchange (ETDEWEB)

Howe, A. C. [Michigan State Univ., East Lansing, MI (United States). Microbiology and Molecular Genetics, Plant Soil and Microbial Sciences; Jansson, J. [USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Earth Sciences Division; Malfatti, S. A. [USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Tringe, S. G. [USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Tiedje, J. M. [Michigan State Univ., East Lansing, MI (United States). Microbiology and Molecular Genetics, Plant Soil and Microbial Sciences; Brown, C. T. [Michigan State Univ., East Lansing, MI (United States). Microbiology and Molecular Genetics, Computer Science and Engineering

2012-12-28

The large volumes of sequencing data required to sample complex environments deeply pose new challenges to sequence analysis approaches. De novo metagenomic assembly effectively reduces the total amount of data to be analyzed but requires significant computational resources. We apply two pre-assembly filtering approaches, digital normalization and partitioning, to make large metagenome assemblies more computationaly tractable. Using a human gut mock community dataset, we demonstrate that these methods result in assemblies nearly identical to assemblies from unprocessed data. We then assemble two large soil metagenomes from matched Iowa corn and native prairie soils. The predicted functional content and phylogenetic origin of the assembled contigs indicate significant taxonomic differences despite similar function. The assembly strategies presented are generic and can be extended to any metagenome; full source code is freely available under a BSD license.

A multi-substrate approach for functional metagenomics-based screening for (hemi)cellulases in two wheat straw-degrading microbial consortia unveils novel thermoalkaliphilic enzymes.

Science.gov (United States)

Maruthamuthu, Mukil; Jiménez, Diego Javier; Stevens, Patricia; van Elsas, Jan Dirk

2016-01-28

Functional metagenomics is a promising strategy for the exploration of the biocatalytic potential of microbiomes in order to uncover novel enzymes for industrial processes (e.g. biorefining or bleaching pulp). Most current methodologies used to screen for enzymes involved in plant biomass degradation are based on the use of single substrates. Moreover, highly diverse environments are used as metagenomic sources. However, such methods suffer from low hit rates of positive clones and hence the discovery of novel enzymatic activities from metagenomes has been hampered. Here, we constructed fosmid libraries from two wheat straw-degrading microbial consortia, denoted RWS (bred on untreated wheat straw) and TWS (bred on heat-treated wheat straw). Approximately 22,000 clones from each library were screened for (hemi)cellulose-degrading enzymes using a multi-chromogenic substrate approach. The screens yielded 71 positive clones for both libraries, giving hit rates of 1:440 and 1:1,047 for RWS and TWS, respectively. Seven clones (NT2-2, T5-5, NT18-17, T4-1, 10BT, NT18-21 and T17-2) were selected for sequence analyses. Their inserts revealed the presence of 18 genes encoding enzymes belonging to twelve different glycosyl hydrolase families (GH2, GH3, GH13, GH17, GH20, GH27, GH32, GH39, GH53, GH58, GH65 and GH109). These encompassed several carbohydrate-active gene clusters traceable mainly to Klebsiella related species. Detailed functional analyses showed that clone NT2-2 (containing a beta-galactosidase of ~116 kDa) had highest enzymatic activity at 55 °C and pH 9.0. Additionally, clone T5-5 (containing a beta-xylosidase of ~86 kDa) showed > 90% of enzymatic activity at 55 °C and pH 10.0. This study employed a high-throughput method for rapid screening of fosmid metagenomic libraries for (hemi)cellulose-degrading enzymes. The approach, consisting of screens on multi-substrates coupled to further analyses, revealed high hit rates, as compared with recent other studies. Two

Metagenomic analysis of the rumen microbial community following inhibition of methane formation by a halogenated methane analogue

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Stuart E Denman

2015-10-01

Full Text Available Japanese goats fed a diet of 50% Timothy grass and 50% concentrate with increasing levels of the anti-methanogenic compound, bromochloromethane (BCM were investigated with respect to the microbial shifts in the rumen. Microbial ecology methods identified many species that exhibited positive and negative responses to the increasing levels of BCM. The methane-inhibited rumen appeared to adapt to the higher H2 levels by shifting fermentation to propionate which was mediated by an increase in the population of hydrogen-consuming Prevotella and Selenomonas spp. Metagenomic analysis of propionate production pathways was dominated by genomic content from these species. Reductive acetogenic marker gene libraries and metagenomics analysis indicate that reductive acetogenic species do not play a major role in the BCM treated rumen.

FMAP: Functional Mapping and Analysis Pipeline for metagenomics and metatranscriptomics studies.

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Kim, Jiwoong; Kim, Min Soo; Koh, Andrew Y; Xie, Yang; Zhan, Xiaowei

2016-10-10

Given the lack of a complete and comprehensive library of microbial reference genomes, determining the functional profile of diverse microbial communities is challenging. The available functional analysis pipelines lack several key features: (i) an integrated alignment tool, (ii) operon-level analysis, and (iii) the ability to process large datasets. Here we introduce our open-sourced, stand-alone functional analysis pipeline for analyzing whole metagenomic and metatranscriptomic sequencing data, FMAP (Functional Mapping and Analysis Pipeline). FMAP performs alignment, gene family abundance calculations, and statistical analysis (three levels of analyses are provided: differentially-abundant genes, operons and pathways). The resulting output can be easily visualized with heatmaps and functional pathway diagrams. FMAP functional predictions are consistent with currently available functional analysis pipelines. FMAP is a comprehensive tool for providing functional analysis of metagenomic/metatranscriptomic sequencing data. With the added features of integrated alignment, operon-level analysis, and the ability to process large datasets, FMAP will be a valuable addition to the currently available functional analysis toolbox. We believe that this software will be of great value to the wider biology and bioinformatics communities.

Critical Assessment of Metagenome Interpretation

DEFF Research Database (Denmark)

Sczyrba, Alexander; Hofmann, Peter; Belmann, Peter

2017-01-01

Methods for assembly, taxonomic profiling and binning are key to interpreting metagenome data, but a lack of consensus about benchmarking complicates performance assessment. The Critical Assessment of Metagenome Interpretation (CAMI) challenge has engaged the global developer community to benchma...

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).

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.

Beyond biodiversity: fish metagenomes.

Science.gov (United States)

Ardura, Alba; Planes, Serge; Garcia-Vazquez, Eva

2011-01-01

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.

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.

Gene Prediction in Metagenomic Fragments with Deep Learning

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Shao-Wu Zhang

2017-01-01

Full Text Available Next generation sequencing technologies used in metagenomics yield numerous sequencing fragments which come from thousands of different species. Accurately identifying genes from metagenomics fragments is one of the most fundamental issues in metagenomics. In this article, by fusing multifeatures (i.e., monocodon usage, monoamino acid usage, ORF length coverage, and Z-curve features and using deep stacking networks learning model, we present a novel method (called Meta-MFDL to predict the metagenomic genes. The results with 10 CV and independent tests show that Meta-MFDL is a powerful tool for identifying genes from metagenomic fragments.

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.

Biotechnological applications of functional metagenomics in the food and pharmaceutical industries

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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.

Human milk metagenome: a functional capacity analysis

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2013-01-01

Background Human milk contains a diverse population of bacteria that likely influences colonization of the infant gastrointestinal tract. Recent studies, however, have been limited to characterization of this microbial community by 16S rRNA analysis. In the present study, a metagenomic approach using Illumina sequencing of a pooled milk sample (ten donors) was employed to determine the genera of bacteria and the types of bacterial open reading frames in human milk that may influence bacterial establishment and stability in this primal food matrix. The human milk metagenome was also compared to that of breast-fed and formula-fed infants’ feces (n = 5, each) and mothers’ feces (n = 3) at the phylum level and at a functional level using open reading frame abundance. Additionally, immune-modulatory bacterial-DNA motifs were also searched for within human milk. Results The bacterial community in human milk contained over 360 prokaryotic genera, with sequences aligning predominantly to the phyla of Proteobacteria (65%) and Firmicutes (34%), and the genera of Pseudomonas (61.1%), Staphylococcus (33.4%) and Streptococcus (0.5%). From assembled human milk-derived contigs, 30,128 open reading frames were annotated and assigned to functional categories. When compared to the metagenome of infants’ and mothers’ feces, the human milk metagenome was less diverse at the phylum level, and contained more open reading frames associated with nitrogen metabolism, membrane transport and stress response (P milk metagenome also contained a similar occurrence of immune-modulatory DNA motifs to that of infants’ and mothers’ fecal metagenomes. Conclusions Our results further expand the complexity of the human milk metagenome and enforce the benefits of human milk ingestion on the microbial colonization of the infant gut and immunity. Discovery of immune-modulatory motifs in the metagenome of human milk indicates more exhaustive analyses of the functionality of the human

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.

Cloning and identification of novel hydrolase genes from a dairy cow rumen metagenomic library and characterization of a cellulase gene

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Gong Xia

2012-10-01

Full Text Available Abstract Background Interest in cellulose degrading enzymes has increased in recent years due to the expansion of the cellulosic biofuel industry. The rumen is a highly adapted environment for the degradation of cellulose and a promising source of enzymes for industrial use. To identify cellulase enzymes that may be of such use we have undertaken a functional metagenomic screen to identify cellulase enzymes from the bacterial community in the rumen of a grass-hay fed dairy cow. Results Twenty five clones specifying cellulose activity were identified. Subcloning and sequence analysis of a subset of these hydrolase-positive clones identified 10 endoglucanase genes. Preliminary characterization of the encoded cellulases was carried out using crude extracts of each of the subclones. Zymogram analysis using carboxymethylcellulose as a substrate showed a single positive band for each subclone, confirming that only one functional cellulase gene was present in each. One cellulase gene, designated Cel14b22, was expressed at a high level in Escherichia coli and purified for further characterization. The purified recombinant enzyme showed optimal activity at pH 6.0 and 50°C. It was stable over a broad pH range, from pH 4.0 to 10.0. The activity was significantly enhanced by Mn2+ and dramatically reduced by Fe3+ or Cu2+. The enzyme hydrolyzed a wide range of beta-1,3-, and beta-1,4-linked polysaccharides, with varying activities. Activities toward microcrystalline cellulose and filter paper were relatively high, while the highest activity was toward Oat Gum. Conclusion The present study shows that a functional metagenomic approach can be used to isolate previously uncharacterized cellulases from the rumen environment.

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.

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.

NeSSM: a Next-generation Sequencing Simulator for Metagenomics.

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Ben Jia

Full Text Available BACKGROUND: Metagenomics can reveal the vast majority of microbes that have been missed by traditional cultivation-based methods. Due to its extremely wide range of application areas, fast metagenome sequencing simulation systems with high fidelity are in great demand to facilitate the development and comparison of metagenomics analysis tools. RESULTS: We present here a customizable metagenome simulation system: NeSSM (Next-generation Sequencing Simulator for Metagenomics. Combining complete genomes currently available, a community composition table, and sequencing parameters, it can simulate metagenome sequencing better than existing systems. Sequencing error models based on the explicit distribution of errors at each base and sequencing coverage bias are incorporated in the simulation. In order to improve the fidelity of simulation, tools are provided by NeSSM to estimate the sequencing error models, sequencing coverage bias and the community composition directly from existing metagenome sequencing data. Currently, NeSSM supports single-end and pair-end sequencing for both 454 and Illumina platforms. In addition, a GPU (graphics processing units version of NeSSM is also developed to accelerate the simulation. By comparing the simulated sequencing data from NeSSM with experimental metagenome sequencing data, we have demonstrated that NeSSM performs better in many aspects than existing popular metagenome simulators, such as MetaSim, GemSIM and Grinder. The GPU version of NeSSM is more than one-order of magnitude faster than MetaSim. CONCLUSIONS: NeSSM is a fast simulation system for high-throughput metagenome sequencing. It can be helpful to develop tools and evaluate strategies for metagenomics analysis and it's freely available for academic users at http://cbb.sjtu.edu.cn/~ccwei/pub/software/NeSSM.php.

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.

Comparative metagenomics of the Red Sea

KAUST Repository

Mineta, Katsuhiko

2016-01-01

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

A viral metagenomic approach on a nonmetagenomic experiment

DEFF Research Database (Denmark)

Bovo, Samuele; Mazzoni, Gianluca; Ribani, Anisa

2017-01-01

Shot-gun next generation sequencing (NGS) on whole DNA extracted from specimens collected from mammals often produces reads that are not mapped (i.e. unmapped reads) on the host reference genome and that are usually discarded as by-products of the experiments. In this study, we mined Ion Torrent...... reads obtained by sequencing DNA isolated from archived blood samples collected from 100 performance tested Italian Large White pigs. Two reduced representation libraries were prepared from two DNA pools constructed each from 50 equimolar DNA samples. Bioinformatic analyses were carried out to mine...... unmapped reads on the reference pig genome that were obtained from the two NGS datasets. In silico analyses included read mapping and sequence assembly approaches for a viral metagenomic analysis using the NCBI Viral Genome Resource. Our approach identified sequences matching several viruses...

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.

Novel Cold-Adapted Esterase MHlip from an Antarctic Soil Metagenome

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Moreno Galleni

2013-01-01

Full Text Available An Antarctic soil metagenomic library was screened for lipolytic enzymes and allowed for the isolation of a new cytosolic esterase from the a/b hydrolase family 6, named MHlip. This enzyme is related to hypothetical genes coding esterases, aryl-esterases and peroxydases, among others. MHlip was produced, purified and its activity was determined. The substrate profile of MHlip reveals a high specificity for short p-nitrophenyl-esters. The apparent optimal activity of MHlip was measured for p-nitrophenyl-acetate, at 33 °C, in the pH range of 6–9. The MHlip thermal unfolding was investigated by spectrophotometric methods, highlighting a transition (Tm at 50 °C. The biochemical characterization of this enzyme showed its adaptation to cold temperatures, even when it did not present evident signatures associated with cold-adapted proteins. Thus, MHlip adaptation to cold probably results from many discrete structural modifications, allowing the protein to remain active at low temperatures. Functional metagenomics is a powerful approach to isolate new enzymes with tailored biophysical properties (e.g., cold adaptation. In addition, beside the ever growing amount of sequenced DNA, the functional characterization of new catalysts derived from environment is still required, especially for poorly characterized protein families like α/b hydrolases.

Interactive metagenomic visualization in a Web browser

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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.

Single Cell and Metagenomic Assemblies: Biology Drives Technical Choices and Goals (Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

Energy Technology Data Exchange (ETDEWEB)

Stepanauskas, Ramunas

2011-10-13

DOE JGI's Tanja Woyke, chair of the Single Cells and Metagenomes session, delivers an introduction, followed by Bigelow Laboratory's Ramunas Stepanauskas on "Single Cell and Metagenomic Assemblies: Biology Drives Technical Choices and Goals" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

Marine metagenomics as a source for bioprospecting

KAUST Repository

Kodzius, Rimantas; Gojobori, Takashi

2015-01-01

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

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.

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.

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. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Screening a novel Na+/H+ antiporter gene from a metagenomic library of halophiles colonizing in the Dagong Ancient Brine Well in China.

Science.gov (United States)

Xiang, Wenliang; Zhang, Jie; Li, Lin; Liang, Huazhong; Luo, Hai; Zhao, Jian; Yang, Zhirong; Sun, Qun

2010-05-01

Metagenomic DNA libraries constructed from the Dagong Ancient Brine Well were screened for genes with Na(+)/H(+) antiporter activity on the antiporter-deficient Escherichia coli KNabc strain. One clone with a stable Na(+)-resistant phenotype was obtained and its Na(+)/H(+) antiporter gene was sequenced and designated as m-nha. The deduced amino acid sequence of M-Nha protein consists of 523 residues with a calculated molecular weight of 58 147 Da and a pI of 5.50, which is homologous with NhaH from Halobacillus dabanensis D-8(T) (92%) and Halobacillus aidingensis AD-6(T) (86%), and with Nhe2 from Bacillus sp. NRRL B-14911 (64%). It had a hydropathy profile with 10 putative transmembrane domains and a long carboxyl terminal hydrophilic tail of 140 amino acid residues, similar to Nhap from Synechocystis sp. and Aphanothece halophytica, as well as NhaG from Bacillus subtilis. The m-nha gene in the antiporter-negative mutant E. coli KNabc conferred resistance to Na(+) and the ability to grow under alkaline conditions. The difference in amino acid sequence and the putative secondary structure suggested that the m-nha isolated from the Dagong Ancient Brine Well in this study was a novel Na(+)/H(+) antiporter gene.

Antibiotic Resistome: Improving Detection and Quantification Accuracy for Comparative Metagenomics.

Science.gov (United States)

Elbehery, Ali H A; Aziz, Ramy K; Siam, Rania

2016-04-01

The unprecedented rise of life-threatening antibiotic resistance (AR), combined with the unparalleled advances in DNA sequencing of genomes and metagenomes, has pushed the need for in silico detection of the resistance potential of clinical and environmental metagenomic samples through the quantification of AR genes (i.e., genes conferring antibiotic resistance). Therefore, determining an optimal methodology to quantitatively and accurately assess AR genes in a given environment is pivotal. Here, we optimized and improved existing AR detection methodologies from metagenomic datasets to properly consider AR-generating mutations in antibiotic target genes. Through comparative metagenomic analysis of previously published AR gene abundance in three publicly available metagenomes, we illustrate how mutation-generated resistance genes are either falsely assigned or neglected, which alters the detection and quantitation of the antibiotic resistome. In addition, we inspected factors influencing the outcome of AR gene quantification using metagenome simulation experiments, and identified that genome size, AR gene length, total number of metagenomics reads and selected sequencing platforms had pronounced effects on the level of detected AR. In conclusion, our proposed improvements in the current methodologies for accurate AR detection and resistome assessment show reliable results when tested on real and simulated metagenomic datasets.

Back to the Future of Soil Metagenomics.\

Czech Academy of Sciences Publication Activity Database

Nesme J, J.; Achouak, W.; Agathos SN, S.N.; Bailey, M.; Baldrian, Petr; Brunel, D.; Frostegård, Å.; Heulin, T.; Jansson JK, J.K.; Jurkevitch, E.; Kruus, K.L.; Kowalchuk, G.A.; Lagares, A.; Lapin-Scott, H.M.; Lemanceau, P.; Le Paslier, D.; Mandic-Mulec, I.; Murrell, J.C.; Myrold, D.D.; Nalin, R.; Nannipieri, P.; Neufeld, J.D.; O'Gara, F.; Parnell, J.J.; Pühler, A.; Pylro, V.; Ramos, J.L.; Roesch, L.F.; Schloter, M.; Schleper, C.; Sczyrba, A.; Sessitsch, A.; Sjöling, S.; Sørensen, J.; Sørensen, S.J.; Tebbe, C.C.; Topp, E.; Tsiamis, G.; van Elsas, J.D.; van Keulen, G.; Widmer, F.; Wagner, M.; Zhang, T.; Zhang, X.; Zhao, L; Zhu, Y-G.; Vogel, T.M.; Simonet, P.

2016-01-01

Roč. 7, FEB 10 (2016), s. 73 ISSN 1664-302X Institutional support: RVO:61388971 Keywords : metagenomic * soil microbiology; terrestrial microbiology * metagenomic; soil microbiology; terrestrial microbiology Subject RIV: EE - Microbiology, Virology Impact factor: 4.076, year: 2016

A Novel Multifunctional β-N-Acetylhexosaminidase Revealed through Metagenomics of an Oil-Spilled Mangrove

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Fábio Lino Soares

2017-07-01

Full Text Available The use of culture-independent approaches, such as metagenomics, provides complementary access to environmental microbial diversity. Mangrove environments represent a highly complex system with plenty of opportunities for finding singular functions. In this study we performed a functional screening of fosmid libraries obtained from an oil contaminated mangrove site, with the purpose of identifying clones expressing hydrolytic activities. A novel gene coding for a β-N-acetylhexosaminidase with 355 amino acids and 43KDa was retrieved and characterized. The translated sequence showed only 38% similarity to a β-N-acetylhexosaminidase gene in the genome of Veillonella sp. CAG:933, suggesting that it might constitute a novel enzyme. The enzyme was expressed, purified, and characterized for its enzymatic activity on carboxymethyl cellulose, p-Nitrophenyl-2acetamide-2deoxy-β-d-glucopyranoside, p-Nitrophenyl-2acetamide-2deoxy-β-d-galactopyranoside, and 4-Nitrophenyl β-d-glucopyranoside, presenting β-N-acetylglucosaminidase, β-glucosidase, and β-1,4-endoglucanase activities. The enzyme showed optimum activity at 30 °C and pH 5.5. The characterization of the putative novel β-N-acetylglucosaminidase enzyme reflects similarities to characteristics of the environment explored, which differs from milder conditions environments. This work exemplifies the application of cultivation-independent molecular techniques to the mangrove microbiome for obtaining a novel biotechnological product.

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/.

Expression sequence tag library derived from peripheral blood mononuclear cells of the chlorocebus sabaeus

Directory of Open Access Journals (Sweden)

Tchitchek Nicolas

2012-06-01

Full Text Available Abstract Background African Green Monkeys (AGM are amongst the most frequently used nonhuman primate models in clinical and biomedical research, nevertheless only few genomic resources exist for this species. Such information would be essential for the development of dedicated new generation technologies in fundamental and pre-clinical research using this model, and would deliver new insights into primate evolution. Results We have exhaustively sequenced an Expression Sequence Tag (EST library made from a pool of Peripheral Blood Mononuclear Cells from sixteen Chlorocebus sabaeus monkeys. Twelve of them were infected with the Simian Immunodeficiency Virus. The mononuclear cells were or not stimulated in vitro with Concanavalin A, with lipopolysacharrides, or through mixed lymphocyte reaction in order to generate a representative and broad library of expressed sequences in immune cells. We report here 37,787 sequences, which were assembled into 14,410 contigs representing an estimated 12% of the C. sabaeus transcriptome. Using data from primate genome databases, 9,029 assembled sequences from C. sabaeus could be annotated. Sequences have been systematically aligned with ten cDNA references of primate species including Homo sapiens, Pan troglodytes, and Macaca mulatta to identify ortholog transcripts. For 506 transcripts, sequences were quasi-complete. In addition, 6,576 transcript fragments are potentially specific to the C. sabaeus or corresponding to not yet described primate genes. Conclusions The EST library we provide here will prove useful in gene annotation efforts for future sequencing of the African Green Monkey genomes. Furthermore, this library, which particularly well represents immunological and hematological gene expression, will be an important resource for the comparative analysis of gene expression in clinically relevant nonhuman primate and human research.

Multiple comparative metagenomics using multiset k-mer counting

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Gaëtan Benoit

2016-11-01

Full Text Available Background Large scale metagenomic projects aim to extract biodiversity knowledge between different environmental conditions. Current methods for comparing microbial communities face important limitations. Those based on taxonomical or functional assignation rely on a small subset of the sequences that can be associated to known organisms. On the other hand, de novo methods, that compare the whole sets of sequences, either do not scale up on ambitious metagenomic projects or do not provide precise and exhaustive results. Methods These limitations motivated the development of a new de novo metagenomic comparative method, called Simka. This method computes a large collection of standard ecological distances by replacing species counts by k-mer counts. Simka scales-up today’s metagenomic projects thanks to a new parallel k-mer counting strategy on multiple datasets. Results Experiments on public Human Microbiome Project datasets demonstrate that Simka captures the essential underlying biological structure. Simka was able to compute in a few hours both qualitative and quantitative ecological distances on hundreds of metagenomic samples (690 samples, 32 billions of reads. We also demonstrate that analyzing metagenomes at the k-mer level is highly correlated with extremely precise de novo comparison techniques which rely on all-versus-all sequences alignment strategy or which are based on taxonomic profiling.

Genome signature analysis of thermal virus metagenomes reveals Archaea and thermophilic signatures.

Science.gov (United States)

Pride, David T; Schoenfeld, Thomas

2008-09-17

Metagenomic analysis provides a rich source of biological information for otherwise intractable viral communities. However, study of viral metagenomes has been hampered by its nearly complete reliance on BLAST algorithms for identification of DNA sequences. We sought to develop algorithms for examination of viral metagenomes to identify the origin of sequences independent of BLAST algorithms. We chose viral metagenomes obtained from two hot springs, Bear Paw and Octopus, in Yellowstone National Park, as they represent simple microbial populations where comparatively large contigs were obtained. Thermal spring metagenomes have high proportions of sequences without significant Genbank homology, which has hampered identification of viruses and their linkage with hosts. To analyze each metagenome, we developed a method to classify DNA fragments using genome signature-based phylogenetic classification (GSPC), where metagenomic fragments are compared to a database of oligonucleotide signatures for all previously sequenced Bacteria, Archaea, and viruses. From both Bear Paw and Octopus hot springs, each assembled contig had more similarity to other metagenome contigs than to any sequenced microbial genome based on GSPC analysis, suggesting a genome signature common to each of these extreme environments. While viral metagenomes from Bear Paw and Octopus share some similarity, the genome signatures from each locale are largely unique. GSPC using a microbial database predicts most of the Octopus metagenome has archaeal signatures, while bacterial signatures predominate in Bear Paw; a finding consistent with those of Genbank BLAST. When using a viral database, the majority of the Octopus metagenome is predicted to belong to archaeal virus Families Globuloviridae and Fuselloviridae, while none of the Bear Paw metagenome is predicted to belong to archaeal viruses. As expected, when microbial and viral databases are combined, each of the Octopus and Bear Paw metagenomic contigs

Compositional profile of α/β-hydrolase fold proteins in mangrove soil metagenomes: prevalence of epoxide hydrolases and haloalkane dehalogenases in oil-contaminated sites

Science.gov (United States)

Jiménez, Diego Javier; Dini-Andreote, Francisco; Ottoni, Júlia Ronzella; de Oliveira, Valéria Maia; van Elsas, Jan Dirk; Andreote, Fernando Dini

2015-01-01

The occurrence of genes encoding biotechnologically relevant α/β-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered based on local amino acid alignments against the Lipase Engineering Database. In total, 5923 unassembled sequences were affiliated with 30 different α/β-hydrolase fold superfamilies. The most abundant predicted proteins encompassed cytosolic hydrolases (abH08; ∼ 23%), microsomal hydrolases (abH09; ∼ 12%) and Moraxella lipase-like proteins (abH04 and abH01; mangroves BrMgv01-02-03. This suggested selection and putative involvement in local degradation/detoxification of the pollutants. Seven sequences that were annotated as genes for putative epoxide hydrolases and five for putative haloalkane dehalogenases were found in a fosmid library generated from BrMgv02 DNA. The latter enzymes were predicted to belong to Actinobacteria, Deinococcus-Thermus, Planctomycetes and Proteobacteria. Our integrated approach thus identified 12 genes (complete and/or partial) that may encode hitherto undescribed enzymes. The low amino acid identity (< 60%) with already-described genes opens perspectives for both production in an expression host and genetic screening of metagenomes. PMID:25171437

Developing a Bacteroides System for Function-Based Screening of DNA from the Human Gut Microbiome.

Science.gov (United States)

Lam, Kathy N; Martens, Eric C; Charles, Trevor C

2018-01-01

Functional metagenomics is a powerful method that allows the isolation of genes whose role may not have been predicted from DNA sequence. In this approach, first, environmental DNA is cloned to generate metagenomic libraries that are maintained in Escherichia coli, and second, the cloned DNA is screened for activities of interest. Typically, functional screens are carried out using E. coli as a surrogate host, although there likely exist barriers to gene expression, such as lack of recognition of native promoters. Here, we describe efforts to develop Bacteroides thetaiotaomicron as a surrogate host for screening metagenomic DNA from the human gut. We construct a B. thetaiotaomicron-compatible fosmid cloning vector, generate a fosmid clone library using DNA from the human gut, and show successful functional complementation of a B. thetaiotaomicron glycan utilization mutant. Though we were unable to retrieve the physical fosmid after complementation, we used genome sequencing to identify the complementing genes derived from the human gut microbiome. Our results demonstrate that the use of B. thetaiotaomicron to express metagenomic DNA is promising, but they also exemplify the challenges that can be encountered in the development of new surrogate hosts for functional screening. IMPORTANCE Human gut microbiome research has been supported by advances in DNA sequencing that make it possible to obtain gigabases of sequence data from metagenomes but is limited by a lack of knowledge of gene function that leads to incomplete annotation of these data sets. There is a need for the development of methods that can provide experimental data regarding microbial gene function. Functional metagenomics is one such method, but functional screens are often carried out using hosts that may not be able to express the bulk of the environmental DNA being screened. We expand the range of current screening hosts and demonstrate that human gut-derived metagenomic libraries can be

Unsupervised Two-Way Clustering of Metagenomic Sequences

Directory of Open Access Journals (Sweden)

Shruthi Prabhakara

2012-01-01

Full Text Available A major challenge facing metagenomics is the development of tools for the characterization of functional and taxonomic content of vast amounts of short metagenome reads. The efficacy of clustering methods depends on the number of reads in the dataset, the read length and relative abundances of source genomes in the microbial community. In this paper, we formulate an unsupervised naive Bayes multispecies, multidimensional mixture model for reads from a metagenome. We use the proposed model to cluster metagenomic reads by their species of origin and to characterize the abundance of each species. We model the distribution of word counts along a genome as a Gaussian for shorter, frequent words and as a Poisson for longer words that are rare. We employ either a mixture of Gaussians or mixture of Poissons to model reads within each bin. Further, we handle the high-dimensionality and sparsity associated with the data, by grouping the set of words comprising the reads, resulting in a two-way mixture model. Finally, we demonstrate the accuracy and applicability of this method on simulated and real metagenomes. Our method can accurately cluster reads as short as 100 bps and is robust to varying abundances, divergences and read lengths.

Metagenomics, metatranscriptomics and single cell genomics reveal functional response of active Oceanospirillales to Gulf oil spill

Energy Technology Data Exchange (ETDEWEB)

Mason, Olivia U.; Hazen, Terry C.; Borglin, Sharon; Chain, Patrick S. G.; Dubinsky, Eric A.; Fortney, Julian L.; Han, James; Holman, Hoi-Ying N.; Hultman, Jenni; Lamendella, Regina; Mackelprang, Rachel; Malfatti, Stephanie; Tom, Lauren M.; Tringe, Susannah G.; Woyke, Tanja; Zhou, Jizhong; Rubin, Edward M.; Jansson, Janet K.

2012-06-12

The Deepwater Horizon oil spill in the Gulf of Mexico resulted in a deep-sea hydrocarbon plume that caused a shift in the indigenous microbial community composition with unknown ecological consequences. Early in the spill history, a bloom of uncultured, thus uncharacterized, members of the Oceanospirillales was previously detected, but their role in oil disposition was unknown. Here our aim was to determine the functional role of the Oceanospirillales and other active members of the indigenous microbial community using deep sequencing of community DNA and RNA, as well as single-cell genomics. Shotgun metagenomic and metatranscriptomic sequencing revealed that genes for motility, chemotaxis and aliphatic hydrocarbon degradation were significantly enriched and expressed in the hydrocarbon plume samples compared with uncontaminated seawater collected from plume depth. In contrast, although genes coding for degradation of more recalcitrant compounds, such as benzene, toluene, ethylbenzene, total xylenes and polycyclic aromatic hydrocarbons, were identified in the metagenomes, they were expressed at low levels, or not at all based on analysis of the metatranscriptomes. Isolation and sequencing of two Oceanospirillales single cells revealed that both cells possessed genes coding for n-alkane and cycloalkane degradation. Specifically, the near-complete pathway for cyclohexane oxidation in the Oceanospirillales single cells was elucidated and supported by both metagenome and metatranscriptome data. The draft genome also included genes for chemotaxis, motility and nutrient acquisition strategies that were also identified in the metagenomes and metatranscriptomes. These data point towards a rapid response of members of the Oceanospirillales to aliphatic hydrocarbons in the deep sea.

Critical Assessment of Metagenome Interpretation-a benchmark of metagenomics software.

Science.gov (United States)

Sczyrba, Alexander; Hofmann, Peter; Belmann, Peter; Koslicki, David; Janssen, Stefan; Dröge, Johannes; Gregor, Ivan; Majda, Stephan; Fiedler, Jessika; Dahms, Eik; Bremges, Andreas; Fritz, Adrian; Garrido-Oter, Ruben; Jørgensen, Tue Sparholt; Shapiro, Nicole; Blood, Philip D; Gurevich, Alexey; Bai, Yang; Turaev, Dmitrij; DeMaere, Matthew Z; Chikhi, Rayan; Nagarajan, Niranjan; Quince, Christopher; Meyer, Fernando; Balvočiūtė, Monika; Hansen, Lars Hestbjerg; Sørensen, Søren J; Chia, Burton K H; Denis, Bertrand; Froula, Jeff L; Wang, Zhong; Egan, Robert; Don Kang, Dongwan; Cook, Jeffrey J; Deltel, Charles; Beckstette, Michael; Lemaitre, Claire; Peterlongo, Pierre; Rizk, Guillaume; Lavenier, Dominique; Wu, Yu-Wei; Singer, Steven W; Jain, Chirag; Strous, Marc; Klingenberg, Heiner; Meinicke, Peter; Barton, Michael D; Lingner, Thomas; Lin, Hsin-Hung; Liao, Yu-Chieh; Silva, Genivaldo Gueiros Z; Cuevas, Daniel A; Edwards, Robert A; Saha, Surya; Piro, Vitor C; Renard, Bernhard Y; Pop, Mihai; Klenk, Hans-Peter; Göker, Markus; Kyrpides, Nikos C; Woyke, Tanja; Vorholt, Julia A; Schulze-Lefert, Paul; Rubin, Edward M; Darling, Aaron E; Rattei, Thomas; McHardy, Alice C

2017-11-01

Methods for assembly, taxonomic profiling and binning are key to interpreting metagenome data, but a lack of consensus about benchmarking complicates performance assessment. The Critical Assessment of Metagenome Interpretation (CAMI) challenge has engaged the global developer community to benchmark their programs on highly complex and realistic data sets, generated from ∼700 newly sequenced microorganisms and ∼600 novel viruses and plasmids and representing common experimental setups. Assembly and genome binning programs performed well for species represented by individual genomes but were substantially affected by the presence of related strains. Taxonomic profiling and binning programs were proficient at high taxonomic ranks, with a notable performance decrease below family level. Parameter settings markedly affected performance, underscoring their importance for program reproducibility. The CAMI results highlight current challenges but also provide a roadmap for software selection to answer specific research questions.

Molecular cloning and characterization of a novel pyrethroid-hydrolyzing esterase originating from the Metagenome

Directory of Open Access Journals (Sweden)

Liu Yu

2008-12-01

, purified and characterized. The molecular mass of the native enzyme was approximately 31 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of the Pye3 indicated molecular mass of 31 kDa and 31.5 kDa, respectively, suggesting that the Pye3 is a monomer. The purified Pye3 not only degraded all pyrethroid pesticides tested, but also hydrolyzed ρ-nitrophenyl esters of medium-short chain fatty acids, indicating that the Pye3 is an esterase with broader specificity. The Km values for trans-Permethrin and cis-permethrin are 0.10 μM and 0.18 μM, respectively, and these catalytic properties were superior to carboxylesterases from resistant insects and mammals. The catalytic activity of the Pye3 was strongly inhibited by Hg2+, Ag+, ρ-chloromercuribenzoate, whereas less pronounced effect was observed in the presence of divalent cations, the chelating agent EDTA and phenanthroline. Conclusion A novel pyrethroid-hydrolyzing esterase gene was successfully cloned using metagenomic DNA combined with activity-based functional screening from soil, the broader substrate specificities and higher activity of the pyrethroid-hydrolyzing esterase (Pye3 make it an ideal candidate for in situ for detoxification of pyrethroids where they cause environmental contamination problems. Consequently, metagenomic DNA clone library offers possibilities to discover novel bio-molecules through the expression of genes from uncultivated bacteria.

Genome signature analysis of thermal virus metagenomes reveals Archaea and thermophilic signatures

Directory of Open Access Journals (Sweden)

Pride David T

2008-09-01

Full Text Available Abstract Background Metagenomic analysis provides a rich source of biological information for otherwise intractable viral communities. However, study of viral metagenomes has been hampered by its nearly complete reliance on BLAST algorithms for identification of DNA sequences. We sought to develop algorithms for examination of viral metagenomes to identify the origin of sequences independent of BLAST algorithms. We chose viral metagenomes obtained from two hot springs, Bear Paw and Octopus, in Yellowstone National Park, as they represent simple microbial populations where comparatively large contigs were obtained. Thermal spring metagenomes have high proportions of sequences without significant Genbank homology, which has hampered identification of viruses and their linkage with hosts. To analyze each metagenome, we developed a method to classify DNA fragments using genome signature-based phylogenetic classification (GSPC, where metagenomic fragments are compared to a database of oligonucleotide signatures for all previously sequenced Bacteria, Archaea, and viruses. Results From both Bear Paw and Octopus hot springs, each assembled contig had more similarity to other metagenome contigs than to any sequenced microbial genome based on GSPC analysis, suggesting a genome signature common to each of these extreme environments. While viral metagenomes from Bear Paw and Octopus share some similarity, the genome signatures from each locale are largely unique. GSPC using a microbial database predicts most of the Octopus metagenome has archaeal signatures, while bacterial signatures predominate in Bear Paw; a finding consistent with those of Genbank BLAST. When using a viral database, the majority of the Octopus metagenome is predicted to belong to archaeal virus Families Globuloviridae and Fuselloviridae, while none of the Bear Paw metagenome is predicted to belong to archaeal viruses. As expected, when microbial and viral databases are combined, each of

Identification and characterization of a novel thermostable pyrethroid-hydrolyzing enzyme isolated through metagenomic approach

Directory of Open Access Journals (Sweden)

Fan Xinjiong

2012-03-01

Full Text Available Abstract Background Pyrethroid pesticides are broad-spectrum pest control agents in agricultural production. Both agricultural and residential usage is continuing to grow, leading to the development of insecticide resistance in the pest and toxic effects on a number of nontarget organisms. Thus, it is necessary to hunt suitable enzymes including hydrolases for degrading pesticide residues, which is an efficient "green" solution to biodegrade polluting chemicals. Although many pyrethroid esterases have consistently been purified and characterized from various resources including metagenomes and organisms, the thermostable pyrethroid esterases have not been reported up to the present. Results In this study, we identified a novel pyrethroid-hydrolyzing enzyme Sys410 belonging to familyV esterases/lipases with activity-based functional screening from Turban Basin metagenomic library. Sys410 contained 280 amino acids with a predicted molecular mass (Mr of 30.8 kDa and was overexpressed in Escherichia coli BL21 (DE3 in soluble form. The optimum pH and temperature of the recombinant Sys410 were 6.5 and 55°C, respectively. The enzyme was stable in the pH range of 4.5-8.5 and at temperatures below 50°C. The activity of Sys410 decreased a little when stored at 4°C for 10 weeks, and the residual activity reached 94.1%. Even after incubation at 25°C for 10 weeks, it kept 68.3% of its activity. The recombinant Sys410 could hydrolyze a wide range of ρ-nitrophenyl esters, but its best substrate is ρ-nitrophenyl acetate with the highest activity (772.9 U/mg. The enzyme efficiently degraded cyhalothrin, cypermethrin, sumicidin, and deltamethrin under assay conditions of 37°C for 15 min, with exceeding 95% hydrolysis rate. Conclusion This is the first report to construct metagenomic libraries from Turban Basin to obtain the thermostable pyrethroid-hydrolyzing enzyme. The recombinant Sys410 with broad substrate specificities and high activity was the most

A viral metagenomic approach on a non-metagenomic experiment: Mining next generation sequencing datasets from pig DNA identified several porcine parvoviruses for a retrospective evaluation of viral infections.

Directory of Open Access Journals (Sweden)

Samuele Bovo

Full Text Available Shot-gun next generation sequencing (NGS on whole DNA extracted from specimens collected from mammals often produces reads that are not mapped (i.e. unmapped reads on the host reference genome and that are usually discarded as by-products of the experiments. In this study, we mined Ion Torrent reads obtained by sequencing DNA isolated from archived blood samples collected from 100 performance tested Italian Large White pigs. Two reduced representation libraries were prepared from two DNA pools constructed each from 50 equimolar DNA samples. Bioinformatic analyses were carried out to mine unmapped reads on the reference pig genome that were obtained from the two NGS datasets. In silico analyses included read mapping and sequence assembly approaches for a viral metagenomic analysis using the NCBI Viral Genome Resource. Our approach identified sequences matching several viruses of the Parvoviridae family: porcine parvovirus 2 (PPV2, PPV4, PPV5 and PPV6 and porcine bocavirus 1-H18 isolate (PBoV1-H18. The presence of these viruses was confirmed by PCR and Sanger sequencing of individual DNA samples. PPV2, PPV4, PPV5, PPV6 and PBoV1-H18 were all identified in samples collected in 1998-2007, 1998-2000, 1997-2000, 1998-2004 and 2003, respectively. For most of these viruses (PPV4, PPV5, PPV6 and PBoV1-H18 previous studies reported their first occurrence much later (from 5 to more than 10 years than our identification period and in different geographic areas. Our study provided a retrospective evaluation of apparently asymptomatic parvovirus infected pigs providing information that could be important to define occurrence and prevalence of different parvoviruses in South Europe. This study demonstrated the potential of mining NGS datasets non-originally derived by metagenomics experiments for viral metagenomics analyses in a livestock species.

Immunoresponse of Coho salmon immunized with a gene expression library from Piscirickettsia salmonis

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ALVARO MIQUEL

2003-01-01

Full Text Available We have used the expression library immunization technology to study the protection of Coho salmon Oncorhynchus kisutch to the infection with Piscirickettsia salmonis. Purified DNA from this bacterium was sonicated and the fragments were cloned in the expression vector pCMV-Bios. Two libraries were obtained containing 22,000 and 28,000 colonies and corresponding to approximately 8 and 10 times the genome of the pathogen, respectively. On average, the size of the inserts ranged between 300 and 1,000 bp. The plasmid DNA isolated from one of these libraries was purified and 20 µg were injected intramuscularly into 60 fish followed by a second dose of 10 µg applied 40 days later. As control, fish were injected with the same amount of DNA of the vector pCMV-Bios without insert. The titer of IgM anti-P. salmonis of vaccinated fish, evaluated 60 days post-injection, was significantly higher than that of the control group injected with the vector alone. Moreover, this response was specific against P. salmonis antigens, since no cross reaction was detected with Renibacterium salmoninarum and Yersinia ruckeri. The vaccinated and control fish were challenged 60 days after the second dose of DNA with 2.5 x 10(7 P. salmonis corresponding to 7.5 times the LD50. At 30 days post-challenge, 100% mortality was obtained with the control fish while 20% of the vaccinated animals survived. All surviving fish exhibited a lower bacterial load in the kidney than control fish. The expression library was also tested in Balb/c mice and it was found that the humoral immune response was specific to P. salmonis and it was dependent on the amount of DNA injected

Exploiting HPC Platforms for Metagenomics: Challenges and Opportunities (MICW - Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

Energy Technology Data Exchange (ETDEWEB)

Canon, Shane

2011-10-12

DOE JGI's Zhong Wang, chair of the High-performance Computing session, gives a brief introduction before Berkeley Lab's Shane Canon talks about "Exploiting HPC Platforms for Metagenomics: Challenges and Opportunities" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

Marine Metagenome as A Resource for Novel Enzymes

KAUST Repository

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

2015-01-01

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

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

Cloning, expression and characterization of a novel esterase from a South China Sea sediment metagenome

Science.gov (United States)

Zhang, Hao; Li, Fuchao; Chen, Huaxin; Zhao, Jin; Yan, Jinfei; Jiang, Peng; Li, Ronggui; Zhu, Baoli

2015-07-01

Lipolytic enzymes, including esterases and lipases, represent a group of hydrolases that catalyze the cleavage and formation of ester bonds. A novel esterase gene, scsEst01, was cloned from a South China Sea sediment metagenome. The scsEst01 gene consisted of 921 bp encoding 307 amino acid residues. The predicted amino acid sequence shared less than 90% identity with other lipolytic enzymes in the NCBI nonredundant protein database. ScsEst01 was successfully co-expressed in Escherichia coli BL21 (DE3) with chaperones (dnaK-dnaJ-grpE) to prevent the formation of inclusion bodies. The recombinant protein was purified on an immobilized metal ion affinity column containing chelating Sepharose charged with Ni2+. The enzyme was characterized using p -nitrophenol butyrate as a substrate. ScsEst01 had the highest lipolytic activity at 35°C and pH 8.0, indicative of a meso-thermophilic alkaline esterase. ScsEst01 was thermostable at 20°C. The lipolytic activity of scsEst01 was strongly increased by Fe2+, Mn2+ and 1% Tween 80 or Tween 20.

Assembly of viral genomes from metagenomes

Directory of Open Access Journals (Sweden)

Saskia L Smits

2014-12-01

Full Text Available Viral infections remain a serious global health issue. Metagenomic approaches are increasingly used in the detection of novel viral pathogens but also to generate complete genomes of uncultivated viruses. In silico identification of complete viral genomes from sequence data would allow rapid phylogenetic characterization of these new viruses. Often, however, complete viral genomes are not recovered, but rather several distinct contigs derived from a single entity, some of which have no sequence homology to any known proteins. De novo assembly of single viruses from a metagenome is challenging, not only because of the lack of a reference genome, but also because of intrapopulation variation and uneven or insufficient coverage. Here we explored different assembly algorithms, remote homology searches, genome-specific sequence motifs, k-mer frequency ranking, and coverage profile binning to detect and obtain viral target genomes from metagenomes. All methods were tested on 454-generated sequencing datasets containing three recently described RNA viruses with a relatively large genome which were divergent to previously known viruses from the viral families Rhabdoviridae and Coronaviridae. Depending on specific characteristics of the target virus and the metagenomic community, different assembly and in silico gap closure strategies were successful in obtaining near complete viral genomes.

BioCreative Workshops for DOE Genome Sciences: Text Mining for Metagenomics

Energy Technology Data Exchange (ETDEWEB)

Wu, Cathy H. [Univ. of Delaware, Newark, DE (United States). Center for Bioinformatics and Computational Biology; Hirschman, Lynette [The MITRE Corporation, Bedford, MA (United States)

2016-10-29

The objective of this project was to host BioCreative workshops to define and develop text mining tasks to meet the needs of the Genome Sciences community, focusing on metadata information extraction in metagenomics. Following the successful introduction of metagenomics at the BioCreative IV workshop, members of the metagenomics community and BioCreative communities continued discussion to identify candidate topics for a BioCreative metagenomics track for BioCreative V. Of particular interest was the capture of environmental and isolation source information from text. The outcome was to form a “community of interest” around work on the interactive EXTRACT system, which supported interactive tagging of environmental and species data. This experiment is included in the BioCreative V virtual issue of Database. In addition, there was broad participation by members of the metagenomics community in the panels held at BioCreative V, leading to valuable exchanges between the text mining developers and members of the metagenomics research community. These exchanges are reflected in a number of the overview and perspective pieces also being captured in the BioCreative V virtual issue. Overall, this conversation has exposed the metagenomics researchers to the possibilities of text mining, and educated the text mining developers to the specific needs of the metagenomics community.

Tentacle: distributed quantification of genes in metagenomes.

Science.gov (United States)

Boulund, Fredrik; Sjögren, Anders; Kristiansson, Erik

2015-01-01

In metagenomics, microbial communities are sequenced at increasingly high resolution, generating datasets with billions of DNA fragments. Novel methods that can efficiently process the growing volumes of sequence data are necessary for the accurate analysis and interpretation of existing and upcoming metagenomes. Here we present Tentacle, which is a novel framework that uses distributed computational resources for gene quantification in metagenomes. Tentacle is implemented using a dynamic master-worker approach in which DNA fragments are streamed via a network and processed in parallel on worker nodes. Tentacle is modular, extensible, and comes with support for six commonly used sequence aligners. It is easy to adapt Tentacle to different applications in metagenomics and easy to integrate into existing workflows. Evaluations show that Tentacle scales very well with increasing computing resources. We illustrate the versatility of Tentacle on three different use cases. Tentacle is written for Linux in Python 2.7 and is published as open source under the GNU General Public License (v3). Documentation, tutorials, installation instructions, and the source code are freely available online at: http://bioinformatics.math.chalmers.se/tentacle.

SmashCommunity: A metagenomic annotation and analysis tool

DEFF Research Database (Denmark)

Arumugam, Manimozhiyan; Harrington, Eoghan D; Foerstner, Konrad U

2010-01-01

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....

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.

Crystallization and preliminary X-ray crystallographic analysis of EstE1, a new and thermostable esterase cloned from a metagenomic library

Energy Technology Data Exchange (ETDEWEB)

Byun, Jung-Sue [Department of Biology, Yonsei University, Seoul 120-749 (Korea, Republic of); Protein Network Research Center, Yonsei University, Seoul 120-749 (Korea, Republic of); Rhee, Jin-Kyu [Department of Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Kim, Dong-Uk [Department of Biology, Yonsei University, Seoul 120-749 (Korea, Republic of); Oh, Jong-Won [Department of Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Cho, Hyun-Soo, E-mail: hscho8@yonsei.ac.kr [Department of Biology, Yonsei University, Seoul 120-749 (Korea, Republic of); Protein Network Research Center, Yonsei University, Seoul 120-749 (Korea, Republic of)

2006-02-01

Recombinant EstE1 protein with a histidine tag at the C-terminus was overexpressed in Escherichia coli strain BL21(DE3) and then purified by affinity chromatography. The protein was then crystallized at 290 K by the hanging-drop vapour-diffusion method. EstE1, a new thermostable esterase, was isolated by functional screening of a metagenomic DNA library from thermal environment samples. This enzyme showed activity towards short-chain acyl derivatives of length C4–C6 at a temperature of 303–363 K and displayed a high thermostability above 353 K. EstE1 has 64 and 57% amino-acid sequence similarity to est{sub pc}-encoded carboxylesterase from Pyrobaculum calidifontis and AFEST from Archaeoglobus fulgidus, respectively. The recombinant protein with a histidine tag at the C-terminus was overexpressed in Escherichia coli strain BL21(DE3) and then purified by affinity chromatography. The protein was crystallized at 290 K by the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 2.3 Å resolution from an EstE1 crystal; the crystal belongs to space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 73.71, c = 234.23 Å. Assuming the presence of four molecules in the asymmetric unit, the Matthews coefficient V{sub M} is calculated to be 2.2 Å{sup 3} Da{sup −1} and the solvent content is 44.1%.

Preparation of a differentially expressed, full-length cDNA expression library by RecA-mediated triple-strand formation with subtractively enriched cDNA fragments

NARCIS (Netherlands)

Hakvoort, T. B.; Spijkers, J. A.; Vermeulen, J. L.; Lamers, W. H.

1996-01-01

We have developed a fast and general method to obtain an enriched, full-length cDNA expression library with subtractively enriched cDNA fragments. The procedure relies on RecA-mediated triple-helix formation of single-stranded cDNA fragments with a double-stranded cDNA plasmid library. The complexes

Tuning protein expression using synonymous codon libraries targeted to the 5' mRNA coding region

DEFF Research Database (Denmark)

Goltermann, Lise; Borch Jensen, Martin; Bentin, Thomas

2011-01-01

intermediate expression levels of green fluorescent protein in Escherichia coli. At least in one case, no apparent effect on protein stability was observed, pointing to RNA level effects as the principal reason for the observed expression differences. Targeting a synonymous codon library to the 5' coding...

Towards diagnostic metagenomics of Campylobacter in fecal samples

DEFF Research Database (Denmark)

Andersen, Sandra Christine; Kiil, Kristoffer; Harder, Christoffer Bugge

2017-01-01

The development of diagnostic metagenomics is driven by the need for universal, culture-independent methods for detection and characterization of pathogens to substitute the time-consuming, organism-specific, and often culture-based laboratory procedures for epidemiological source-tracing. Some...... of the challenges in diagnostic metagenomics are, that it requires a great next-generation sequencing depth and unautomated data analysis. DNA from human fecal samples spiked with 7.75 × 101-7.75 × 107 colony forming unit (CFU)/ml Campylobacter jejuni and chicken fecal samples spiked with 1 × 102-1 × 106 CFU...... Campylobacter in all the clinical samples. Sensitivity in diagnostic metagenomics is improving and has reached a clinically relevant level. There are still challenges to overcome before real-time diagnostic metagenomics can replace quantitative polymerase chain reaction (qPCR) or culture-based surveillance...

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

Expressing Intellectual Freedom: A Content Analysis of Catholic Library World from 1980 to 2015

Directory of Open Access Journals (Sweden)

Megan E. Welsh

2016-12-01

Full Text Available Objective – Professional librarians have varying values relating to the topic of intellectual freedom that may or may not align with the American Library Association’s (ALA policies defining professional expectations on the topic. The personally held values and beliefs of Roman Catholic librarians and those working in libraries affiliated with Roman Catholicism are worthy of study to determine how personal religious values may translate into professional practice. The objective of this paper is to ascertain how frequently and in what context the topics of intellectual freedom and censorship were expressed in articles published in Catholic Library World (CLW, the professional journal of the Catholic Library Association (CLA from 1980 to 2015. Published content on these topics can be used as evidence to determine how this population discusses the concept of intellectual freedom. Methods – Articles relevant to these topics were retrieved from the American Theological Library Association Catholic Periodical and Literature Index (ATLA CPLI and Library, Information Science & Technology Abstracts (LISTA databases by conducting keyword searches using the terms “intellectual freedom” and censorship. Each retrieved publication was analyzed by counting the number of times the phrase “intellectual freedom” and the root censor* occurred. Through a deep reading of each publication, statements containing these search terms were then coded as positive, negative, or neutral, establishing a context for each occurrence. Results – The majority of published content supported intellectual freedom and opposed censorship. Negative content typically occurred in publications about children or school libraries. Additionally, CLW contributors did express a certain level of conflict between personally held religious values and professional values. Conclusions – This study adds to the limited research available on the intersection of personally held

Effective Analysis of NGS Metagenomic Data with Ultra-Fast Clustering Algorithms (MICW - Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

Energy Technology Data Exchange (ETDEWEB)

Li, Weizhong

2011-10-12

San Diego Supercomputer Center's Weizhong Li on "Effective Analysis of NGS Metagenomic Data with Ultra-fast Clustering Algorithms" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

Challenges and Opportunities of Airborne Metagenomics

OpenAIRE

Behzad, Hayedeh; Gojobori, Takashi; Mineta, Katsuhiko

2015-01-01

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 su...

Metagenomic analysis of permafrost microbial community response to thaw

Energy Technology Data Exchange (ETDEWEB)

Mackelprang, R.; Waldrop, M.P.; DeAngelis, K.M.; David, M.M.; Chavarria, K.L.; Blazewicz, S.J.; Rubin, E.M.; Jansson, J.K.

2011-07-01

We employed deep metagenomic sequencing to determine the impact of thaw on microbial phylogenetic and functional genes and related this data to measurements of methane emissions. Metagenomics, the direct sequencing of DNA from the environment, allows for the examination of whole biochemical pathways and associated processes, as opposed to individual pieces of the metabolic puzzle. Our metagenome analyses revealed that during transition from a frozen to a thawed state there were rapid shifts in many microbial, phylogenetic and functional gene abundances and pathways. After one week of incubation at 5°C, permafrost metagenomes converged to be more similar to each other than while they were frozen. We found that multiple genes involved in cycling of C and nitrogen shifted rapidly during thaw. We also constructed the first draft genome from a complex soil metagenome, which corresponded to a novel methanogen. Methane previously accumulated in permafrost was released during thaw and subsequently consumed by methanotrophic bacteria. Together these data point towards the importance of rapid cycling of methane and nitrogen in thawing permafrost.

Revealing the uncultivated majority: combining DNA stable-isotope probing, multiple displacement amplification and metagenomic analyses of uncultivated Methylocystis in acidic peatlands.

Science.gov (United States)

Chen, Yin; Dumont, Marc G; Neufeld, Josh D; Bodrossy, Levente; Stralis-Pavese, Nancy; McNamara, Niall P; Ostle, Nick; Briones, Maria J I; Murrell, J Colin

2008-10-01

Peatlands represent an enormous carbon reservoir and have a potential impact on the global climate because of the active methanogenesis and methanotrophy in these soils. Uncultivated methanotrophs from seven European peatlands were studied using a combination of molecular methods. Screening for methanotroph diversity using a particulate methane monooxygenase-based diagnostic gene array revealed that Methylocystis-related species were dominant in six of the seven peatlands studied. The abundance and methane oxidation activity of Methylocystis spp. were further confirmed by DNA stable-isotope probing analysis of a sample taken from the Moor House peatland (England). After ultracentrifugation, (13)C-labelled DNA, containing genomic DNA of these Methylocystis spp., was separated from (12)C DNA and subjected to multiple displacement amplification (MDA) to generate sufficient DNA for the preparation of a fosmid metagenomic library. Potential bias of MDA was detected by fingerprint analysis of 16S rRNA using denaturing gradient gel electrophoresis for low-template amplification (0.01 ng template). Sufficient template (1-5 ng) was used in MDA to circumvent this bias and chimeric artefacts were minimized by using an enzymatic treatment of MDA-generated DNA with S1 nuclease and DNA polymerase I. Screening of the metagenomic library revealed one fosmid containing methanol dehydrogenase and two fosmids containing 16S rRNA genes from these Methylocystis-related species as well as one fosmid containing a 16S rRNA gene related to that of Methylocella/Methylocapsa. Sequencing of the 14 kb methanol dehydrogenase-containing fosmid allowed the assembly of a gene cluster encoding polypeptides involved in bacterial methanol utilization (mxaFJGIRSAC). This combination of DNA stable-isotope probing, MDA and metagenomics provided access to genomic information of a relatively large DNA fragment of these thus far uncultivated, predominant and active methanotrophs in peatland soil.

DOE JGI Quality Metrics; Approaches to Scaling and Improving Metagenome Assembly (Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

Energy Technology Data Exchange (ETDEWEB)

Copeland, Alex; Brown, C. Titus

2011-10-13

DOE JGI's Alex Copeland on "DOE JGI Quality Metrics" and Michigan State University's C. Titus Brown on "Approaches to Scaling and Improving Metagenome Assembly" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

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.

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.

Bracken: estimating species abundance in metagenomics data

Directory of Open Access Journals (Sweden)

Jennifer Lu

2017-01-01

Full Text Available Metagenomic experiments attempt to characterize microbial communities using high-throughput DNA sequencing. Identification of the microorganisms in a sample provides information about the genetic profile, population structure, and role of microorganisms within an environment. Until recently, most metagenomics studies focused on high-level characterization at the level of phyla, or alternatively sequenced the 16S ribosomal RNA gene that is present in bacterial species. As the cost of sequencing has fallen, though, metagenomics experiments have increasingly used unbiased shotgun sequencing to capture all the organisms in a sample. This approach requires a method for estimating abundance directly from the raw read data. Here we describe a fast, accurate new method that computes the abundance at the species level using the reads collected in a metagenomics experiment. Bracken (Bayesian Reestimation of Abundance after Classification with KrakEN uses the taxonomic assignments made by Kraken, a very fast read-level classifier, along with information about the genomes themselves to estimate abundance at the species level, the genus level, or above. We demonstrate that Bracken can produce accurate species- and genus-level abundance estimates even when a sample contains multiple near-identical species.

Parallel Sequencing of Expressed Sequence Tags from Two Complementary DNA Libraries for High and Low Phosphorus Adaptation in Common Beans

Directory of Open Access Journals (Sweden)

Matthew W. Blair

2011-11-01

Full Text Available Expressed sequence tags (ESTs have proven useful for gene discovery in many crops. In this work, our objective was to construct complementary DNA (cDNA libraries from root tissues of common beans ( L. grown under low and high P hydroponic conditions and to conduct EST sequencing and comparative analyses of the libraries. Expressed sequence tag analysis of 3648 clones identified 2372 unigenes, of which 1591 were annotated as known genes while a total of 465 unigenes were not associated with any known gene. Unigenes with hits were categorized according to biological processes, molecular function, and cellular compartmentalization. Given the young tissue used to make the root libraries, genes for catalytic activity and binding were highly expressed. Comparisons with previous root EST sequencing and between the two libraries made here resulted in a set of genes to study further for differential gene expression and adaptation to low P, such as a 14 kDa praline-rich protein, a metallopeptidase, tonoplast intrinsic protein, adenosine triphosphate (ATP citrate synthase, and cell proliferation genes expressed in the low P treated plants. Given that common beans are often grown on acid soils of the tropics and subtropics that are usually low in P these genes and the two parallel libraries will be useful for selection for better uptake of this essential macronutrient. The importance of EST generation for common bean root tissues under low P and other abiotic soil stresses is also discussed.

MetaQUAST: evaluation of metagenome assemblies.

Science.gov (United States)

Mikheenko, Alla; Saveliev, Vladislav; Gurevich, Alexey

2016-04-01

During the past years we have witnessed the rapid development of new metagenome assembly methods. Although there are many benchmark utilities designed for single-genome assemblies, there is no well-recognized evaluation and comparison tool for metagenomic-specific analogues. In this article, we present MetaQUAST, a modification of QUAST, the state-of-the-art tool for genome assembly evaluation based on alignment of contigs to a reference. MetaQUAST addresses such metagenome datasets features as (i) unknown species content by detecting and downloading reference sequences, (ii) huge diversity by giving comprehensive reports for multiple genomes and (iii) presence of highly relative species by detecting chimeric contigs. We demonstrate MetaQUAST performance by comparing several leading assemblers on one simulated and two real datasets. http://bioinf.spbau.ru/metaquast aleksey.gurevich@spbu.ru Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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.

MetaStorm: A Public Resource for Customizable Metagenomics Annotation.

Directory of Open Access Journals (Sweden)

Gustavo Arango-Argoty

Full Text Available 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.

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.

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

Culture-Independent Identification of Manganese-Oxidizing Genes from Deep-Sea Hydrothermal Vent Chemoautotrophic Ferromanganese Microbial Communities Using a Metagenomic Approach

Science.gov (United States)

Davis, R.; Tebo, B. M.

2013-12-01

Microbial activity has long been recognized as being important to the fate of manganese (Mn) in hydrothermal systems, yet we know very little about the organisms that catalyze Mn oxidation, the mechanisms by which Mn is oxidized or the physiological function that Mn oxidation serves in these hydrothermal systems. Hydrothermal vents with thick ferromanganese microbial mats and Mn oxide-coated rocks observed throughout the Pacific Ring of Fire are ideal models to study the mechanisms of microbial Mn oxidation, as well as primary productivity in these metal-cycling ecosystems. We sampled ferromanganese microbial mats from Vai Lili Vent Field (Tmax=43°C) located on the Eastern Lau Spreading Center and Mn oxide-encrusted rhyolytic pumice (4°C) from Niua South Seamount on the Tonga Volcanic Arc. Metagenomic libraries were constructed and assembled from these samples and key genes known to be involved in Mn oxidation and carbon fixation pathways were identified in the reconstructed genomes. The Vai Lili metagenome assembled to form 121,157 contiguous sequences (contigs) greater than 1000bp in length, with an N50 of 8,261bp and a total metagenome size of 593 Mbp. Contigs were binned using an emergent self-organizing map of tetranucleotide frequencies. Putative homologs of the multicopper Mn-oxidase MnxG were found in the metagenome that were related to both the Pseudomonas-like and Bacillus-like forms of the enzyme. The bins containing the Pseudomonas-like mnxG genes are most closely related to uncultured Deltaproteobacteria and Chloroflexi. The Deltaproteobacteria bin appears to be an obligate anaerobe with possible chemoautotrophic metabolisms, while the Chloroflexi appears to be a heterotrophic organism. The metagenome from the Mn-stained pumice was assembled into 122,092 contigs greater than 1000bp in length with an N50 of 7635 and a metagenome size of 385 Mbp. Both forms of mnxG genes are present in this metagenome as well as the genes encoding the putative Mn

Computational workflow for the fine-grained analysis of metagenomic samples.

Science.gov (United States)

Pérez-Wohlfeil, Esteban; Arjona-Medina, Jose A; Torreno, Oscar; Ulzurrun, Eugenia; Trelles, Oswaldo

2016-10-25

The field of metagenomics, defined as the direct genetic analysis of uncultured samples of genomes contained within an environmental sample, is gaining increasing popularity. The aim of studies of metagenomics is to determine the species present in an environmental community and identify changes in the abundance of species under different conditions. Current metagenomic analysis software faces bottlenecks due to the high computational load required to analyze complex samples. A computational open-source workflow has been developed for the detailed analysis of metagenomes. This workflow provides new tools and datafile specifications that facilitate the identification of differences in abundance of reads assigned to taxa (mapping), enables the detection of reads of low-abundance bacteria (producing evidence of their presence), provides new concepts for filtering spurious matches, etc. Innovative visualization ideas for improved display of metagenomic diversity are also proposed to better understand how reads are mapped to taxa. Illustrative examples are provided based on the study of two collections of metagenomes from faecal microbial communities of adult female monozygotic and dizygotic twin pairs concordant for leanness or obesity and their mothers. The proposed workflow provides an open environment that offers the opportunity to perform the mapping process using different reference databases. Additionally, this workflow shows the specifications of the mapping process and datafile formats to facilitate the development of new plugins for further post-processing. This open and extensible platform has been designed with the aim of enabling in-depth analysis of metagenomic samples and better understanding of the underlying biological processes.

Computational workflow for the fine-grained analysis of metagenomic samples

Directory of Open Access Journals (Sweden)

Esteban Pérez-Wohlfeil

2016-10-01

Full Text Available Abstract Background The field of metagenomics, defined as the direct genetic analysis of uncultured samples of genomes contained within an environmental sample, is gaining increasing popularity. The aim of studies of metagenomics is to determine the species present in an environmental community and identify changes in the abundance of species under different conditions. Current metagenomic analysis software faces bottlenecks due to the high computational load required to analyze complex samples. Results A computational open-source workflow has been developed for the detailed analysis of metagenomes. This workflow provides new tools and datafile specifications that facilitate the identification of differences in abundance of reads assigned to taxa (mapping, enables the detection of reads of low-abundance bacteria (producing evidence of their presence, provides new concepts for filtering spurious matches, etc. Innovative visualization ideas for improved display of metagenomic diversity are also proposed to better understand how reads are mapped to taxa. Illustrative examples are provided based on the study of two collections of metagenomes from faecal microbial communities of adult female monozygotic and dizygotic twin pairs concordant for leanness or obesity and their mothers. Conclusions The proposed workflow provides an open environment that offers the opportunity to perform the mapping process using different reference databases. Additionally, this workflow shows the specifications of the mapping process and datafile formats to facilitate the development of new plugins for further post-processing. This open and extensible platform has been designed with the aim of enabling in-depth analysis of metagenomic samples and better understanding of the underlying biological processes.

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.

Functional Metagenomic Investigations of the Human Intestinal Microbiota

Directory of Open Access Journals (Sweden)

Aimee Marguerite Moore

2011-10-01

Full Text Available The human intestinal microbiota encode multiple critical functions impacting human health, including, metabolism of dietary substrate, prevention of pathogen invasion, immune system modulation, and provision of a reservoir of antibiotic resistance genes accessible to pathogens. The complexity of this microbial community, its recalcitrance to standard cultivation and the immense diversity of its encoded genes has necessitated the development of novel molecular, microbiological, and genomic tools. Functional metagenomics is one such culture-independent technique used for decades to study environmental microorganisms but relatively recently applied to the study of the human commensal microbiota. Metagenomic functional screens characterize the functional capacity of a microbial community independent of identity to known genes by subjecting the metagenome to functional assays in a genetically tractable host. Here we highlight recent work applying this technique to study the functional diversity of the intestinal microbiota, and discuss how an approach combining high-throughput sequencing, cultivation, and metagenomic functional screens can improve our understanding of interactions between this complex community and its human host.

Functional Metagenomic Investigations of the Human Intestinal Microbiota

DEFF Research Database (Denmark)

Moore, Aimee M.; Munck, Christian; Sommer, Morten Otto Alexander

2011-01-01

The human intestinal microbiota encode multiple critical functions impacting human health, including metabolism of dietary substrate, prevention of pathogen invasion, immune system modulation, and provision of a reservoir of antibiotic resistance genes accessible to pathogens. The complexity...... microorganisms, but relatively recently applied to the study of the human commensal microbiota. Metagenomic functional screens characterize the functional capacity of a microbial community, independent of identity to known genes, by subjecting the metagenome to functional assays in a genetically tractable host....... Here we highlight recent work applying this technique to study the functional diversity of the intestinal microbiota, and discuss how an approach combining high-throughput sequencing, cultivation, and metagenomic functional screens can improve our understanding of interactions between this complex...

Molecular cloning of a novel bioH gene from an environmental metagenome encoding a carboxylesterase with exceptional tolerance to organic solvents

DEFF Research Database (Denmark)

Shi, Yuping; Pan, Yingjie; Li, Bailin

2013-01-01

with a strong potential in industrial applications. CONCLUSIONS: This study constituted the first investigation of a novel bioHx gene in a biotin biosynthetic gene cluster cloned from an environmental metagenome. The bioHx gene was successfully cloned, expressed and characterized. The results demonstrated...... that BioHx is a novel carboxylesterase, displaying distinct biochemical properties with strong application potential in industry. Our results also provided the evidence for the effectiveness of functional metagenomic approach for identifying novel bioH genes from complex ecosystem.......ABSTRACT: BACKGROUND: BioH is one of the key enzymes to produce the precursor pimeloyl-ACP to initiate biotin biosynthesis de novo in bacteria. To date, very few bioH genes have been characterized. In this study, we cloned and identified a novel bioH gene, bioHx, from an environmental metagenome...

WebMGA: a customizable web server for fast metagenomic sequence analysis.

Science.gov (United States)

Wu, Sitao; Zhu, Zhengwei; Fu, Liming; Niu, Beifang; Li, Weizhong

2011-09-07

The new field of metagenomics studies microorganism communities by culture-independent sequencing. With the advances in next-generation sequencing techniques, researchers are facing tremendous challenges in metagenomic data analysis due to huge quantity and high complexity of sequence data. Analyzing large datasets is extremely time-consuming; also metagenomic annotation involves a wide range of computational tools, which are difficult to be installed and maintained by common users. The tools provided by the few available web servers are also limited and have various constraints such as login requirement, long waiting time, inability to configure pipelines etc. We developed WebMGA, a customizable web server for fast metagenomic analysis. WebMGA includes over 20 commonly used tools such as ORF calling, sequence clustering, quality control of raw reads, removal of sequencing artifacts and contaminations, taxonomic analysis, functional annotation etc. WebMGA provides users with rapid metagenomic data analysis using fast and effective tools, which have been implemented to run in parallel on our local computer cluster. Users can access WebMGA through web browsers or programming scripts to perform individual analysis or to configure and run customized pipelines. WebMGA is freely available at http://weizhongli-lab.org/metagenomic-analysis. WebMGA offers to researchers many fast and unique tools and great flexibility for complex metagenomic data analysis.

WebMGA: a customizable web server for fast metagenomic sequence analysis

Directory of Open Access Journals (Sweden)

Niu Beifang

2011-09-01

Full Text Available Abstract Background The new field of metagenomics studies microorganism communities by culture-independent sequencing. With the advances in next-generation sequencing techniques, researchers are facing tremendous challenges in metagenomic data analysis due to huge quantity and high complexity of sequence data. Analyzing large datasets is extremely time-consuming; also metagenomic annotation involves a wide range of computational tools, which are difficult to be installed and maintained by common users. The tools provided by the few available web servers are also limited and have various constraints such as login requirement, long waiting time, inability to configure pipelines etc. Results We developed WebMGA, a customizable web server for fast metagenomic analysis. WebMGA includes over 20 commonly used tools such as ORF calling, sequence clustering, quality control of raw reads, removal of sequencing artifacts and contaminations, taxonomic analysis, functional annotation etc. WebMGA provides users with rapid metagenomic data analysis using fast and effective tools, which have been implemented to run in parallel on our local computer cluster. Users can access WebMGA through web browsers or programming scripts to perform individual analysis or to configure and run customized pipelines. WebMGA is freely available at http://weizhongli-lab.org/metagenomic-analysis. Conclusions WebMGA offers to researchers many fast and unique tools and great flexibility for complex metagenomic data analysis.

Metagenomics at Grass Roots

Indian Academy of Sciences (India)

Metagenomics is a robust, interdisciplinary approach for studyingmicrobial community composition, function, and dynamics.It typically involves a core of molecular biology, microbiology,ecology, statistics, and computational biology. Excitingoutcomes anticipated from these studies include unravelingof complex interactions ...

Balancing gene expression without library construction via a reusable sRNA pool.

Science.gov (United States)

Ghodasara, Amar; Voigt, Christopher A

2017-07-27

Balancing protein expression is critical when optimizing genetic systems. Typically, this requires library construction to vary the genetic parts controlling each gene, which can be expensive and time-consuming. Here, we develop sRNAs corresponding to 15nt 'target' sequences that can be inserted upstream of a gene. The targeted gene can be repressed from 1.6- to 87-fold by controlling sRNA expression using promoters of different strength. A pool is built where six sRNAs are placed under the control of 16 promoters that span a ∼103-fold range of strengths, yielding ∼107 combinations. This pool can simultaneously optimize up to six genes in a system. This requires building only a single system-specific construct by placing a target sequence upstream of each gene and transforming it with the pre-built sRNA pool. The resulting library is screened and the top clone is sequenced to determine the promoter controlling each sRNA, from which the fold-repression of the genes can be inferred. The system is then rebuilt by rationally selecting parts that implement the optimal expression of each gene. We demonstrate the versatility of this approach by using the same pool to optimize a metabolic pathway (β-carotene) and genetic circuit (XNOR logic gate). © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Purification, crystallization and preliminary crystallographic analysis of Est-Y29: a novel oligomeric β-lactamase

International Nuclear Information System (INIS)

Kim, SeungBum; Joo, Sangbum; Yoon, Sangyoung; Kim, Sungsoo; Moon, Jongkook; Ryu, Yeonwoo; Kim, Kyeong Kyu; Kim, T. Doohun

2009-01-01

Est-Y29, a novel oligomeric class C β-lactamase from a metagenomic library, was crystallized in space group I4 1 and diffraction data were collected to 1.49 Å resolution. β-Lactam antibiotics such as penicillins and cephalosporins have a four-atom ring as a common element in their structure. The β-lactamases, which catalyze the inactivation of these antibiotics, are of great interest because of their high incidence in pathogenic bacteria. A novel oligomeric class C β-lactamase (Est-Y29) from a metagenomic library was expressed, purified and crystallized. The recombinant protein was expressed in Escherichia coli with an N-terminal 6×His tag and purified to homogeneity. EstY-29 was crystallized and X-ray intensity data were collected to 1.49 Å resolution using synchrotron radiation

Comparison of normalization methods for the analysis of metagenomic gene abundance data.

Science.gov (United States)

Pereira, Mariana Buongermino; Wallroth, Mikael; Jonsson, Viktor; Kristiansson, Erik

2018-04-20

In shotgun metagenomics, microbial communities are studied through direct sequencing of DNA without any prior cultivation. By comparing gene abundances estimated from the generated sequencing reads, functional differences between the communities can be identified. However, gene abundance data is affected by high levels of systematic variability, which can greatly reduce the statistical power and introduce false positives. Normalization, which is the process where systematic variability is identified and removed, is therefore a vital part of the data analysis. A wide range of normalization methods for high-dimensional count data has been proposed but their performance on the analysis of shotgun metagenomic data has not been evaluated. Here, we present a systematic evaluation of nine normalization methods for gene abundance data. The methods were evaluated through resampling of three comprehensive datasets, creating a realistic setting that preserved the unique characteristics of metagenomic data. Performance was measured in terms of the methods ability to identify differentially abundant genes (DAGs), correctly calculate unbiased p-values and control the false discovery rate (FDR). Our results showed that the choice of normalization method has a large impact on the end results. When the DAGs were asymmetrically present between the experimental conditions, many normalization methods had a reduced true positive rate (TPR) and a high false positive rate (FPR). The methods trimmed mean of M-values (TMM) and relative log expression (RLE) had the overall highest performance and are therefore recommended for the analysis of gene abundance data. For larger sample sizes, CSS also showed satisfactory performance. This study emphasizes the importance of selecting a suitable normalization methods in the analysis of data from shotgun metagenomics. Our results also demonstrate that improper methods may result in unacceptably high levels of false positives, which in turn may lead

Metagenomics at Grass Roots

Indian Academy of Sciences (India)

CAMERA (Community Cyber-infrastructure for Advanced Mi- crobial Ecology .... Acidobacteria known to metabolize a variety of car- bon sources .... [7] J Nesme et al., Back to the future of soil metagenomics, Frontiers in Microbi- ology, Vol.7 ...

Unlocking the potential of metagenomics through replicated experimental design

NARCIS (Netherlands)

Knight, R.; Jansson, J.; Field, D.; Fierer, N.; Desai, N.; Fuhrman, J.A.; Hugenholtz, P.; Van der Lelie, D.; Meyer, F.; Stevens, R.; Bailey, M.J.; Gordon, J.I.; Kowalchuk, G.A.; Gilbert, J.A.

2012-01-01

Metagenomics holds enormous promise for discovering novel enzymes and organisms that are biomarkers or drivers of processes relevant to disease, industry and the environment. In the past two years, we have seen a paradigm shift in metagenomics to the application of cross-sectional and longitudinal

Unlocking the potential of metagenomics through replicated experimental design.

NARCIS (Netherlands)

Knight, R.; Jansson, J.; Field, D.; Fierer, N.; Desai, N.; Fuhrman, J.A.; Hugenholtz, P.; van der Lelie, D.; Meyer, F.; Stevens, R.; Bailey, M.J.; Gordon, J.I.; Kowalchuk, G.A.; Gilbert, J.A.

2012-01-01

Metagenomics holds enormous promise for discovering novel enzymes and organisms that are biomarkers or drivers of processes relevant to disease, industry and the environment. In the past two years, we have seen a paradigm shift in metagenomics to the application of cross-sectional and longitudinal

Compositional profile of α / β-hydrolase fold proteins in mangrove soil metagenomes: prevalence of epoxide hydrolases and haloalkane dehalogenases in oil-contaminated sites.

Science.gov (United States)

Jiménez, Diego Javier; Dini-Andreote, Francisco; Ottoni, Júlia Ronzella; de Oliveira, Valéria Maia; van Elsas, Jan Dirk; Andreote, Fernando Dini

2015-05-01

The occurrence of genes encoding biotechnologically relevant α/β-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered based on local amino acid alignments against the Lipase Engineering Database. In total, 5923 unassembled sequences were affiliated with 30 different α/β-hydrolase fold superfamilies. The most abundant predicted proteins encompassed cytosolic hydrolases (abH08; ∼ 23%), microsomal hydrolases (abH09; ∼ 12%) and Moraxella lipase-like proteins (abH04 and abH01; mangroves BrMgv01-02-03. This suggested selection and putative involvement in local degradation/detoxification of the pollutants. Seven sequences that were annotated as genes for putative epoxide hydrolases and five for putative haloalkane dehalogenases were found in a fosmid library generated from BrMgv02 DNA. The latter enzymes were predicted to belong to Actinobacteria, Deinococcus-Thermus, Planctomycetes and Proteobacteria. Our integrated approach thus identified 12 genes (complete and/or partial) that may encode hitherto undescribed enzymes. The low amino acid identity (< 60%) with already-described genes opens perspectives for both production in an expression host and genetic screening of metagenomes. © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Metagenomics: The Next Culture-Independent Game Changer

Directory of Open Access Journals (Sweden)

Jessica D. Forbes

2017-07-01

Full Text Available A trend towards the abandonment of obtaining pure culture isolates in frontline laboratories is at a crossroads with the ability of public health agencies to perform their basic mandate of foodborne disease surveillance and response. The implementation of culture-independent diagnostic tests (CIDTs including nucleic acid and antigen-based assays for acute gastroenteritis is leaving public health agencies without laboratory evidence to link clinical cases to each other and to food or environmental substances. This limits the efficacy of public health epidemiology and surveillance as well as outbreak detection and investigation. Foodborne outbreaks have the potential to remain undetected or have insufficient evidence to support source attribution and may inadvertently increase the incidence of foodborne diseases. Next-generation sequencing of pure culture isolates in clinical microbiology laboratories has the potential to revolutionize the fields of food safety and public health. Metagenomics and other ‘omics’ disciplines could provide the solution to a cultureless future in clinical microbiology, food safety and public health. Data mining of information obtained from metagenomics assays can be particularly useful for the identification of clinical causative agents or foodborne contamination, detection of AMR and/or virulence factors, in addition to providing high-resolution subtyping data. Thus, metagenomics assays may provide a universal test for clinical diagnostics, foodborne pathogen detection, subtyping and investigation. This information has the potential to reform the field of enteric disease diagnostics and surveillance and also infectious diseases as a whole. The aim of this review will be to present the current state of CIDTs in diagnostic and public health laboratories as they relate to foodborne illness and food safety. Moreover, we will also discuss the diagnostic and subtyping utility and concomitant bias limitations of

Porcine transcriptome analysis based on 97 non-normalized cDNA libraries and assembly of 1,021,891 expressed sequence tags

DEFF Research Database (Denmark)

Gorodkin, Jan; Cirera, Susanna; Hedegaard, Jacob

2007-01-01

public databases. The Sino-Danish ESTs were generated from one normalized and 97 non-normalized cDNA libraries representing 35 different tissues and three developmental stages. RESULTS: Using the Distiller package, the ESTs were assembled to roughly 48,000 contigs and 73,000 singletons, of which...... with the greatest number of different expressed genes, whereas tissues with more specialized function, such as developing liver, have fewer expressed genes. There are at least 65 high confidence housekeeping gene candidates and 876 cDNA library-specific gene candidates. We identified differential expression...

Methods for the Isolation of Genes Encoding Novel PHA Metabolism Enzymes from Complex Microbial Communities.

Science.gov (United States)

Cheng, Jiujun; Nordeste, Ricardo; Trainer, Maria A; Charles, Trevor C

2017-01-01

Development of different PHAs as alternatives to petrochemically derived plastics can be facilitated by mining metagenomic libraries for diverse PHA cycle genes that might be useful for synthesis of bio-plastics. The specific phenotypes associated with mutations of the PHA synthesis pathway genes in Sinorhizobium meliloti and Pseudomonas putida, allows the use of powerful selection and screening tools to identify complementing novel PHA synthesis genes. Identification of novel genes through their function rather than sequence facilitates the functional proteins that may otherwise have been excluded through sequence-only screening methodology. We present here methods that we have developed for the isolation of clones expressing novel PHA metabolism genes from metagenomic libraries.

Methods for the isolation of genes encoding novel PHB cycle enzymes from complex microbial communities.

Science.gov (United States)

Nordeste, Ricardo F; Trainer, Maria A; Charles, Trevor C

2010-01-01

Development of different PHAs as alternatives to petrochemically derived plastics can be facilitated by mining metagenomic libraries for diverse PHA cycle genes that might be useful for synthesis of bioplastics. The specific phenotypes associated with mutations of the PHA synthesis pathway genes in Sinorhizobium meliloti allows for the use of powerful selection and screening tools to identify complementing novel PHA synthesis genes. Identification of novel genes through their function rather than sequence facilitates finding functional proteins that may otherwise have been excluded through sequence-only screening methodology. We present here methods that we have developed for the isolation of clones expressing novel PHA metabolism genes from metagenomic libraries.

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)

SPHINX--an algorithm for taxonomic binning of metagenomic sequences.

Science.gov (United States)

Mohammed, Monzoorul Haque; Ghosh, Tarini Shankar; Singh, Nitin Kumar; Mande, Sharmila S

2011-01-01

Compared with composition-based binning algorithms, the binning accuracy and specificity of alignment-based binning algorithms is significantly higher. However, being alignment-based, the latter class of algorithms require enormous amount of time and computing resources for binning huge metagenomic datasets. The motivation was to develop a binning approach that can analyze metagenomic datasets as rapidly as composition-based approaches, but nevertheless has the accuracy and specificity of alignment-based algorithms. This article describes a hybrid binning approach (SPHINX) that achieves high binning efficiency by utilizing the principles of both 'composition'- and 'alignment'-based binning algorithms. Validation results with simulated sequence datasets indicate that SPHINX is able to analyze metagenomic sequences as rapidly as composition-based algorithms. Furthermore, the binning efficiency (in terms of accuracy and specificity of assignments) of SPHINX is observed to be comparable with results obtained using alignment-based algorithms. A web server for the SPHINX algorithm is available at http://metagenomics.atc.tcs.com/SPHINX/.

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

A catalog of the mouse gut metagenome

DEFF Research Database (Denmark)

Xiao, Liang; Feng, Qiang; Liang, Suisha

2015-01-01

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......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...... counterpart, with 95.2% of its Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologous groups in common. However, only 4.0% of the mouse gut microbial genes were shared (95% identity, 90% coverage) with those of the human gut microbiome. This catalog provides a useful reference for future studies....

Development of library preparation method able to correct gene expression levels in rice anther and isolate a trace expression gene mediated in cold-resistance

Energy Technology Data Exchange (ETDEWEB)

Yamaguchi, Tomoya; Koike, Setsuo [Tohoku National Agricultural Experiment Station, Morioka (Japan)

2000-02-01

When cDNA library is prepared by a previously developed method, genes of which expression level is high are apt to be cloned at a high frequency, whereas genes of which expression level are low, are difficult to be cloned. A low-expression gene has been cloned at very low frequency. Therefore, the gene encoding the key enzyme that is involved in growth disturbance of rice pollen has not been identified. In this study, development of a library preparing method able to correct the expression level was attempted using highly sensitive detection method with radioisotope and some genes related to cold-resistance of rice were isolated. Double strand DNAs were synthesized using mRNA extract from rice anthers and annealed following heat-denaturation. It has been known that single strand DNA molecules abundantly existing in DNA solution can easily aggregate to form double strand DNA, but single stranded DNA molecules poor in the solution are apt to still remain as single strand after annealing. Thus, the amount of single strand DNA would be balanced in the solution between abundant DNA and poor DNA species. The authors succeeded to prepare a gene library including low and high expression genes at similar proportions. Moreover, spin trap method that allows RI labeling of DNA bound to latex particle, was developed to detect with high sensitivity, especially for genes that are expressed at low level. The present method could be used for recovery, detection and quantitative analysis of radiolabeled single strand DNA. Thus, it was demonstrated that the stage from tetrad sperm to small sperm might be easily affected by cold stress. The present results suggest that the expressions of {beta}-1 and {beta}-3 glucanase, which are involved in the release of small sperms following meiosis in the pollen formation, might be easily affected by cold stress. (M.N.)

Development of library preparation method able to correct gene expression levels in rice anther and isolate a trace expression gene mediated in cold-resistance

International Nuclear Information System (INIS)

Yamaguchi, Tomoya; Koike, Setsuo

2000-01-01

When cDNA library is prepared by a previously developed method, genes of which expression level is high are apt to be cloned at a high frequency, whereas genes of which expression level are low, are difficult to be cloned. A low-expression gene has been cloned at very low frequency. Therefore, the gene encoding the key enzyme that is involved in growth disturbance of rice pollen has not been identified. In this study, development of a library preparing method able to correct the expression level was attempted using highly sensitive detection method with radioisotope and some genes related to cold-resistance of rice were isolated. Double strand DNAs were synthesized using mRNA extract from rice anthers and annealed following heat-denaturation. It has been known that single strand DNA molecules abundantly existing in DNA solution can easily aggregate to form double strand DNA, but single stranded DNA molecules poor in the solution are apt to still remain as single strand after annealing. Thus, the amount of single strand DNA would be balanced in the solution between abundant DNA and poor DNA species. The authors succeeded to prepare a gene library including low and high expression genes at similar proportions. Moreover, spin trap method that allows RI labeling of DNA bound to latex particle, was developed to detect with high sensitivity, especially for genes that are expressed at low level. The present method could be used for recovery, detection and quantitative analysis of radiolabeled single strand DNA. Thus, it was demonstrated that the stage from tetrad sperm to small sperm might be easily affected by cold stress. The present results suggest that the expressions of β-1 and β-3 glucanase, which are involved in the release of small sperms following meiosis in the pollen formation, might be easily affected by cold stress. (M.N.)

Moleculo Long-Read Sequencing Facilitates Assembly and Genomic Binning from Complex Soil Metagenomes

Energy Technology Data Exchange (ETDEWEB)

White, Richard Allen; Bottos, Eric M.; Roy Chowdhury, Taniya; Zucker, Jeremy D.; Brislawn, Colin J.; Nicora, Carrie D.; Fansler, Sarah J.; Glaesemann, Kurt R.; Glass, Kevin; Jansson, Janet K.; Langille, Morgan

2016-06-28

Soil metagenomics has been touted as the “grand challenge” for metagenomics, as the high microbial diversity and spatial heterogeneity of soils make them unamenable to current assembly platforms. Here, we aimed to improve soil metagenomic sequence assembly by applying the Moleculo synthetic long-read sequencing technology. In total, we obtained 267 Gbp of raw sequence data from a native prairie soil; these data included 109.7 Gbp of short-read data (~100 bp) from the Joint Genome Institute (JGI), an additional 87.7 Gbp of rapid-mode read data (~250 bp), plus 69.6 Gbp (>1.5 kbp) from Moleculo sequencing. The Moleculo data alone yielded over 5,600 reads of >10 kbp in length, and over 95% of the unassembled reads mapped to contigs of >1.5 kbp. Hybrid assembly of all data resulted in more than 10,000 contigs over 10 kbp in length. We mapped three replicate metatranscriptomes derived from the same parent soil to the Moleculo subassembly and found that 95% of the predicted genes, based on their assignments to Enzyme Commission (EC) numbers, were expressed. The Moleculo subassembly also enabled binning of >100 microbial genome bins. We obtained via direct binning the first complete genome, that of “CandidatusPseudomonas sp. strain JKJ-1” from a native soil metagenome. By mapping metatranscriptome sequence reads back to the bins, we found that several bins corresponding to low-relative-abundanceAcidobacteriawere highly transcriptionally active, whereas bins corresponding to high-relative-abundanceVerrucomicrobiawere not. These results demonstrate that Moleculo sequencing provides a significant advance for resolving complex soil microbial communities.

IMPORTANCESoil microorganisms carry out key processes for life on our planet, including cycling of carbon and other nutrients and supporting growth of plants. However, there is poor molecular-level understanding of their

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

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.

Analysis of a viral metagenomic library from 200 m depth in Monterey Bay, California constructed by direct shotgun cloning

OpenAIRE

Preston Christina M; Steward Grieg F

2011-01-01

Abstract Background Viruses have a profound influence on both the ecology and evolution of marine plankton, but the genetic diversity of viral assemblages, particularly those in deeper ocean waters, remains poorly described. Here we report on the construction and analysis of a viral metagenome prepared from below the euphotic zone in a temperate, eutrophic bay of coastal California. Methods We purified viruses from approximately one cubic meter of seawater collected from 200m depth in Montere...

[Mini review] metagenomic studies of the Red Sea

KAUST Repository

Behzad, Hayedeh

2015-10-23

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 amongst 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 osmolyte C1 oxidation 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

[Mini review] metagenomic studies of the Red Sea

KAUST Repository

Behzad, Hayedeh; Ibarra, Martin Augusto; Mineta, Katsuhiko; Gojobori, Takashi

2015-01-01

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 amongst 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 osmolyte C1 oxidation 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

Meta-IDBA: a de Novo assembler for metagenomic data.

Science.gov (United States)

Peng, Yu; Leung, Henry C M; Yiu, S M; Chin, Francis Y L

2011-07-01

Next-generation sequencing techniques allow us to generate reads from a microbial environment in order to analyze the microbial community. However, assembling of a set of mixed reads from different species to form contigs is a bottleneck of metagenomic research. Although there are many assemblers for assembling reads from a single genome, there are no assemblers for assembling reads in metagenomic data without reference genome sequences. Moreover, the performances of these assemblers on metagenomic data are far from satisfactory, because of the existence of common regions in the genomes of subspecies and species, which make the assembly problem much more complicated. We introduce the Meta-IDBA algorithm for assembling reads in metagenomic data, which contain multiple genomes from different species. There are two core steps in Meta-IDBA. It first tries to partition the de Bruijn graph into isolated components of different species based on an important observation. Then, for each component, it captures the slight variants of the genomes of subspecies from the same species by multiple alignments and represents the genome of one species, using a consensus sequence. Comparison of the performances of Meta-IDBA and existing assemblers, such as Velvet and Abyss for different metagenomic datasets shows that Meta-IDBA can reconstruct longer contigs with similar accuracy. Meta-IDBA toolkit is available at our website http://www.cs.hku.hk/~alse/metaidba. chin@cs.hku.hk.

Students lead the library the importance of student contributions to the academic library

CERN Document Server

Arnold-Garza, Sara

2017-01-01

In six parts-Students as Employees, Students as Curators, Students as Ambassadors, the Library as Client, Student Groups as Library Leaders, and Students as Library Designers-Students Lead the Library provides case studies of programs and initiatives that seek student input, assistance, and leadership in the academic library. Through the library, students can develop leadership skills, cultivate high levels of engagement, and offer peer learning opportunities. Through the students, libraries can create participatory design processes, enhancement and transformation of the library's core functions, and expressed library value for stakeholders.

Phylogeny and phylogeography of functional genes shared among seven terrestrial subsurface metagenomes reveal N-cycling and microbial evolutionary relationships

Directory of Open Access Journals (Sweden)

Maggie CY Lau

2014-10-01

Full Text Available Comparative studies on community phylogenetics and phylogeography of microorganisms living in extreme environments are rare. Terrestrial subsurface habitats are valuable for studying microbial biogeographical patterns due to their isolation and the restricted dispersal mechanisms. Since the taxonomic identity of a microorganism does not always correspond well with its functional role in a particular community, the use of taxonomic assignments or patterns may give limited inference on how microbial functions are affected by historical, geographical and environmental factors. With seven metagenomic libraries generated from fracture water samples collected from five South African mines, this study was carried out to (1 screen for ubiquitous functions or pathways of biogeochemical cycling of CH4, S and N; (2 to characterize the biodiversity represented by the common functional genes; (3 to investigate the subsurface biogeography as revealed by this subset of genes; and (4 to explore the possibility of using metagenomic data for evolutionary study. The ubiquitous functional genes are NarV, NPD, PAP reductase, NifH, NifD, NifK, NifE and NifN genes. Although these 8 common functional genes were taxonomically and phylogenetically diverse and distinct from each other, the dissimilarity between samples did not correlate strongly with either geographical, environmental or residence time of the water. Por genes homologous to those of Thermodesulfovibrio yellowstonii detected in all metagenomes were deep lineages of Nitrospirae, suggesting that subsurface habitats have preserved ancestral genetic signatures that inform the study of the origin and evolution of prokaryotes.

Cell-free translational screening of an expression sequence tag library of Clonorchis sinensis for novel antigen discovery.

Science.gov (United States)

Kasi, Devi; Catherine, Christy; Lee, Seung-Won; Lee, Kyung-Ho; Kim, Yu Jung; Ro Lee, Myeong; Ju, Jung Won; Kim, Dong-Myung

2017-05-01

The rapidly evolving cloning and sequencing technologies have enabled understanding of genomic structure of parasite genomes, opening up new ways of combatting parasite-related diseases. To make the most of the exponentially accumulating genomic data, however, it is crucial to analyze the proteins encoded by these genomic sequences. In this study, we adopted an engineered cell-free protein synthesis system for large-scale expression screening of an expression sequence tag (EST) library of Clonorchis sinensis to identify potential antigens that can be used for diagnosis and treatment of clonorchiasis. To allow high-throughput expression and identification of individual genes comprising the library, a cell-free synthesis reaction was designed such that both the template DNA and the expressed proteins were co-immobilized on the same microbeads, leading to microbead-based linkage of the genotype and phenotype. This reaction configuration allowed streamlined expression, recovery, and analysis of proteins. This approach enabled us to identify 21 antigenic proteins. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:832-837, 2017. © 2017 American Institute of Chemical Engineers.

Methods for transforming and expression screening of filamentous fungal cells with a DNA library

Science.gov (United States)

Teter, Sarah; Lamsa, Michael; Cherry, Joel; Ward, Connie

2015-06-02

The present invention relates to methods for expression screening of filamentous fungal transformants, comprising: (a) isolating single colony transformants of a DNA library introduced into E. coli; (b) preparing DNA from each of the single colony E. coli transformants; (c) introducing a sample of each of the DNA preparations of step (b) into separate suspensions of protoplasts of a filamentous fungus to obtain transformants thereof, wherein each transformant contains one or more copies of an individual polynucleotide from the DNA library; (d) growing the individual filamentous fungal transformants of step (c) on selective growth medium, thereby permitting growth of the filamentous fungal transformants, while suppressing growth of untransformed filamentous fungi; and (e) measuring activity or a property of each polypeptide encoded by the individual polynucleotides. The present invention also relates to isolated polynucleotides encoding polypeptides of interest obtained by such methods, to nucleic acid constructs, expression vectors, and recombinant host cells comprising the isolated polynucleotides, and to methods of producing the polypeptides encoded by the isolated polynucleotides.

Metagenomic analysis of planktonic microbial consortia from a non-tidal urban-impacted segment of James River.

Science.gov (United States)

Brown, Bonnie L; LePrell, Rebecca V; Franklin, Rima B; Rivera, Maria C; Cabral, Francine M; Eaves, Hugh L; Gardiakos, Vicki; Keegan, Kevin P; King, Timothy L

2015-01-01

Knowledge of the diversity and ecological function of the microbial consortia of James River in Virginia, USA, is essential to developing a more complete understanding of the ecology of this model river system. Metagenomic analysis of James River's planktonic microbial community was performed for the first time using an unamplified genomic library and a 16S rDNA amplicon library prepared and sequenced by Ion PGM and MiSeq, respectively. From the 0.46-Gb WGS library (GenBank:SRR1146621; MG-RAST:4532156.3), 4 × 10(6) reads revealed >3 × 10(6) genes, 240 families of prokaryotes, and 155 families of eukaryotes. From the 0.68-Gb 16S library (GenBank:SRR2124995; MG-RAST:4631271.3; EMB:2184), 4 × 10(6) reads revealed 259 families of eubacteria. Results of the WGS and 16S analyses were highly consistent and indicated that more than half of the bacterial sequences were Proteobacteria, predominantly Comamonadaceae. The most numerous genera in this group were Acidovorax (including iron oxidizers, nitrotolulene degraders, and plant pathogens), which accounted for 10 % of assigned bacterial reads. Polaromonas were another 6 % of all bacterial reads, with many assignments to groups capable of degrading polycyclic aromatic hydrocarbons. Albidiferax (iron reducers) and Variovorax (biodegraders of a variety of natural biogenic compounds as well as anthropogenic contaminants such as polycyclic aromatic hydrocarbons and endocrine disruptors) each accounted for an additional 3 % of bacterial reads. Comparison of these data to other publically-available aquatic metagenomes revealed that this stretch of James River is highly similar to the upper Mississippi River, and that these river systems are more similar to aquaculture and sludge ecosystems than they are to lakes or to a pristine section of the upper Amazon River. Taken together, these analyses exposed previously unknown aspects of microbial biodiversity, documented the ecological responses of microbes to urban

Metagenomes from two microbial consortia associated with Santa Barbara seep oil.

Science.gov (United States)

Hawley, Erik R; Malfatti, Stephanie A; Pagani, Ioanna; Huntemann, Marcel; Chen, Amy; Foster, Brian; Copeland, Alexander; del Rio, Tijana Glavina; Pati, Amrita; Jansson, Janet R; Gilbert, Jack A; Tringe, Susannah Green; Lorenson, Thomas D; Hess, Matthias

2014-12-01

The metagenomes from two microbial consortia associated with natural oils seeping into the Pacific Ocean offshore the coast of Santa Barbara (California, USA) were determined to complement already existing metagenomes generated from microbial communities associated with hydrocarbons that pollute the marine ecosystem. This genomics resource article is the first of two publications reporting a total of four new metagenomes from oils that seep into the Santa Barbara Channel. Copyright © 2014 Elsevier B.V. All rights reserved.

The metagenome-derived enzymes LipS and LipT increase the diversity of known lipases.

Directory of Open Access Journals (Sweden)

Jennifer Chow

Full Text Available Triacylglycerol lipases (EC 3.1.1.3 catalyze both hydrolysis and synthesis reactions with a broad spectrum of substrates rendering them especially suitable for many biotechnological applications. Most lipases used today originate from mesophilic organisms and are susceptible to thermal denaturation whereas only few possess high thermotolerance. Here, we report on the identification and characterization of two novel thermostable bacterial lipases identified by functional metagenomic screenings. Metagenomic libraries were constructed from enrichment cultures maintained at 65 to 75 °C and screened resulting in the identification of initially 10 clones with lipolytic activities. Subsequently, two ORFs were identified encoding lipases, LipS and LipT. Comparative sequence analyses suggested that both enzymes are members of novel lipase families. LipS is a 30.2 kDa protein and revealed a half-life of 48 h at 70 °C. The lipT gene encoded for a multimeric enzyme with a half-life of 3 h at 70 °C. LipS had an optimum temperature at 70 °C and LipT at 75 °C. Both enzymes catalyzed hydrolysis of long-chain (C(12 and C(14 fatty acid esters and additionally hydrolyzed a number of industry-relevant substrates. LipS was highly specific for (R-ibuprofen-phenyl ester with an enantiomeric excess (ee of 99%. Furthermore, LipS was able to synthesize 1-propyl laurate and 1-tetradecyl myristate at 70 °C with rates similar to those of the lipase CalB from Candida antarctica. LipS represents the first example of a thermostable metagenome-derived lipase with significant synthesis activities. Its X-ray structure was solved with a resolution of 1.99 Å revealing an unusually compact lid structure.

Genetic variability of psychrotolerant Acidithiobacillus ferrivorans revealed by (meta)genomic analysis.

Science.gov (United States)

González, Carolina; Yanquepe, María; Cardenas, Juan Pablo; Valdes, Jorge; Quatrini, Raquel; Holmes, David S; Dopson, Mark

2014-11-01

Acidophilic microorganisms inhabit low pH environments such as acid mine drainage that is generated when sulfide minerals are exposed to air. The genome sequence of the psychrotolerant Acidithiobacillus ferrivorans SS3 was compared to a metagenome from a low temperature acidic stream dominated by an A. ferrivorans-like strain. Stretches of genomic DNA characterized by few matches to the metagenome, termed 'metagenomic islands', encoded genes associated with metal efflux and pH homeostasis. The metagenomic islands were enriched in mobile elements such as phage proteins, transposases, integrases and in one case, predicted to be flanked by truncated tRNAs. Cus gene clusters predicted to be involved in copper efflux and further Cus-like RND systems were predicted to be located in metagenomic islands and therefore, constitute part of the flexible gene complement of the species. Phylogenetic analysis of Cus clusters showed both lineage specificity within the Acidithiobacillus genus as well as niche specificity associated with an acidic environment. The metagenomic islands also contained a predicted copper efflux P-type ATPase system and a polyphosphate kinase potentially involved in polyphosphate mediated copper resistance. This study identifies genetic variability of low temperature acidophiles that likely reflects metal resistance selective pressures in the copper rich environment. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Metagenomics and the protein universe

Science.gov (United States)

Godzik, Adam

2011-01-01

Metagenomics sequencing projects have dramatically increased our knowledge of the protein universe and provided over one-half of currently known protein sequences; they have also introduced a much broader phylogenetic diversity into the protein databases. The full analysis of metagenomic datasets is only beginning, but it has already led to the discovery of thousands of new protein families, likely representing novel functions specific to given environments. At the same time, a deeper analysis of such novel families, including experimental structure determination of some representatives, suggests that most of them represent distant homologs of already characterized protein families, and thus most of the protein diversity present in the new environments are due to functional divergence of the known protein families rather than the emergence of new ones. PMID:21497084

Metagenomic Detection Methods in Biopreparedness Outbreak Scenarios

DEFF Research Database (Denmark)

Karlsson, Oskar Erik; Hansen, Trine; Knutsson, Rickard

2013-01-01

In the field of diagnostic microbiology, rapid molecular methods are critically important for detecting pathogens. With rapid and accurate detection, preventive measures can be put in place early, thereby preventing loss of life and further spread of a disease. From a preparedness perspective...... of a clinical sample, creating a metagenome, in a single week of laboratory work. As new technologies emerge, their dissemination and capacity building must be facilitated, and criteria for use, as well as guidelines on how to report results, must be established. This article focuses on the use of metagenomics...

Comparative fecal metagenomics unveils unique functional capacity of the swine gut

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Martinson John

2011-05-01

Full Text Available Abstract Background Uncovering the taxonomic composition and functional capacity within the swine gut microbial consortia is of great importance to animal physiology and health as well as to food and water safety due to the presence of human pathogens in pig feces. Nonetheless, limited information on the functional diversity of the swine gut microbiome is available. Results Analysis of 637, 722 pyrosequencing reads (130 megabases generated from Yorkshire pig fecal DNA extracts was performed to help better understand the microbial diversity and largely unknown functional capacity of the swine gut microbiome. Swine fecal metagenomic sequences were annotated using both MG-RAST and JGI IMG/M-ER pipelines. Taxonomic analysis of metagenomic reads indicated that swine fecal microbiomes were dominated by Firmicutes and Bacteroidetes phyla. At a finer phylogenetic resolution, Prevotella spp. dominated the swine fecal metagenome, while some genes associated with Treponema and Anareovibrio species were found to be exclusively within the pig fecal metagenomic sequences analyzed. Functional analysis revealed that carbohydrate metabolism was the most abundant SEED subsystem, representing 13% of the swine metagenome. Genes associated with stress, virulence, cell wall and cell capsule were also abundant. Virulence factors associated with antibiotic resistance genes with highest sequence homology to genes in Bacteroidetes, Clostridia, and Methanosarcina were numerous within the gene families unique to the swine fecal metagenomes. Other abundant proteins unique to the distal swine gut shared high sequence homology to putative carbohydrate membrane transporters. Conclusions The results from this metagenomic survey demonstrated the presence of genes associated with resistance to antibiotics and carbohydrate metabolism suggesting that the swine gut microbiome may be shaped by husbandry practices.

A metagenomic snapshot of taxonomic and functional diversity in an alpine glacier cryoconite ecosystem

International Nuclear Information System (INIS)

Edwards, Arwyn; Pachebat, Justin A; Swain, Martin; Hegarty, Matt; Rassner, Sara M E; Hodson, Andrew J; Irvine-Fynn, Tristram D L; Sattler, Birgit

2013-01-01

Cryoconite is a microbe–mineral aggregate which darkens the ice surface of glaciers. Microbial process and marker gene PCR-dependent measurements reveal active and diverse cryoconite microbial communities on polar glaciers. Here, we provide the first report of a cryoconite metagenome and culture-independent study of alpine cryoconite microbial diversity. We assembled 1.2 Gbp of metagenomic DNA sequenced using an Illumina HiScanSQ from cryoconite holes across the ablation zone of Rotmoosferner in the Austrian Alps. The metagenome revealed a bacterially-dominated community, with Proteobacteria (62% of bacterial-assigned contigs) and Bacteroidetes (14%) considerably more abundant than Cyanobacteria (2.5%). Streptophyte DNA dominated the eukaryotic metagenome. Functional genes linked to N, Fe, S and P cycling illustrated an acquisitive trend and a nitrogen cycle based upon efficient ammonia recycling. A comparison of 32 metagenome datasets revealed a similarity in functional profiles between the cryoconite and metagenomes characterized from other cold microbe–mineral aggregates. Overall, the metagenomic snapshot reveals the cryoconite ecosystem of this alpine glacier as dependent on scavenging carbon and nutrients from allochthonous sources, in particular mosses transported by wind from ice-marginal habitats, consistent with net heterotrophy indicated by productivity measurements. A transition from singular snapshots of cryoconite metagenomes to comparative analyses is advocated. (letter)

In-depth resistome analysis by targeted metagenomics.

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Lanza, Val F; Baquero, Fernando; Martínez, José Luís; Ramos-Ruíz, Ricardo; González-Zorn, Bruno; Andremont, Antoine; Sánchez-Valenzuela, Antonio; Ehrlich, Stanislav Dusko; Kennedy, Sean; Ruppé, Etienne; van Schaik, Willem; Willems, Rob J; de la Cruz, Fernando; Coque, Teresa M

2018-01-15

Antimicrobial resistance is a major global health challenge. Metagenomics allows analyzing the presence and dynamics of "resistomes" (the ensemble of genes encoding antimicrobial resistance in a given microbiome) in disparate microbial ecosystems. However, the low sensitivity and specificity of available metagenomic methods preclude the detection of minority populations (often present below their detection threshold) and/or the identification of allelic variants that differ in the resulting phenotype. Here, we describe a novel strategy that combines targeted metagenomics using last generation in-solution capture platforms, with novel bioinformatics tools to establish a standardized framework that allows both quantitative and qualitative analyses of resistomes. We developed ResCap, a targeted sequence capture platform based on SeqCapEZ (NimbleGene) technology, which includes probes for 8667 canonical resistance genes (7963 antibiotic resistance genes and 704 genes conferring resistance to metals or biocides), and 2517 relaxase genes (plasmid markers) and 78,600 genes homologous to the previous identified targets (47,806 for antibiotics and 30,794 for biocides or metals). Its performance was compared with metagenomic shotgun sequencing (MSS) for 17 fecal samples (9 humans, 8 swine). ResCap significantly improves MSS to detect "gene abundance" (from 2.0 to 83.2%) and "gene diversity" (26 versus 14.9 genes unequivocally detected per sample per million of reads; the number of reads unequivocally mapped increasing up to 300-fold by using ResCap), which were calculated using novel bioinformatic tools. ResCap also facilitated the analysis of novel genes potentially involved in the resistance to antibiotics, metals, biocides, or any combination thereof. ResCap, the first targeted sequence capture, specifically developed to analyze resistomes, greatly enhances the sensitivity and specificity of available metagenomic methods and offers the possibility to analyze genes

MetaVelvet: An Extension of Velvet Assembler to de novo Metagenome Assembly from Short Sequence Reads (Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

Energy Technology Data Exchange (ETDEWEB)

Sakakibara, Yasumbumi

2011-10-13

Keio University's Yasumbumi Sakakibara on "MetaVelvet: An Extension of Velvet Assembler to de novo Metagenome Assembly from Short Sequence Reads" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

Novel high-performance metagenome β-galactosidases for lactose hydrolysis in the dairy industry.

Science.gov (United States)

Erich, Sarah; Kuschel, Beatrice; Schwarz, Thilo; Ewert, Jacob; Böhmer, Nico; Niehaus, Frank; Eck, Jürgen; Lutz-Wahl, Sabine; Stressler, Timo; Fischer, Lutz

2015-09-20

The industrially utilised β-galactosidases from Kluyveromyces spp. and Aspergillus spp. feature undesirable kinetic properties in praxis, such as an unsatisfactory lactose affinity (KM) and product inhibition (KI) by galactose. In this study, a metagenome library of about 1.3 million clones was investigated with a three-step activity-based screening strategy in order to find new β-galactosidases with more favourable kinetic properties. Six novel metagenome β-galactosidases (M1-M6) were found with an improved lactose hydrolysis performance in original milk when directly compared to the commercial β-galactosidase from Kluyveromyces lactis (GODO-YNL2). The best metagenome candidate, called "M1", was recombinantly produced in Escherichia coli BL21(DE3) in a bioreactor (volume 35 L), resulting in a total β-galactosidase M1 activity of about 1100 μkatoNPGal,37 °C L(-1). Since milk is a sensitive and complex medium, it has to be processed at 5-10 °C in the dairy industry. Therefore, the β-galactosidase M1 was tested at 8 °C in milk and possessed a good stability (t1/2=21.8 d), a desirably low apparent KM,lactose,8 °C value of 3.8±0.7 mM and a high apparent KI,galactose,8 °C value of 196.6±55.5 mM. A lactose hydrolysis process (milk, 40 nkatlactose mLmilk,8 °C(-1)) was conducted at a scale of 0.5L to compare the performance of M1 with the commercial β-galactosidase from K. lactis (GODO-YNL2). Lactose was completely (>99.99%) hydrolysed by M1 and to 99.6% (w/v) by K. lactis β-galactosidase after 25 h process time. Thus, M1 was able to achieve the limit of lactose per litre milk, which is recommended for dairy products labelled as "lactose-free". Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

The potential of viral metagenomics in blood transfusion safety.

Science.gov (United States)

Sauvage, V; Gomez, J; Boizeau, L; Laperche, S

2017-09-01

Thanks to the significant advent of high throughput sequencing in the last ten years, it is now possible via metagenomics to define the spectrum of the microbial sequences present in human blood samples. Therefore, metagenomics sequencing appears as a promising approach for the identification and global surveillance of new, emerging and/or unexpected viruses that could impair blood transfusion safety. However, despite considerable advantages compared to the traditional methods of pathogen identification, this non-targeted approach presents several drawbacks including a lack of sensitivity and sequence contaminant issues. With further improvements, especially to increase sensitivity, metagenomics sequencing should become in a near future an additional diagnostic tool in infectious disease field and especially in blood transfusion safety. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Challenges and Opportunities of Airborne Metagenomics

KAUST Repository

Behzad, H.; Gojobori, Takashi; Mineta, K.

2015-01-01

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.

MG-Digger: an automated pipeline to search for giant virus-related sequences in metagenomes

Directory of Open Access Journals (Sweden)

Jonathan eVerneau

2016-03-01

Full Text Available The number of metagenomic studies conducted each year is growing dramatically. Storage and analysis of such big data is difficult and time-consuming. Interestingly, analysis shows that environmental and human metagenomes include a significant amount of non-annotated sequences, representing a ‘dark matter’. We established a bioinformatics pipeline that automatically detects metagenome reads matching query sequences from a given set and applied this tool to the detection of sequences matching large and giant DNA viral members of the proposed order Megavirales or virophages. A total of 1,045 environmental and human metagenomes (≈ 1 Terabase pairs were collected, processed and stored on our bioinformatics server. In addition, nucleotide and protein sequences from 93 Megavirales representatives, including 19 giant viruses of amoeba, and five virophages, were collected. The pipeline was generated by scripts written in Python language and entitled MG-Digger. Metagenomes previously found to contain megavirus-like sequences were tested as controls. MG-Digger was able to annotate hundreds of metagenome sequences as best matching those of giant viruses. These sequences were most often found to be similar to phycodnavirus or mimivirus sequences, but included reads related to recently available pandoraviruses, Pithovirus sibericum, and faustoviruses. Compared to other tools, MG-Digger combined stand-alone use on Linux or Windows operating systems through a user-friendly interface, implementation of ready-to-use customized metagenome databases and query sequence databases, adjustable parameters for BLAST searches, and creation of output files containing selected reads with best match identification. Compared to Metavir 2, a reference tool in viral metagenome analysis, MG-Digger detected 8% more true positive Megavirales-related reads in a control metagenome. The present work shows that massive, automated and recurrent analyses of metagenomes are

Integrative Workflows for Metagenomic Analysis

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Efthymios eLadoukakis

2014-11-01

Full Text Available The rapid evolution of all sequencing technologies, described by the term Next Generation Sequencing (NGS, have revolutionized metagenomic analysis. They constitute a combination of high-throughput analytical protocols, coupled to delicate measuring techniques, in order to potentially discover, properly assemble and map allelic sequences to the correct genomes, achieving particularly high yields for only a fraction of the cost of traditional processes (i.e. Sanger. From a bioinformatic perspective, this boils down to many gigabytes of data being generated from each single sequencing experiment, rendering the management or even the storage, critical bottlenecks with respect to the overall analytical endeavor. The enormous complexity is even more aggravated by the versatility of the processing steps available, represented by the numerous bioinformatic tools that are essential, for each analytical task, in order to fully unveil the genetic content of a metagenomic dataset. These disparate tasks range from simple, nonetheless non-trivial, quality control of raw data to exceptionally complex protein annotation procedures, requesting a high level of expertise for their proper application or the neat implementation of the whole workflow. Furthermore, a bioinformatic analysis of such scale, requires grand computational resources, imposing as the sole realistic solution, the utilization of cloud computing infrastructures. In this review article we discuss different, integrative, bioinformatic solutions available, which address the aforementioned issues, by performing a critical assessment of the available automated pipelines for data management, quality control and annotation of metagenomic data, embracing various, major sequencing technologies and applications.

Metaviz: interactive statistical and visual analysis of metagenomic data.

Science.gov (United States)

Wagner, Justin; Chelaru, Florin; Kancherla, Jayaram; Paulson, Joseph N; Zhang, Alexander; Felix, Victor; Mahurkar, Anup; Elmqvist, Niklas; Corrada Bravo, Héctor

2018-04-06

Large studies profiling microbial communities and their association with healthy or disease phenotypes are now commonplace. Processed data from many of these studies are publicly available but significant effort is required for users to effectively organize, explore and integrate it, limiting the utility of these rich data resources. Effective integrative and interactive visual and statistical tools to analyze many metagenomic samples can greatly increase the value of these data for researchers. We present Metaviz, a tool for interactive exploratory data analysis of annotated microbiome taxonomic community profiles derived from marker gene or whole metagenome shotgun sequencing. Metaviz is uniquely designed to address the challenge of browsing the hierarchical structure of metagenomic data features while rendering visualizations of data values that are dynamically updated in response to user navigation. We use Metaviz to provide the UMD Metagenome Browser web service, allowing users to browse and explore data for more than 7000 microbiomes from published studies. Users can also deploy Metaviz as a web service, or use it to analyze data through the metavizr package to interoperate with state-of-the-art analysis tools available through Bioconductor. Metaviz is free and open source with the code, documentation and tutorials publicly accessible.

Diversity Indices as Measures of Functional Annotation Methods in Metagenomics Studies

KAUST Repository

Jankovic, Boris R.

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.

Phylogenetic convolutional neural networks in metagenomics.

Science.gov (United States)

Fioravanti, Diego; Giarratano, Ylenia; Maggio, Valerio; Agostinelli, Claudio; Chierici, Marco; Jurman, Giuseppe; Furlanello, Cesare

2018-03-08

Convolutional Neural Networks can be effectively used only when data are endowed with an intrinsic concept of neighbourhood in the input space, as is the case of pixels in images. We introduce here Ph-CNN, a novel deep learning architecture for the classification of metagenomics data based on the Convolutional Neural Networks, with the patristic distance defined on the phylogenetic tree being used as the proximity measure. The patristic distance between variables is used together with a sparsified version of MultiDimensional Scaling to embed the phylogenetic tree in a Euclidean space. Ph-CNN is tested with a domain adaptation approach on synthetic data and on a metagenomics collection of gut microbiota of 38 healthy subjects and 222 Inflammatory Bowel Disease patients, divided in 6 subclasses. Classification performance is promising when compared to classical algorithms like Support Vector Machines and Random Forest and a baseline fully connected neural network, e.g. the Multi-Layer Perceptron. Ph-CNN represents a novel deep learning approach for the classification of metagenomics data. Operatively, the algorithm has been implemented as a custom Keras layer taking care of passing to the following convolutional layer not only the data but also the ranked list of neighbourhood of each sample, thus mimicking the case of image data, transparently to the user.

Metagenomic sequence of saline desert microbiota from wild ass sanctuary, Little Rann of Kutch, Gujarat, India.

Science.gov (United States)

Patel, Rajesh; Mevada, Vishal; Prajapati, Dhaval; Dudhagara, Pravin; Koringa, Prakash; Joshi, C G

2015-03-01

We report Metagenome from the saline desert soil sample of Little Rann of Kutch, Gujarat State, India. Metagenome consisted of 633,760 sequences with size 141,307,202 bp and 56% G + C content. Metagenome sequence data are available at EBI under EBI Metagenomics database with accession no. ERP005612. Community metagenomics revealed total 1802 species belonged to 43 different phyla with dominating Marinobacter (48.7%) and Halobacterium (4.6%) genus in bacterial and archaeal domain respectively. Remarkably, 18.2% sequences in a poorly characterized group and 4% gene for various stress responses along with versatile presence of commercial enzyme were evident in a functional metagenome analysis.

Improved cultivation and metagenomics as new tools for bioprospecting in cold environments

DEFF Research Database (Denmark)

Vester, Jan Kjølhede; Glaring, Mikkel Andreas; Stougaard, Peter

2015-01-01

Only a small minority of microorganisms from an environmental sample can be cultured in the laboratory leaving the enormous bioprospecting potential of the uncultured diversity unexplored. This resource can be accessed by improved cultivation methods in which the natural environment is brought...... be limited as few hosts are available for expression of genes with extremophilic properties. This review summarizes the methods developed for improved cultivation as well as the metagenomic approaches for bioprospecting with focus on the challenges faced by bioprospecting in cold environments....

Metagenomic screening for aromatic compound-responsive transcriptional regulators.

Directory of Open Access Journals (Sweden)

Taku Uchiyama

Full Text Available We applied a metagenomics approach to screen for transcriptional regulators that sense aromatic compounds. The library was constructed by cloning environmental DNA fragments into a promoter-less vector containing green fluorescence protein. Fluorescence-based screening was then performed in the presence of various aromatic compounds. A total of 12 clones were isolated that fluoresced in response to salicylate, 3-methyl catechol, 4-chlorocatechol and chlorohydroquinone. Sequence analysis revealed at least 1 putative transcriptional regulator, excluding 1 clone (CHLO8F. Deletion analysis identified compound-specific transcriptional regulators; namely, 8 LysR-types, 2 two-component-types and 1 AraC-type. Of these, 9 representative clones were selected and their reaction specificities to 18 aromatic compounds were investigated. Overall, our transcriptional regulators were functionally diverse in terms of both specificity and induction rates. LysR- and AraC- type regulators had relatively narrow specificities with high induction rates (5-50 fold, whereas two-component-types had wide specificities with low induction rates (3 fold. Numerous transcriptional regulators have been deposited in sequence databases, but their functions remain largely unknown. Thus, our results add valuable information regarding the sequence-function relationship of transcriptional regulators.

Metagenomic survey of bacterial diversity in the atmosphere of Mexico City using different sampling methods.

Science.gov (United States)

Serrano-Silva, N; Calderón-Ezquerro, M C

2018-04-01

The identification of airborne bacteria has traditionally been performed by retrieval in culture media, but the bacterial diversity in the air is underestimated using this method because many bacteria are not readily cultured. Advances in DNA sequencing technology have produced a broad knowledge of genomics and metagenomics, which can greatly improve our ability to identify and study the diversity of airborne bacteria. However, researchers are facing several challenges, particularly the efficient retrieval of low-density microorganisms from the air and the lack of standardized protocols for sample collection and processing. In this study, we tested three methods for sampling bioaerosols - a Durham-type spore trap (Durham), a seven-day recording volumetric spore trap (HST), and a high-throughput 'Jet' spore and particle sampler (Jet) - and recovered metagenomic DNA for 16S rDNA sequencing. Samples were simultaneously collected with the three devices during one week, and the sequencing libraries were analyzed. A simple and efficient method for collecting bioaerosols and extracting good quality DNA for high-throughput sequencing was standardized. The Durham sampler collected preferentially Cyanobacteria, the HST Actinobacteria, Proteobacteria and Firmicutes, and the Jet mainly Proteobacteria and Firmicutes. The HST sampler collected the largest amount of airborne bacterial diversity. More experiments are necessary to select the right sampler, depending on study objectives, which may require monitoring and collecting specific airborne bacteria. Copyright © 2017 Elsevier Ltd. All rights reserved.

Evaluation of the Cow Rumen Metagenome: Assembly by Single Copy Gene Analysis and Single Cell Genome Assemblies (Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

Energy Technology Data Exchange (ETDEWEB)

Sczyrba, Alex

2011-10-13

DOE JGI's Alex Sczyrba on "Evaluation of the Cow Rumen Metagenome" and "Assembly by Single Copy Gene Analysis and Single Cell Genome Assemblies" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

Reconstruction of ribosomal RNA genes from metagenomic data.

Directory of Open Access Journals (Sweden)

Lu Fan

Full Text Available Direct sequencing of environmental DNA (metagenomics has a great potential for describing the 16S rRNA gene diversity of microbial communities. However current approaches using this 16S rRNA gene information to describe community diversity suffer from low taxonomic resolution or chimera problems. Here we describe a new strategy that involves stringent assembly and data filtering to reconstruct full-length 16S rRNA genes from metagenomicpyrosequencing data. Simulations showed that reconstructed 16S rRNA genes provided a true picture of the community diversity, had minimal rates of chimera formation and gave taxonomic resolution down to genus level. The strategy was furthermore compared to PCR-based methods to determine the microbial diversity in two marine sponges. This showed that about 30% of the abundant phylotypes reconstructed from metagenomic data failed to be amplified by PCR. Our approach is readily applicable to existing metagenomic datasets and is expected to lead to the discovery of new microbial phylotypes.

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

Metagenomic frameworks for monitoring antibiotic resistance in aquatic environments.

Science.gov (United States)

Port, Jesse A; Cullen, Alison C; Wallace, James C; Smith, Marissa N; Faustman, Elaine M

2014-03-01

High-throughput genomic technologies offer new approaches for environmental health monitoring, including metagenomic surveillance of antibiotic resistance determinants (ARDs). Although natural environments serve as reservoirs for antibiotic resistance genes that can be transferred to pathogenic and human commensal bacteria, monitoring of these determinants has been infrequent and incomplete. Furthermore, surveillance efforts have not been integrated into public health decision making. We used a metagenomic epidemiology-based approach to develop an ARD index that quantifies antibiotic resistance potential, and we analyzed this index for common modal patterns across environmental samples. We also explored how metagenomic data such as this index could be conceptually framed within an early risk management context. We analyzed 25 published data sets from shotgun pyrosequencing projects. The samples consisted of microbial community DNA collected from marine and freshwater environments across a gradient of human impact. We used principal component analysis to identify index patterns across samples. We observed significant differences in the overall index and index subcategory levels when comparing ecosystems more proximal versus distal to human impact. The selection of different sequence similarity thresholds strongly influenced the index measurements. Unique index subcategory modes distinguished the different metagenomes. Broad-scale screening of ARD potential using this index revealed utility for framing environmental health monitoring and surveillance. This approach holds promise as a screening tool for establishing baseline ARD levels that can be used to inform and prioritize decision making regarding management of ARD sources and human exposure routes. Port JA, Cullen AC, Wallace JC, Smith MN, Faustman EM. 2014. Metagenomic frameworks for monitoring antibiotic resistance in aquatic environments. Environ Health Perspect 122:222–228; http://dx.doi.org/10.1289/ehp

Comprehensive benchmarking and ensemble approaches for metagenomic classifiers.

Science.gov (United States)

McIntyre, Alexa B R; Ounit, Rachid; Afshinnekoo, Ebrahim; Prill, Robert J; Hénaff, Elizabeth; Alexander, Noah; Minot, Samuel S; Danko, David; Foox, Jonathan; Ahsanuddin, Sofia; Tighe, Scott; Hasan, Nur A; Subramanian, Poorani; Moffat, Kelly; Levy, Shawn; Lonardi, Stefano; Greenfield, Nick; Colwell, Rita R; Rosen, Gail L; Mason, Christopher E

2017-09-21

One of the main challenges in metagenomics is the identification of microorganisms in clinical and environmental samples. While an extensive and heterogeneous set of computational tools is available to classify microorganisms using whole-genome shotgun sequencing data, comprehensive comparisons of these methods are limited. In this study, we use the largest-to-date set of laboratory-generated and simulated controls across 846 species to evaluate the performance of 11 metagenomic classifiers. Tools were characterized on the basis of their ability to identify taxa at the genus, species, and strain levels, quantify relative abundances of taxa, and classify individual reads to the species level. Strikingly, the number of species identified by the 11 tools can differ by over three orders of magnitude on the same datasets. Various strategies can ameliorate taxonomic misclassification, including abundance filtering, ensemble approaches, and tool intersection. Nevertheless, these strategies were often insufficient to completely eliminate false positives from environmental samples, which are especially important where they concern medically relevant species. Overall, pairing tools with different classification strategies (k-mer, alignment, marker) can combine their respective advantages. This study provides positive and negative controls, titrated standards, and a guide for selecting tools for metagenomic analyses by comparing ranges of precision, accuracy, and recall. We show that proper experimental design and analysis parameters can reduce false positives, provide greater resolution of species in complex metagenomic samples, and improve the interpretation of results.

Metagenomic Analysis of Dairy Bacteriophages

DEFF Research Database (Denmark)

Muhammed, Musemma K.; Kot, Witold; Neve, Horst

2017-01-01

Despite their huge potential for characterizing the biodiversity of phages, metagenomic studies are currently not available for dairy bacteriophages, partly due to the lack of a standard procedure for phage extraction. We optimized an extraction method that allows to remove the bulk protein from...

ELIXIR pilot action: Marine metagenomics - towards a domain specific set of sustainable services.

Science.gov (United States)

Robertsen, Espen Mikal; Denise, Hubert; Mitchell, Alex; Finn, Robert D; Bongo, Lars Ailo; Willassen, Nils Peder

2017-01-01

Metagenomics, the study of genetic material recovered directly from environmental samples, has the potential to provide insight into the structure and function of heterogeneous microbial communities.  There has been an increased use of metagenomics to discover and understand the diverse biosynthetic capacities of marine microbes, thereby allowing them to be exploited for industrial, food, and health care products. This ELIXIR pilot action was motivated by the need to establish dedicated data resources and harmonized metagenomics pipelines for the marine domain, in order to enhance the exploration and exploitation of marine genetic resources. In this paper, we summarize some of the results from the ELIXIR pilot action "Marine metagenomics - towards user centric services".

Challenges and opportunities in understanding microbial communities with metagenome assembly (accompanied by IPython Notebook tutorial)

Science.gov (United States)

Howe, Adina; Chain, Patrick S. G.

2015-01-01

Metagenomic investigations hold great promise for informing the genetics, physiology, and ecology of environmental microorganisms. Current challenges for metagenomic analysis are related to our ability to connect the dots between sequencing reads, their population of origin, and their encoding functions. Assembly-based methods reduce dataset size by extending overlapping reads into larger contiguous sequences (contigs), providing contextual information for genetic sequences that does not rely on existing references. These methods, however, tend to be computationally intensive and are again challenged by sequencing errors as well as by genomic repeats While numerous tools have been developed based on these methodological concepts, they present confounding choices and training requirements to metagenomic investigators. To help with accessibility to assembly tools, this review also includes an IPython Notebook metagenomic assembly tutorial. This tutorial has instructions for execution any operating system using Amazon Elastic Cloud Compute and guides users through downloading, assembly, and mapping reads to contigs of a mock microbiome metagenome. Despite its challenges, metagenomic analysis has already revealed novel insights into many environments on Earth. As software, training, and data continue to emerge, metagenomic data access and its discoveries will to grow. PMID:26217314

Challenges and opportunities in understanding microbial communities with metagenome assembly (accompanied by IPython Notebook tutorial

Directory of Open Access Journals (Sweden)

Adina eHowe

2015-07-01

Full Text Available Metagenomic investigations hold great promise for informing the genetics, physiology, and ecology of environmental microorganisms. Current challenges for metagenomic analysis are related to our ability to connect the dots between sequencing reads, their population of origin, and their encoding functions. Assembly-based methods reduce dataset size by extending overlapping reads into larger contiguous sequences (contigs, providing contextual information for genetic sequences that does not rely on existing references. These methods, however, tend to be computationally intensive and are again challenged by sequencing errors as well as by genomic repeats While numerous tools have been developed based on these methodological concepts, they present confounding choices and training requirements to metagenomic investigators. To help with accessibility to assembly tools, this review also includes an IPython Notebook metagenomic assembly tutorial. This tutorial has instructions for execution any operating system using Amazon Elastic Cloud Compute and guides users through downloading, assembly, and mapping reads to contigs of a mock microbiome metagenome. Despite its challenges, metagenomic analysis has already revealed novel insights into many environments on Earth. As software, training, and data continue to emerge, metagenomic data access and its discoveries will to grow.

Metagenomic Analysis of Antibiotic Resistance Genes in Dairy Cow Feces following Therapeutic Administration of Third Generation Cephalosporin.

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Lindsey Chambers

Full Text Available Although dairy manure is widely applied to land, it is relatively understudied compared to other livestock as a potential source of antibiotic resistance genes (ARGs to the environment and ultimately to human pathogens. Ceftiofur, the most widely used antibiotic used in U.S. dairy cows, is a 3rd generation cephalosporin, a critically important class of antibiotics to human health. The objective of this study was to evaluate the effect of typical ceftiofur antibiotic treatment on the prevalence of ARGs in the fecal microbiome of dairy cows using a metagenomics approach. β-lactam ARGs were found to be elevated in feces from Holstein cows administered ceftiofur (n = 3 relative to control cows (n = 3. However, total numbers of ARGs across all classes were not measurably affected by ceftiofur treatment, likely because of dominance of unaffected tetracycline ARGs in the metagenomics libraries. Functional analysis via MG-RAST further revealed that ceftiofur treatment resulted in increases in gene sequences associated with "phages, prophages, transposable elements, and plasmids", suggesting that this treatment also enriched the ability to horizontally transfer ARGs. Additional functional shifts were noted with ceftiofur treatment (e.g., increase in genes associated with stress, chemotaxis, and resistance to toxic compounds; decrease in genes associated with metabolism of aromatic compounds and cell division and cell cycle, along with measureable taxonomic shifts (increase in Bacterioidia and decrease in Actinobacteria. This study demonstrates that ceftiofur has a broad, measureable and immediate effect on the cow fecal metagenome. Given the importance of 3rd generation cephalospirins to human medicine, their continued use in dairy cattle should be carefully considered and waste treatment strategies to slow ARG dissemination from dairy cattle manure should be explored.

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. Copyright © 2013 Elsevier B.V. All rights reserved.

Comparative analysis of metagenomes of Italian top soil improvers

International Nuclear Information System (INIS)

Gigliucci, Federica; Brambilla, Gianfranco; Tozzoli, Rosangela; Michelacci, Valeria; Morabito, Stefano

2017-01-01

Biosolids originating from Municipal Waste Water Treatment Plants are proposed as top soil improvers (TSI) for their beneficial input of organic carbon on agriculture lands. Their use to amend soil is controversial, as it may lead to the presence of emerging hazards of anthropogenic or animal origin in the environment devoted to food production. In this study, we used a shotgun metagenomics sequencing as a tool to perform a characterization of the hazards related with the TSIs. The samples showed the presence of many virulence genes associated to different diarrheagenic E. coli pathotypes as well as of different antimicrobial resistance-associated genes. The genes conferring resistance to Fluoroquinolones was the most relevant class of antimicrobial resistance genes observed in all the samples tested. To a lesser extent traits associated with the resistance to Methicillin in Staphylococci and genes conferring resistance to Streptothricin, Fosfomycin and Vancomycin were also identified. The most represented metal resistance genes were cobalt-zinc-cadmium related, accounting for 15–50% of the sequence reads in the different metagenomes out of the total number of those mapping on the class of resistance to compounds determinants. Moreover the taxonomic analysis performed by comparing compost-based samples and biosolids derived from municipal sewage-sludges treatments divided the samples into separate populations, based on the microbiota composition. The results confirm that the metagenomics is efficient to detect genomic traits associated with pathogens and antimicrobial resistance in complex matrices and this approach can be efficiently used for the traceability of TSI samples using the microorganisms’ profiles as indicators of their origin. - Highlights: • Sludge- and green- based biosolids analysed by metagenomics. • Biosolids may introduce microbial hazards in the food chain. • Metagenomics enables tracking biosolids’ sources.

Comparative analysis of metagenomes of Italian top soil improvers

Energy Technology Data Exchange (ETDEWEB)

Gigliucci, Federica, E-mail: Federica.gigliucci@libero.it [Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Viale Regina Elena, 299 00161 Rome (Italy); Department of Sciences, University Roma,Tre, Viale Marconi, 446, 00146 Rome (Italy); Brambilla, Gianfranco; Tozzoli, Rosangela; Michelacci, Valeria; Morabito, Stefano [Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Viale Regina Elena, 299 00161 Rome (Italy)

2017-05-15

Biosolids originating from Municipal Waste Water Treatment Plants are proposed as top soil improvers (TSI) for their beneficial input of organic carbon on agriculture lands. Their use to amend soil is controversial, as it may lead to the presence of emerging hazards of anthropogenic or animal origin in the environment devoted to food production. In this study, we used a shotgun metagenomics sequencing as a tool to perform a characterization of the hazards related with the TSIs. The samples showed the presence of many virulence genes associated to different diarrheagenic E. coli pathotypes as well as of different antimicrobial resistance-associated genes. The genes conferring resistance to Fluoroquinolones was the most relevant class of antimicrobial resistance genes observed in all the samples tested. To a lesser extent traits associated with the resistance to Methicillin in Staphylococci and genes conferring resistance to Streptothricin, Fosfomycin and Vancomycin were also identified. The most represented metal resistance genes were cobalt-zinc-cadmium related, accounting for 15–50% of the sequence reads in the different metagenomes out of the total number of those mapping on the class of resistance to compounds determinants. Moreover the taxonomic analysis performed by comparing compost-based samples and biosolids derived from municipal sewage-sludges treatments divided the samples into separate populations, based on the microbiota composition. The results confirm that the metagenomics is efficient to detect genomic traits associated with pathogens and antimicrobial resistance in complex matrices and this approach can be efficiently used for the traceability of TSI samples using the microorganisms’ profiles as indicators of their origin. - Highlights: • Sludge- and green- based biosolids analysed by metagenomics. • Biosolids may introduce microbial hazards in the food chain. • Metagenomics enables tracking biosolids’ sources.

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

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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

MOCAT: a metagenomics assembly and gene prediction toolkit.

Science.gov (United States)

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. Relevant statistics for each processing step can be summarized into multi-sheet Excel documents and queryable SQL databases. MOCAT runs on UNIX machines and integrates seamlessly with the SGE and PBS queuing systems, commonly used to process large datasets. The open source code and modular architecture allow users to modify or exchange the programs that are utilized in the various processing steps. Individual processing steps and parameters were benchmarked and tested on artificial, real, and simulated metagenomes resulting in an improvement of selected quality metrics. MOCAT can be freely downloaded at http://www.bork.embl.de/mocat/.

MOCAT: a metagenomics assembly and gene prediction toolkit.

Directory of Open Access Journals (Sweden)

Jens Roat Kultima

Full Text Available 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. Relevant statistics for each processing step can be summarized into multi-sheet Excel documents and queryable SQL databases. MOCAT runs on UNIX machines and integrates seamlessly with the SGE and PBS queuing systems, commonly used to process large datasets. The open source code and modular architecture allow users to modify or exchange the programs that are utilized in the various processing steps. Individual processing steps and parameters were benchmarked and tested on artificial, real, and simulated metagenomes resulting in an improvement of selected quality metrics. MOCAT can be freely downloaded at http://www.bork.embl.de/mocat/.

Computational analysis of the human HSPH/HSPA/DNAJ family and cloning of a human HSPH/HSPA/DNAJ expression library

NARCIS (Netherlands)

Hageman, Jurre; Kampinga, Harm H.

In this manuscript, we describe the generation of a gene library for the expression of HSP110/HSPH, HSP70/HSPA and HSP40/DNAJ members. First, the heat shock protein (HSP) genes were collected from the gene databases and the gene families were analyzed for expression patterns, heat inducibility,

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 < 0.0001) than individuals with Ruminococcus- and Prevotella-driven enterotypes. This is the first study to investigate the relationship between Blastocystis and communities of gut bacteria using a 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. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Resolving the Complexity of Human Skin Metagenomes Using Single-Molecule Sequencing

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Yu-Chih Tsai

2016-02-01

Full Text Available Deep metagenomic shotgun sequencing has emerged as a powerful tool to interrogate composition and function of complex microbial communities. Computational approaches to assemble genome fragments have been demonstrated to be an effective tool for de novo reconstruction of genomes from these communities. However, the resultant “genomes” are typically fragmented and incomplete due to the limited ability of short-read sequence data to assemble complex or low-coverage regions. Here, we use single-molecule, real-time (SMRT sequencing to reconstruct a high-quality, closed genome of a previously uncharacterized Corynebacterium simulans and its companion bacteriophage from a skin metagenomic sample. Considerable improvement in assembly quality occurs in hybrid approaches incorporating short-read data, with even relatively small amounts of long-read data being sufficient to improve metagenome reconstruction. Using short-read data to evaluate strain variation of this C. simulans in its skin community at single-nucleotide resolution, we observed a dominant C. simulans strain with moderate allelic heterozygosity throughout the population. We demonstrate the utility of SMRT sequencing and hybrid approaches in metagenome quantitation, reconstruction, and annotation.

Resolving the Complexity of Human Skin Metagenomes Using Single-Molecule Sequencing

Science.gov (United States)

Tsai, Yu-Chih; Deming, Clayton; Segre, Julia A.; Kong, Heidi H.; Korlach, Jonas

2016-01-01

ABSTRACT Deep metagenomic shotgun sequencing has emerged as a powerful tool to interrogate composition and function of complex microbial communities. Computational approaches to assemble genome fragments have been demonstrated to be an effective tool for de novo reconstruction of genomes from these communities. However, the resultant “genomes” are typically fragmented and incomplete due to the limited ability of short-read sequence data to assemble complex or low-coverage regions. Here, we use single-molecule, real-time (SMRT) sequencing to reconstruct a high-quality, closed genome of a previously uncharacterized Corynebacterium simulans and its companion bacteriophage from a skin metagenomic sample. Considerable improvement in assembly quality occurs in hybrid approaches incorporating short-read data, with even relatively small amounts of long-read data being sufficient to improve metagenome reconstruction. Using short-read data to evaluate strain variation of this C. simulans in its skin community at single-nucleotide resolution, we observed a dominant C. simulans strain with moderate allelic heterozygosity throughout the population. We demonstrate the utility of SMRT sequencing and hybrid approaches in metagenome quantitation, reconstruction, and annotation. PMID:26861018

ELIXIR pilot action: Marine metagenomics – towards a domain specific set of sustainable services

Science.gov (United States)

Robertsen, Espen Mikal; Denise, Hubert; Mitchell, Alex; Finn, Robert D.; Bongo, Lars Ailo; Willassen, Nils Peder

2017-01-01

Metagenomics, the study of genetic material recovered directly from environmental samples, has the potential to provide insight into the structure and function of heterogeneous microbial communities.  There has been an increased use of metagenomics to discover and understand the diverse biosynthetic capacities of marine microbes, thereby allowing them to be exploited for industrial, food, and health care products. This ELIXIR pilot action was motivated by the need to establish dedicated data resources and harmonized metagenomics pipelines for the marine domain, in order to enhance the exploration and exploitation of marine genetic resources. In this paper, we summarize some of the results from the ELIXIR pilot action “Marine metagenomics – towards user centric services”. PMID:28620454

Synthetic promoter libraries for Corynebacterium glutamicum

DEFF Research Database (Denmark)

Rytter, Jakob Vang; Helmark, Søren; Chen, Jun

2014-01-01

The ability to modulate gene expression is an important genetic tool in systems biology and biotechnology. Here, we demonstrate that a previously published easy and fast PCR-based method for modulating gene expression in lactic acid bacteria is also applicable to Corynebacterium glutamicum. We co...... promoter library (SPL) technology is convenient for modulating gene expression in C. glutamicum and should have many future applications, within basic research as well as for optimizing industrial production organisms....... constructed constitutive promoter libraries based on various combinations of a previously reported C. glutamicum -10 consensus sequence (gngnTA(c/t)aaTgg) and the Escherichia coli -35 consensus, either with or without an AT-rich region upstream. A promoter library based on consensus sequences frequently found...... in low-GC Gram-positive microorganisms was also included. The strongest promoters were found in the library with a -35 region and a C. glutamicum -10 consensus, and this library also represents the largest activity span. Using the alternative -10 consensus TATAAT, which can be found in many other...

MetaBAT: Metagenome Binning based on Abundance and Tetranucleotide frequence

Energy Technology Data Exchange (ETDEWEB)

Kang, Dongwan; Froula, Jeff; Egan, Rob; Wang, Zhong

2014-03-21

Grouping large fragments assembled from shotgun metagenomic sequences to deconvolute complex microbial communities, or metagenome binning, enables the study of individual organisms and their interactions. Here we developed automated metagenome binning software, called MetaBAT, which integrates empirical probabilistic distances of genome abundance and tetranucleotide frequency. On synthetic datasets MetaBAT on average achieves 98percent precision and 90percent recall at the strain level with 281 near complete unique genomes. Applying MetaBAT to a human gut microbiome data set we recovered 176 genome bins with 92percent precision and 80percent recall. Further analyses suggest MetaBAT is able to recover genome fragments missed in reference genomes up to 19percent, while 53 genome bins are novel. In summary, we believe MetaBAT is a powerful tool to facilitate comprehensive understanding of complex microbial communities.

MALINA: a web service for visual analytics of human gut microbiota whole-genome metagenomic reads.

Science.gov (United States)

Tyakht, Alexander V; Popenko, Anna S; Belenikin, Maxim S; Altukhov, Ilya A; Pavlenko, Alexander V; Kostryukova, Elena S; Selezneva, Oksana V; Larin, Andrei K; Karpova, Irina Y; Alexeev, Dmitry G

2012-12-07

MALINA is a web service for bioinformatic analysis of whole-genome metagenomic data obtained from human gut microbiota sequencing. As input data, it accepts metagenomic reads of various sequencing technologies, including long reads (such as Sanger and 454 sequencing) and next-generation (including SOLiD and Illumina). It is the first metagenomic web service that is capable of processing SOLiD color-space reads, to authors' knowledge. The web service allows phylogenetic and functional profiling of metagenomic samples using coverage depth resulting from the alignment of the reads to the catalogue of reference sequences which are built into the pipeline and contain prevalent microbial genomes and genes of human gut microbiota. The obtained metagenomic composition vectors are processed by the statistical analysis and visualization module containing methods for clustering, dimension reduction and group comparison. Additionally, the MALINA database includes vectors of bacterial and functional composition for human gut microbiota samples from a large number of existing studies allowing their comparative analysis together with user samples, namely datasets from Russian Metagenome project, MetaHIT and Human Microbiome Project (downloaded from http://hmpdacc.org). MALINA is made freely available on the web at http://malina.metagenome.ru. The website is implemented in JavaScript (using Ext JS), Microsoft .NET Framework, MS SQL, Python, with all major browsers supported.

Metagenomic Analysis of the Gut Microbiome of the Common Black Slug Arion ater in Search of Novel Lignocellulose Degrading Enzymes

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Ryan Joynson

2017-11-01

Full Text Available Some eukaryotes are able to gain access to well-protected carbon sources in plant biomass by exploiting microorganisms in the environment or harbored in their digestive system. One is the land pulmonate Arion ater, which takes advantage of a gut microbial consortium that can break down the widely available, but difficult to digest, carbohydrate polymers in lignocellulose, enabling them to digest a broad range of fresh and partially degraded plant material efficiently. This ability is considered one of the major factors that have enabled A. ater to become one of the most widespread plant pest species in Western Europe and North America. Using metagenomic techniques we have characterized the bacterial diversity and functional capability of the gut microbiome of this notorious agricultural pest. Analysis of gut metagenomic community sequences identified abundant populations of known lignocellulose-degrading bacteria, along with well-characterized bacterial plant pathogens. This also revealed a repertoire of more than 3,383 carbohydrate active enzymes (CAZymes including multiple enzymes associated with lignin degradation, demonstrating a microbial consortium capable of degradation of all components of lignocellulose. This would allow A. ater to make extensive use of plant biomass as a source of nutrients through exploitation of the enzymatic capabilities of the gut microbial consortia. From this metagenome assembly we also demonstrate the successful amplification of multiple predicted gene sequences from metagenomic DNA subjected to whole genome amplification and expression of functional proteins, facilitating the low cost acquisition and biochemical testing of the many thousands of novel genes identified in metagenomics studies. These findings demonstrate the importance of studying Gastropod microbial communities. Firstly, with respect to understanding links between feeding and evolutionary success and, secondly, as sources of novel enzymes with

Bioprospecting of functional cellulases from metagenome for second generation biofuel production: a review.

Science.gov (United States)

Tiwari, Rameshwar; Nain, Lata; Labrou, Nikolaos E; Shukla, Pratyoosh

2018-03-01

Second generation biofuel production has been appeared as a sustainable and alternative energy option. The ultimate aim is the development of an industrially feasible and economic conversion process of lignocellulosic biomass into biofuel molecules. Since, cellulose is the most abundant biopolymer and also represented as the photosynthetically fixed form of carbon, the efficient hydrolysis of cellulose is the most important step towards the development of a sustainable biofuel production process. The enzymatic hydrolysis of cellulose by suites of hydrolytic enzymes underlines the importance of cellulase enzyme system in whole hydrolysis process. However, the selection of the suitable cellulolytic enzymes with enhanced activities remains a challenge for the biorefinery industry to obtain efficient enzymatic hydrolysis of biomass. The present review focuses on deciphering the novel and effective cellulases from different environmental niches by unculturable metagenomic approaches. Furthermore, a comprehensive functional aspect of cellulases is also presented and evaluated by assessing the structural and catalytic properties as well as sequence identities and expression patterns. This review summarizes the recent development in metagenomics based approaches for identifying and exploring novel cellulases which open new avenues for their successful application in biorefineries.

Analysis of composition-based metagenomic classification.

Science.gov (United States)

Higashi, Susan; Barreto, André da Motta Salles; Cantão, Maurício Egidio; de Vasconcelos, Ana Tereza Ribeiro

2012-01-01

An essential step of a metagenomic study is the taxonomic classification, that is, the identification of the taxonomic lineage of the organisms in a given sample. The taxonomic classification process involves a series of decisions. Currently, in the context of metagenomics, such decisions are usually based on empirical studies that consider one specific type of classifier. In this study we propose a general framework for analyzing the impact that several decisions can have on the classification problem. Instead of focusing on any specific classifier, we define a generic score function that provides a measure of the difficulty of the classification task. Using this framework, we analyze the impact of the following parameters on the taxonomic classification problem: (i) the length of n-mers used to encode the metagenomic sequences, (ii) the similarity measure used to compare sequences, and (iii) the type of taxonomic classification, which can be conventional or hierarchical, depending on whether the classification process occurs in a single shot or in several steps according to the taxonomic tree. We defined a score function that measures the degree of separability of the taxonomic classes under a given configuration induced by the parameters above. We conducted an extensive computational experiment and found out that reasonable values for the parameters of interest could be (i) intermediate values of n, the length of the n-mers; (ii) any similarity measure, because all of them resulted in similar scores; and (iii) the hierarchical strategy, which performed better in all of the cases. As expected, short n-mers generate lower configuration scores because they give rise to frequency vectors that represent distinct sequences in a similar way. On the other hand, large values for n result in sparse frequency vectors that represent differently metagenomic fragments that are in fact similar, also leading to low configuration scores. Regarding the similarity measure, in

Tapping uncultured microorganisms through metagenomics for drug ...

African Journals Online (AJOL)

African Journal of Biotechnology ... Microorganisms are major source of bioactive natural products, and several ... This review highlights the recent methodologies, limitations, and applications of metagenomics for the discovery of new drugs.

Glucose-tolerant β-glucosidase retrieved from the metagenome

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Taku eUchiyama

2015-06-01

Full Text Available β-glucosidases (BGLs hydrolyze cellooligosaccharides to glucose and play a crucial role in the enzymatic saccharification of cellulosic biomass. Despite their significance for the production of glucose, most identified BGLs are commonly inhibited by low (~mM concentrations of glucose. Therefore, BGLs that are insensitive to glucose inhibition have great biotechnological merit. We applied a metagenomic approach to screen for such rare glucose-tolerant BGLs. A metagenomic library was created in Escherichia coli (approximately 10,000 colonies and grown on LB agar plates containing 5-bromo-4-chloro-3-indolyl-β-D-glucoside, yielding 828 positive (blue colonies. These were then arrayed in 96-well plates, grown in LB, and secondarily screened for activity in the presence of 10% (w/v glucose. Seven glucose-tolerant clones were identified, each of which contained a single bgl gene. The genes were classified into two groups, differing by two nucleotides. The deduced amino acid sequences of these genes were identical (452 aa and found to belong to the glycosyl hydrolase family 1. The recombinant protein (Ks5A7 was overproduced in E. coli as a C-terminal 6 × His-tagged protein and purified to apparent homogeneity. The molecular mass of the purified Ks5A7 was determined to be 54 kDa by SDS-PAGE, and 160 kDa by gel filtration analysis. The enzyme was optimally active at 45°C and pH 5.0–6.5 and retained full or 1.5–2-fold enhanced activity in the presence of 0.1–0.5 M glucose. It had a low KM (78 µM with p-nitrophenyl β-D-glucoside; 0.36 mM with cellobiose and high Vmax (91 µmol min-1 mg-1 with p-nitrophenyl β-D-glucoside; 155 µmol min-1 mg-1 with cellobiose among known glucose-tolerant BGLs and was free from substrate (0.1 M cellobiose inhibition. The efficient use of Ks5A7 in conjunction with Trichoderma reesei cellulases in enzymatic saccharification of alkaline-treated rice straw was demonstrated by increased production of glucose.

Natural history bycatch: a pipeline for identifying metagenomic sequences in RADseq data

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Iris Holmes

2018-04-01

Full Text Available Background Reduced representation genomic datasets are increasingly becoming available from a variety of organisms. These datasets do not target specific genes, and so may contain sequences from parasites and other organisms present in the target tissue sample. In this paper, we demonstrate that (1 RADseq datasets can be used for exploratory analysis of tissue-specific metagenomes, and (2 tissue collections house complete metagenomic communities, which can be investigated and quantified by a variety of techniques. Methods We present an exploratory method for mining metagenomic “bycatch” sequences from a range of host tissue types. We use a combination of the pyRAD assembly pipeline, NCBI’s blastn software, and custom R scripts to isolate metagenomic sequences from RADseq type datasets. Results When we focus on sequences that align with existing references in NCBI’s GenBank, we find that between three and five percent of identifiable double-digest restriction site associated DNA (ddRAD sequences from host tissue samples are from phyla to contain known blood parasites. In addition to tissue samples, we examine ddRAD sequences from metagenomic DNA extracted snake and lizard hind-gut samples. We find that the sequences recovered from these samples match with expected bacterial and eukaryotic gut microbiome phyla. Discussion Our results suggest that (1 museum tissue banks originally collected for host DNA archiving are also preserving valuable parasite and microbiome communities, (2 that publicly available RADseq datasets may include metagenomic sequences that could be explored, and (3 that restriction site approaches are a useful exploratory technique to identify microbiome lineages that could be missed by primer-based approaches.

Untangling Genomes from Metagenomes: Revealing an Uncultured Class of Marine Euryarchaeota

Science.gov (United States)

Iverson, Vaughn; Morris, Robert M.; Frazar, Christian D.; Berthiaume, Chris T.; Morales, Rhonda L.; Armbrust, E. Virginia

2012-02-01

Ecosystems are shaped by complex communities of mostly unculturable microbes. Metagenomes provide a fragmented view of such communities, but the ecosystem functions of major groups of organisms remain mysterious. To better characterize members of these communities, we developed methods to reconstruct genomes directly from mate-paired short-read metagenomes. We closed a genome representing the as-yet uncultured marine group II Euryarchaeota, assembled de novo from 1.7% of a metagenome sequenced from surface seawater. The genome describes a motile, photo-heterotrophic cell focused on degradation of protein and lipids and clarifies the origin of proteorhodopsin. It also demonstrates that high-coverage mate-paired sequence can overcome assembly difficulties caused by interstrain variation in complex microbial communities, enabling inference of ecosystem functions for uncultured members.

Assembly of 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)

2014-01-01

textabstractViral infections remain a serious global health issue. Metagenomic approaches are increasingly used in the detection of novel viral pathogens but also to generate complete genomes of uncultivated viruses. In silico identification of complete viral genomes from sequence data would allow

Metagenomes obtained by "deep sequencing" - what do they tell about the EBPR communities?

DEFF Research Database (Denmark)

Albertsen, Mads; Saunders, Aaron Marc; Nielsen, Kåre Lehmann

2013-01-01

Metagenomics enables studies of the genomic potential of complex microbial communities by sequencing bulk genomic DNA directly from the environment. Knowledge of the genetic potential of a community can be used to formulate and test ecological hypotheses about stability and performance...... demonstrate that metagenomics can be used as a powerful tool for system wide characterization of the EBPR community as well as for a deeper understanding of the function of specific community members. Furthermore, we discuss and illustrate some of the general pitfalls in metagenomics and stress the need...

A novel feruloyl esterase from rumen microbial metagenome: Gene cloning and enzyme characterization in the release of mono- and diferulic acids

Science.gov (United States)

A feruloyl esterase (FAE) gene was isolated from a rumen microbial metagenome, cloned into E. coli, and expressed in active form. The enzyme (RuFae4) was classified as a Type D feruloyl esterase based on its action on synthetic substrates and ability to release diferulates. The RuFae4 alone releas...

Purification, crystallization and preliminary crystallographic analysis of Est25: a ketoprofen-specific hormone-sensitive lipase

International Nuclear Information System (INIS)

Kim, SeungBum; Joo, Sangbum; Yoon, Hyun C.; Ryu, Yeonwoo; Kim, Kyeong Kyu; Kim, T. Doohun

2007-01-01

Est25, a ketoprofen-specific hormone-sensitive lipase from a metagenomic library, was crystallized and diffraction data were collected to 1.49 Å resolution. Ketoprofen, a nonsteroidal anti-inflammatory drug, inhibits the synthesis of prostaglandin. A novel hydrolase (Est25) with high ketoprofen specificity has previously been identified using a metagenomic library from environmental samples. Recombinant Est25 protein with a histidine tag at the N-terminus was expressed in Escherichia coli and purified in a homogenous form. Est25 was crystallized from 2.4 M sodium malonate pH 7.0 and X-ray diffraction data were collected to 1.49 Å using synchrotron radiation. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 197.8, b = 95.2, c = 99.4 Å, β = 97.1°

Bayesian mixture analysis for metagenomic community profiling.

Science.gov (United States)

Morfopoulou, Sofia; Plagnol, Vincent

2015-09-15

Deep sequencing of clinical samples is now an established tool for the detection of infectious pathogens, with direct medical applications. The large amount of data generated produces an opportunity to detect species even at very low levels, provided that computational tools can effectively profile the relevant metagenomic communities. Data interpretation is complicated by the fact that short sequencing reads can match multiple organisms and by the lack of completeness of existing databases, in particular for viral pathogens. Here we present metaMix, a Bayesian mixture model framework for resolving complex metagenomic mixtures. We show that the use of parallel Monte Carlo Markov chains for the exploration of the species space enables the identification of the set of species most likely to contribute to the mixture. We demonstrate the greater accuracy of metaMix compared with relevant methods, particularly for profiling complex communities consisting of several related species. We designed metaMix specifically for the analysis of deep transcriptome sequencing datasets, with a focus on viral pathogen detection; however, the principles are generally applicable to all types of metagenomic mixtures. metaMix is implemented as a user friendly R package, freely available on CRAN: http://cran.r-project.org/web/packages/metaMix sofia.morfopoulou.10@ucl.ac.uk Supplementary data are available at Bionformatics online. © The Author 2015. Published by Oxford University Press.

Validation of Metagenomic Next-Generation Sequencing Tests for Universal Pathogen Detection.

Science.gov (United States)

Schlaberg, Robert; Chiu, Charles Y; Miller, Steve; Procop, Gary W; Weinstock, George

2017-06-01

- Metagenomic sequencing can be used for detection of any pathogens using unbiased, shotgun next-generation sequencing (NGS), without the need for sequence-specific amplification. Proof-of-concept has been demonstrated in infectious disease outbreaks of unknown causes and in patients with suspected infections but negative results for conventional tests. Metagenomic NGS tests hold great promise to improve infectious disease diagnostics, especially in immunocompromised and critically ill patients. - To discuss challenges and provide example solutions for validating metagenomic pathogen detection tests in clinical laboratories. A summary of current regulatory requirements, largely based on prior guidance for NGS testing in constitutional genetics and oncology, is provided. - Examples from 2 separate validation studies are provided for steps from assay design, and validation of wet bench and bioinformatics protocols, to quality control and assurance. - Although laboratory and data analysis workflows are still complex, metagenomic NGS tests for infectious diseases are increasingly being validated in clinical laboratories. Many parallels exist to NGS tests in other fields. Nevertheless, specimen preparation, rapidly evolving data analysis algorithms, and incomplete reference sequence databases are idiosyncratic to the field of microbiology and often overlooked.

Long-Term Protective Immune Response Elicited by Vaccination with an Expression Genomic Library of Toxoplasma gondii

OpenAIRE

Fachado, Alberto; Rodriguez, Alexandro; Molina, Judith; Silvério, Jaline C.; Marino, Ana P. M. P.; Pinto, Luzia M. O.; Angel, Sergio O.; Infante, Juan F.; Traub-Cseko, Yara; Amendoeira, Regina R.; Lannes-Vieira, Joseli

2003-01-01

Immunization of BALB/c mice with an expression genomic library of Toxoplasma gondii induces a Th1-type immune response, with recognition of several T. gondii proteins (21 to 117 kDa) and long-term protective immunity against a lethal challenge. These results support further investigations to achieve a multicomponent anti-T. gondii DNA vaccine.

A highly abundant bacteriophage discovered in the unknown sequences of human faecal metagenomes

NARCIS (Netherlands)

Dutilh, Bas E; Cassman, Noriko; McNair, Katelyn; Sanchez, Savannah E; Silva, Genivaldo G Z; Boling, Lance; Barr, Jeremy J; Speth, Daan R; Seguritan, Victor; Aziz, Ramy K; Felts, Ben; Dinsdale, Elizabeth A; Mokili, John L; Edwards, Robert A

2014-01-01

Metagenomics, or sequencing of the genetic material from a complete microbial community, is a promising tool to discover novel microbes and viruses. Viral metagenomes typically contain many unknown sequences. Here we describe the discovery of a previously unidentified bacteriophage present in the

Gene identification and protein classification in microbial metagenomic sequence data via incremental clustering

Directory of Open Access Journals (Sweden)

Li Weizhong

2008-04-01

Full Text Available Abstract Background The identification and study of proteins from metagenomic datasets can shed light on the roles and interactions of the source organisms in their communities. However, metagenomic datasets are characterized by the presence of organisms with varying GC composition, codon usage biases etc., and consequently gene identification is challenging. The vast amount of sequence data also requires faster protein family classification tools. Results We present a computational improvement to a sequence clustering approach that we developed previously to identify and classify protein coding genes in large microbial metagenomic datasets. The clustering approach can be used to identify protein coding genes in prokaryotes, viruses, and intron-less eukaryotes. The computational improvement is based on an incremental clustering method that does not require the expensive all-against-all compute that was required by the original approach, while still preserving the remote homology detection capabilities. We present evaluations of the clustering approach in protein-coding gene identification and classification, and also present the results of updating the protein clusters from our previous work with recent genomic and metagenomic sequences. The clustering results are available via CAMERA, (http://camera.calit2.net. Conclusion The clustering paradigm is shown to be a very useful tool in the analysis of microbial metagenomic data. The incremental clustering method is shown to be much faster than the original approach in identifying genes, grouping sequences into existing protein families, and also identifying novel families that have multiple members in a metagenomic dataset. These clusters provide a basis for further studies of protein families.

Crystallization and preliminary crystallographic analysis of LipC12, a true lipase isolated through a metagenomics approach

International Nuclear Information System (INIS)

Martini, V. P.; Glogauer, A.; Iulek, J.; Souza, E. M.; Pedrosa, F. O.; Krieger, N.

2012-01-01

The preliminary X-ray analysis of LipC12, the first lipase isolated through a metagenomics approach to be crystallized, is reported. LipC12, a true lipase from family I.1 of bacterial lipases which was previously isolated through a metagenomics approach, contains 293 amino acids. Among lipases of known three-dimensional structure, it has a sequence identity of 47% to the lipase from Pseudomonas aeruginosa PAO1. Recombinant N-terminally His 6 -tagged LipC12 protein was expressed in Escherichia coli, purified in a homogenous form and crystallized in several conditions, with the best crystals being obtained using 2.0 M sodium formate and 0.1 M bis-tris propane pH 7.0. X-ray diffraction data were collected to 2.70 Å resolution. The crystals belonged to the tetragonal space group P4 1 22, with unit-cell parameters a = b = 58.62, c = 192.60 Å

Preliminary exploration and thought of promoting library science Indigenization

International Nuclear Information System (INIS)

Liu Wenping; Du Jingling

2014-01-01

The article explains the significance of Library Science Indigenization, Answer some misunderstanding of Library Science Indigenization,reveals express form of Library Science Indigenization, Discusses criteria of Library Science Indigenization, finally give some suggestions and methods of Library Science Indigenization. (authors)

A Combined Bioinformatics and Functional Metagenomics Approach to Discovering Lipolytic Biocatalysts

Directory of Open Access Journals (Sweden)

Thorsten eMasuch

2015-10-01

Full Text Available The majority of protein sequence data published today is of metagenomic origin. However, our ability to assign functions to these sequences is often hampered by our general inability to cultivate the larger part of microbial species and the sheer amount of sequence data generated in these projects. Here we present a combination of bioinformatics, synthetic biology and Escherichia coli genetics to discover biocatalysts in metagenomic datasets. We created a subset of the Global Ocean Sampling dataset, the largest metagenomic project published to date, by removing all proteins that matched Hidden Markov Models of known protein families from PFAM and TIGRFAM with high confidence (e-value > 10-5. This essentially left us with proteins with low or no homology to known protein families, still encompassing ~1.7 million different sequences. In this subset, we then identified protein families de novo with a Markov clustering algorithm. For each protein family, we defined a single representative based on its phylogenetic relationship to all other members in that family. This reduced the dataset to ~17,000 representatives of protein families with more than 10 members. Based on conserved regions typical for lipases and esterases, we selected a representative gene from a family of 27 members for synthesis. This protein, when expressed in E. coli, showed lipolytic activity towards para-nitrophenyl (pNP esters. The Km value of the enzyme was 66.68 µM for pNP-butyrate and 68.08 µM for pNP-palmitate with kcat/Km values at 3.4 x 106 and 6.6 x 105 M-1s-1, respectively. Hydrolysis of model substrates showed enantiopreference for the R-form. Reactions yielded 43% and 61% enantiomeric excess of products with ibuprofen methyl ester and 2-phenylpropanoic acid ethyl ester, respectively. The enzyme retains 50 % of its maximum activity at temperatures as low as 10 °C, its activity is enhanced in artificial seawater and buffers with higher salt concentrations with an

Extremozymes from metagenome: Potential applications in food processing.

Science.gov (United States)

Khan, Mahejibin; Sathya, T A

2017-06-12

The long-established use of enzymes for food processing and product formulation has resulted in an increased enzyme market compounding to 7.0% annual growth rate. Advancements in molecular biology and recognition that enzymes with specific properties have application for industrial production of infant, baby and functional foods boosted research toward sourcing the genes of microorganisms for enzymes with distinctive properties. In this regard, functional metagenomics for extremozymes has gained attention on the premise that such enzymes can catalyze specific reactions. Hence, metagenomics that can isolate functional genes of unculturable extremophilic microorganisms has expanded attention as a promising tool. Developments in this field of research in relation to food sector are reviewed.

Metagenomes provide valuable comparative information on soil microeukaryotes

DEFF Research Database (Denmark)

Jacquiod, Samuel Jehan Auguste; Stenbæk, Jonas; Santos, Susana

2016-01-01

has been identified. Our analyses suggest that publicly available metagenome data can provide valuable information on soil microeukaryotes for comparative purposes when handled appropriately, complementing the current view provided by ribosomal amplicon sequencing methods......., providing microbiologists with substantial amounts of accessible information. We took advantage of public metagenomes in order to investigate microeukaryote communities in a well characterized grassland soil. The data gathered allowed the evaluation of several factors impacting the community structure......, including the DNA extraction method, the database choice and also the annotation procedure. While most studies on soil microeukaryotes are based on sequencing of PCR-amplified taxonomic markers (18S rRNA genes, ITS regions), this work represents, to our knowledge, the first report based solely...

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.

BeerDeCoded: the open beer metagenome project.

Science.gov (United States)

Sobel, Jonathan; Henry, Luc; Rotman, Nicolas; Rando, Gianpaolo

2017-01-01

Next generation sequencing has radically changed research in the life sciences, in both academic and corporate laboratories. The potential impact is tremendous, yet a majority of citizens have little or no understanding of the technological and ethical aspects of this widespread adoption. We designed BeerDeCoded as a pretext to discuss the societal issues related to genomic and metagenomic data with fellow citizens, while advancing scientific knowledge of the most popular beverage of all. In the spirit of citizen science, sample collection and DNA extraction were carried out with the participation of non-scientists in the community laboratory of Hackuarium, a not-for-profit organisation that supports unconventional research and promotes the public understanding of science. The dataset presented herein contains the targeted metagenomic profile of 39 bottled beers from 5 countries, based on internal transcribed spacer (ITS) sequencing of fungal species. A preliminary analysis reveals the presence of a large diversity of wild yeast species in commercial brews. With this project, we demonstrate that coupling simple laboratory procedures that can be carried out in a non-professional environment with state-of-the-art sequencing technologies and targeted metagenomic analyses, can lead to the detection and identification of the microbial content in bottled beer.

PhyloSift: phylogenetic analysis of genomes and metagenomes.

Science.gov (United States)

Darling, Aaron E; Jospin, Guillaume; Lowe, Eric; Matsen, Frederick A; Bik, Holly M; Eisen, Jonathan A

2014-01-01

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).

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.

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...

The binning of metagenomic contigs for microbial physiology of mixed cultures.

Science.gov (United States)

Strous, Marc; Kraft, Beate; Bisdorf, Regina; Tegetmeyer, Halina E

2012-01-01

So far, microbial physiology has dedicated itself mainly to pure cultures. In nature, cross feeding and competition are important aspects of microbial physiology and these can only be addressed by studying complete communities such as enrichment cultures. Metagenomic sequencing is a powerful tool to characterize such mixed cultures. In the analysis of metagenomic data, well established algorithms exist for the assembly of short reads into contigs and for the annotation of predicted genes. However, the binning of the assembled contigs or unassembled reads is still a major bottleneck and required to understand how the overall metabolism is partitioned over different community members. Binning consists of the clustering of contigs or reads that apparently originate from the same source population. In the present study eight metagenomic samples from the same habitat, a laboratory enrichment culture, were sequenced. Each sample contained 13-23 Mb of assembled contigs and up to eight abundant populations. Binning was attempted with existing methods but they were found to produce poor results, were slow, dependent on non-standard platforms or produced errors. A new binning procedure was developed based on multivariate statistics of tetranucleotide frequencies combined with the use of interpolated Markov models. Its performance was evaluated by comparison of the results between samples with BLAST and in comparison to existing algorithms for four publicly available metagenomes and one previously published artificial metagenome. The accuracy of the new approach was comparable or higher than existing methods. Further, it was up to a 100 times faster. It was implemented in Java Swing as a complete open source graphical binning application available for download and further development (http://sourceforge.net/projects/metawatt).

The binning of metagenomic contigs for microbial physiology of mixed cultures

Directory of Open Access Journals (Sweden)

Marc eStrous

2012-12-01

Full Text Available So far, microbial physiology has dedicated itself mainly to pure cultures. In nature, cross feeding and competition are important aspects of microbial physiology and these can only be addressed by studying complete communities such as enrichment cultures. Metagenomic sequencing is a powerful tool to characterize such mixed cultures. In the analysis of metagenomic data, well established algorithms exist for the assembly of short reads into contigs and for the annotation of predicted genes. However, the binning of the assembled contigs or unassembled reads is still a major bottleneck and required to understand how the overall metabolism is partitioned over different community members. Binning consists of the clustering of contigs or reads that apparently originate from the same source population.In the present study eight metagenomic samples originating from the same habitat, a laboratory enrichment culture, were sequenced. Each sample contained 13-23 Mb of assembled contigs and up to eight abundant populations. Binning was attempted with existing methods but they were found to produce poor results, were slow, dependent on non-standard platforms or produced errors. A new binning procedure was developed based on multivariate statistics of tetranucleotide frequencies combined with the use of interpolated Markov models. Its performance was evaluated by comparison of the results between samples with BLAST and in comparison to exisiting algorithms for four publicly available metagenomes and one previously published artificial metagenome. The accuracy of the new approach was comparable or higher than existing methods. Further, it was up to a hunderd times faster. It was implemented in Java Swing as a complete open source graphical binning application available for download and further development (http://sourceforge.net/projects/metawatt.

High-throughput expression of animal venom toxins in Escherichia coli to generate a large library of oxidized disulphide-reticulated peptides for drug discovery.

Science.gov (United States)

Turchetto, Jeremy; Sequeira, Ana Filipa; Ramond, Laurie; Peysson, Fanny; Brás, Joana L A; Saez, Natalie J; Duhoo, Yoan; Blémont, Marilyne; Guerreiro, Catarina I P D; Quinton, Loic; De Pauw, Edwin; Gilles, Nicolas; Darbon, Hervé; Fontes, Carlos M G A; Vincentelli, Renaud

2017-01-17

Animal venoms are complex molecular cocktails containing a wide range of biologically active disulphide-reticulated peptides that target, with high selectivity and efficacy, a variety of membrane receptors. Disulphide-reticulated peptides have evolved to display improved specificity, low immunogenicity and to show much higher resistance to degradation than linear peptides. These properties make venom peptides attractive candidates for drug development. However, recombinant expression of reticulated peptides containing disulphide bonds is challenging, especially when associated with the production of large libraries of bioactive molecules for drug screening. To date, as an alternative to artificial synthetic chemical libraries, no comprehensive recombinant libraries of natural venom peptides are accessible for high-throughput screening to identify novel therapeutics. In the accompanying paper an efficient system for the expression and purification of oxidized disulphide-reticulated venom peptides in Escherichia coli is described. Here we report the development of a high-throughput automated platform, that could be adapted to the production of other families, to generate the largest ever library of recombinant venom peptides. The peptides were produced in the periplasm of E. coli using redox-active DsbC as a fusion tag, thus allowing the efficient formation of correctly folded disulphide bridges. TEV protease was used to remove fusion tags and recover the animal venom peptides in the native state. Globally, within nine months, out of a total of 4992 synthetic genes encoding a representative diversity of venom peptides, a library containing 2736 recombinant disulphide-reticulated peptides was generated. The data revealed that the animal venom peptides produced in the bacterial host were natively folded and, thus, are putatively biologically active. Overall this study reveals that high-throughput expression of animal venom peptides in E. coli can generate large

Metagenomics, metaMicrobesOnline and Kbase Data Integration (MICW - Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

Energy Technology Data Exchange (ETDEWEB)

Dehal, Paramvir

2011-10-12

Berkeley Lab's Paramvir Dehal on "Managing and Storing large Datasets in MicrobesOnline, metaMicrobesOnline and the DOE Knowledgebase" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

Metagenomics of the Svalbard reindeer rumen microbiome reveals abundance of polysaccharide utilization loci.

Directory of Open Access Journals (Sweden)

Phillip B Pope

Full Text Available Lignocellulosic biomass remains a largely untapped source of renewable energy predominantly due to its recalcitrance and an incomplete understanding of how this is overcome in nature. We present here a compositional and comparative analysis of metagenomic data pertaining to a natural biomass-converting ecosystem adapted to austere arctic nutritional conditions, namely the rumen microbiome of Svalbard reindeer (Rangifer tarandus platyrhynchus. Community analysis showed that deeply-branched cellulolytic lineages affiliated to the Bacteroidetes and Firmicutes are dominant, whilst sequence binning methods facilitated the assemblage of metagenomic sequence for a dominant and novel Bacteroidales clade (SRM-1. Analysis of unassembled metagenomic sequence as well as metabolic reconstruction of SRM-1 revealed the presence of multiple polysaccharide utilization loci-like systems (PULs as well as members of more than 20 glycoside hydrolase and other carbohydrate-active enzyme families targeting various polysaccharides including cellulose, xylan and pectin. Functional screening of cloned metagenome fragments revealed high cellulolytic activity and an abundance of PULs that are rich in endoglucanases (GH5 but devoid of other common enzymes thought to be involved in cellulose degradation. Combining these results with known and partly re-evaluated metagenomic data strongly indicates that much like the human distal gut, the digestive system of herbivores harbours high numbers of deeply branched and as-yet uncultured members of the Bacteroidetes that depend on PUL-like systems for plant biomass degradation.

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 th...... that on average 43% of the species abundance and 58% of the richness cannot be captured by current reference genome-based methods. An implementation of the method is available at http://www.bork.embl.de/software/mOTU/.......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...

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.

Bioinformatic approaches reveal metagenomic characterization of soil microbial community.

Directory of Open Access Journals (Sweden)

Zhuofei Xu

Full Text Available As is well known, soil is a complex ecosystem harboring the most prokaryotic biodiversity on the Earth. In recent years, the advent of high-throughput sequencing techniques has greatly facilitated the progress of soil ecological studies. However, how to effectively understand the underlying biological features of large-scale sequencing data is a new challenge. In the present study, we used 33 publicly available metagenomes from diverse soil sites (i.e. grassland, forest soil, desert, Arctic soil, and mangrove sediment and integrated some state-of-the-art computational tools to explore the phylogenetic and functional characterizations of the microbial communities in soil. Microbial composition and metabolic potential in soils were comprehensively illustrated at the metagenomic level. A spectrum of metagenomic biomarkers containing 46 taxa and 33 metabolic modules were detected to be significantly differential that could be used as indicators to distinguish at least one of five soil communities. The co-occurrence associations between complex microbial compositions and functions were inferred by network-based approaches. Our results together with the established bioinformatic pipelines should provide a foundation for future research into the relation between soil biodiversity and ecosystem function.

Introduction to Metagenomics at DOE JGI (Opening Remarks for the Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

Energy Technology Data Exchange (ETDEWEB)

Kyrpides, Nikos [DOE JGI

2011-10-12

After a quick introduction by DOE JGI Director Eddy Rubin, DOE JGI's Nikos Kyrpides delivers the opening remarks at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011

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

Science.gov (United States)

Nielsen, H Bjørn; Almeida, Mathieu; Juncker, Agnieszka Sierakowska; Rasmussen, Simon; Li, Junhua; Sunagawa, Shinichi; Plichta, Damian R; Gautier, Laurent; Pedersen, Anders G; Le Chatelier, Emmanuelle; Pelletier, Eric; Bonde, Ida; Nielsen, Trine; Manichanh, Chaysavanh; Arumugam, Manimozhiyan; Batto, Jean-Michel; Quintanilha Dos Santos, Marcelo B; Blom, Nikolaj; Borruel, Natalia; Burgdorf, Kristoffer S; Boumezbeur, Fouad; Casellas, Francesc; Doré, Joël; Dworzynski, Piotr; Guarner, Francisco; Hansen, Torben; Hildebrand, Falk; Kaas, Rolf S; Kennedy, Sean; Kristiansen, Karsten; Kultima, Jens Roat; Léonard, Pierre; Levenez, Florence; Lund, Ole; Moumen, Bouziane; Le Paslier, Denis; Pons, Nicolas; Pedersen, Oluf; Prifti, Edi; Qin, Junjie; Raes, Jeroen; Sørensen, Søren; Tap, Julien; Tims, Sebastian; Ussery, David W; Yamada, Takuji; Renault, Pierre; Sicheritz-Ponten, Thomas; Bork, Peer; Wang, Jun; Brunak, Søren; Ehrlich, S Dusko

2014-08-01

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 affiliations between MGS and hundreds of viruses or genetic entities. Our method provides the means for comprehensive profiling of the diversity within complex metagenomic samples.

High-resolution metagenomics targets major functional types in complex microbial communities

Energy Technology Data Exchange (ETDEWEB)

Kalyuzhnaya, Marina G.; Lapidus, Alla; Ivanova, Natalia; Copeland, Alex C.; McHardy, Alice C.; Szeto, Ernest; Salamov, Asaf; Grigoriev, Igor V.; Suciu, Dominic; Levine, Samuel R.; Markowitz, Victor M.; Rigoutsos, Isidore; Tringe, Susannah G.; Bruce, David C.; Richardson, Paul M.; Lidstrom, Mary E.; Chistoserdova, Ludmila

2009-08-01

Most microbes in the biosphere remain uncultured and unknown. Whole genome shotgun (WGS) sequencing of environmental DNA (metagenomics) allows glimpses into genetic and metabolic potentials of natural microbial communities. However, in communities of high complexity metagenomics fail to link specific microbes to specific ecological functions. To overcome this limitation, we selectively targeted populations involved in oxidizing single-carbon (C{sub 1}) compounds in Lake Washington (Seattle, USA) by labeling their DNA via stable isotope probing (SIP), followed by WGS sequencing. Metagenome analysis demonstrated specific sequence enrichments in response to different C{sub 1} substrates, highlighting ecological roles of individual phylotypes. We further demonstrated the utility of our approach by extracting a nearly complete genome of a novel methylotroph Methylotenera mobilis, reconstructing its metabolism and conducting genome-wide analyses. This approach allowing high-resolution genomic analysis of ecologically relevant species has the potential to be applied to a wide variety of ecosystems.

Genome diversity of marine phages recovered from Mediterranean metagenomes: Size matters.

Directory of Open Access Journals (Sweden)

Mario López-Pérez

2017-09-01

Full Text Available Marine viruses play a critical role not only in the global geochemical cycles but also in the biology and evolution of their hosts. Despite their importance, viral diversity remains underexplored mostly due to sampling and cultivation challenges. Direct sequencing approaches such as viromics has provided new insights into the marine viral world. As a complementary approach, we analysed 24 microbial metagenomes (>0.2 μm size range obtained from six sites in the Mediterranean Sea that vary by depth, season and filter used to retrieve the fraction. Filter-size comparison showed a significant number of viral sequences that were retained on the larger-pore filters and were different from those found in the viral fraction from the same sample, indicating that some important viral information is missing using only assembly from viromes. Besides, we were able to describe 1,323 viral genomic fragments that were more than 10Kb in length, of which 36 represented complete viral genomes including some of them retrieved from a cross-assembly from different metagenomes. Host prediction based on sequence methods revealed new phage groups belonging to marine prokaryotes like SAR11, Cyanobacteria or SAR116. We also identified the first complete virophage from deep seawater and a new endemic clade of the recently discovered Marine group II Euryarchaeota virus. Furthermore, analysis of viral distribution using metagenomes and viromes indicated that most of the new phages were found exclusively in the Mediterranean Sea and some of them, mostly the ones recovered from deep metagenomes, do not recruit in any database probably indicating higher variability and endemicity in Mediterranean bathypelagic waters. Together these data provide the first detailed picture of genomic diversity, spatial and depth variations of viral communities within the Mediterranean Sea using metagenome assembly.

Pitfalls using tributyrin agar screening to detect lipolytic activity in ...

African Journals Online (AJOL)

The metagenomics approach is an efficient method for obtaining novel biocatalysts and useful genes from uncultured microorganisms within diverse environments. In this study, we constructed a metagenomic library using a South African deep mine biofilm sample. The library was screened for lipolytic activity using LB ...

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. Copyright © 2016 Elsevier B.V. All rights reserved.

Ten years of maintaining and expanding a microbial genome and metagenome analysis system.

Science.gov (United States)

Markowitz, Victor M; Chen, I-Min A; Chu, Ken; Pati, Amrita; Ivanova, Natalia N; Kyrpides, Nikos C

2015-11-01

Launched in March 2005, the Integrated Microbial Genomes (IMG) system is a comprehensive data management system that supports multidimensional comparative analysis of genomic data. At the core of the IMG system is a data warehouse that contains genome and metagenome datasets sequenced at the Joint Genome Institute or provided by scientific users, as well as public genome datasets available at the National Center for Biotechnology Information Genbank sequence data archive. Genomes and metagenome datasets are processed using IMG's microbial genome and metagenome sequence data processing pipelines and are integrated into the data warehouse using IMG's data integration toolkits. Microbial genome and metagenome application specific data marts and user interfaces provide access to different subsets of IMG's data and analysis toolkits. This review article revisits IMG's original aims, highlights key milestones reached by the system during the past 10 years, and discusses the main challenges faced by a rapidly expanding system, in particular the complexity of maintaining such a system in an academic setting with limited budgets and computing and data management infrastructure. Copyright © 2015 Elsevier Ltd. All rights reserved.

Generation of thermostable Moloney murine leukemia virus reverse transcriptase variants using site saturation mutagenesis library and cell-free protein expression system.

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Katano, Yuta; Li, Tongyang; Baba, Misato; Nakamura, Miyo; Ito, Masaaki; Kojima, Kenji; Takita, Teisuke; Yasukawa, Kiyoshi

2017-12-01

We attempted to increase the thermostability of Moloney murine leukemia virus (MMLV) reverse transcriptase (RT). The eight-site saturation mutagenesis libraries corresponding to Ala70-Arg469 in the whole MMLV RT (Thr24-Leu671), in each of which 1 out of 50 amino acid residues was replaced with other amino acid residue, were constructed. Seven-hundred and sixty eight MMLV RT clones were expressed using a cell-free protein expression system, and their thermostabilities were assessed by the temperature of thermal treatment at which they retained cDNA synthesis activity. One clone D200C was selected as the most thermostable variant. The highest temperature of thermal treatment at which D200C exhibited cDNA synthesis activity was 57ºC, which was higher than for WT (53ºC). Our results suggest that a combination of site saturation mutagenesis library and cell-free protein expression system might be useful for generation of thermostable MMLV RT in a short period of time for expression and selection.

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.

Metagenomic analysis of viral diversity in respiratory samples from patients with respiratory tract infections in Kuwait.

Science.gov (United States)

Madi, Nada; Al-Nakib, Widad; Mustafa, Abu Salim; Habibi, Nazima

2018-03-01

A metagenomic approach based on target independent next-generation sequencing has become a known method for the detection of both known and novel viruses in clinical samples. This study aimed to use the metagenomic sequencing approach to characterize the viral diversity in respiratory samples from patients with respiratory tract infections. We have investigated 86 respiratory samples received from various hospitals in Kuwait between 2015 and 2016 for the diagnosis of respiratory tract infections. A metagenomic approach using the next-generation sequencer to characterize viruses was used. According to the metagenomic analysis, an average of 145, 019 reads were identified, and 2% of these reads were of viral origin. Also, metagenomic analysis of the viral sequences revealed many known respiratory viruses, which were detected in 30.2% of the clinical samples. Also, sequences of non-respiratory viruses were detected in 14% of the clinical samples, while sequences of non-human viruses were detected in 55.8% of the clinical samples. The average genome coverage of the viruses was 12% with the highest genome coverage of 99.2% for respiratory syncytial virus, and the lowest was 1% for torque teno midi virus 2. Our results showed 47.7% agreement between multiplex Real-Time PCR and metagenomics sequencing in the detection of respiratory viruses in the clinical samples. Though there are some difficulties in using this method to clinical samples such as specimen quality, these observations are indicative of the promising utility of the metagenomic sequencing approach for the identification of respiratory viruses in patients with respiratory tract infections. © 2017 Wiley Periodicals, Inc.

Introduction to Metagenomics at DOE JGI: Program Overview and Program Informatics (Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

Energy Technology Data Exchange (ETDEWEB)

Tringe, Susannah

2011-10-12

Susannah Tringe of the DOE Joint Genome Institute talks about the Program Overview and Program Informatics at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

Strain-Level Metagenomic Analysis of the Fermented Dairy Beverage Nunu Highlights Potential Food Safety Risks.

Science.gov (United States)

Walsh, Aaron M; Crispie, Fiona; Daari, Kareem; O'Sullivan, Orla; Martin, Jennifer C; Arthur, Cornelius T; Claesson, Marcus J; Scott, Karen P; Cotter, Paul D

2017-08-15

The rapid detection of pathogenic strains in food products is essential for the prevention of disease outbreaks. It has already been demonstrated that whole-metagenome shotgun sequencing can be used to detect pathogens in food but, until recently, strain-level detection of pathogens has relied on whole-metagenome assembly, which is a computationally demanding process. Here we demonstrated that three short-read-alignment-based methods, i.e., MetaMLST, PanPhlAn, and StrainPhlAn, could accurately and rapidly identify pathogenic strains in spinach metagenomes that had been intentionally spiked with Shiga toxin-producing Escherichia coli in a previous study. Subsequently, we employed the methods, in combination with other metagenomics approaches, to assess the safety of nunu, a traditional Ghanaian fermented milk product that is produced by the spontaneous fermentation of raw cow milk. We showed that nunu samples were frequently contaminated with bacteria associated with the bovine gut and, worryingly, we detected putatively pathogenic E. coli and Klebsiella pneumoniae strains in a subset of nunu samples. Ultimately, our work establishes that short-read-alignment-based bioinformatics approaches are suitable food safety tools, and we describe a real-life example of their utilization. IMPORTANCE Foodborne pathogens are responsible for millions of illnesses each year. Here we demonstrate that short-read-alignment-based bioinformatics tools can accurately and rapidly detect pathogenic strains in food products by using shotgun metagenomics data. The methods used here are considerably faster than both traditional culturing methods and alternative bioinformatics approaches that rely on metagenome assembly; therefore, they can potentially be used for more high-throughput food safety testing. Overall, our results suggest that whole-metagenome sequencing can be used as a practical food safety tool to prevent diseases or to link outbreaks to specific food products. Copyright �

A metagenomic viral discovery approach identifies potential zoonotic and novel mammalian viruses in Neoromicia bats within South Africa.

Science.gov (United States)

Geldenhuys, Marike; Mortlock, Marinda; Weyer, Jacqueline; Bezuidt, Oliver; Seamark, Ernest C J; Kearney, Teresa; Gleasner, Cheryl; Erkkila, Tracy H; Cui, Helen; Markotter, Wanda

2018-01-01

Species within the Neoromicia bat genus are abundant and widely distributed in Africa. It is common for these insectivorous bats to roost in anthropogenic structures in urban regions. Additionally, Neoromicia capensis have previously been identified as potential hosts for Middle East respiratory syndrome (MERS)-related coronaviruses. This study aimed to ascertain the gastrointestinal virome of these bats, as viruses excreted in fecal material or which may be replicating in rectal or intestinal tissues have the greatest opportunities of coming into contact with other hosts. Samples were collected in five regions of South Africa over eight years. Initial virome composition was determined by viral metagenomic sequencing by pooling samples and enriching for viral particles. Libraries were sequenced on the Illumina MiSeq and NextSeq500 platforms, producing a combined 37 million reads. Bioinformatics analysis of the high throughput sequencing data detected the full genome of a novel species of the Circoviridae family, and also identified sequence data from the Adenoviridae, Coronaviridae, Herpesviridae, Parvoviridae, Papillomaviridae, Phenuiviridae, and Picornaviridae families. Metagenomic sequencing data was insufficient to determine the viral diversity of certain families due to the fragmented coverage of genomes and lack of suitable sequencing depth, as some viruses were detected from the analysis of reads-data only. Follow up conventional PCR assays targeting conserved gene regions for the Adenoviridae, Coronaviridae, and Herpesviridae families were used to confirm metagenomic data and generate additional sequences to determine genetic diversity. The complete coding genome of a MERS-related coronavirus was recovered with additional amplicon sequencing on the MiSeq platform. The new genome shared 97.2% overall nucleotide identity to a previous Neoromicia-associated MERS-related virus, also from South Africa. Conventional PCR analysis detected diverse adenovirus and

Estimating P-coverage of biosynthetic pathways in DNA libraries and screening by genetic selection: biotin biosynthesis in the marine microorganism Chromohalobacter.

Science.gov (United States)

Kim, Eun Jin; Angell, Scott; Janes, Jeff; Watanabe, Coran M H

2008-06-01

Traditional approaches to natural product discovery involve cell-based screening of natural product extracts followed by compound isolation and characterization. Their importance notwithstanding, continued mining leads to depletion of natural resources and the reisolation of previously identified metabolites. Metagenomic strategies aimed at localizing the biosynthetic cluster genes and expressing them in surrogate hosts offers one possible alternative. A fundamental question that naturally arises when pursuing such a strategy is, how large must the genomic library be to effectively represent the genome of an organism(s) and the biosynthetic gene clusters they harbor? Such an issue is certainly augmented in the absence of expensive robotics to expedite colony picking and/or screening of clones. We have developed an algorism, named BPC (biosynthetic pathway coverage), supported by molecular simulations to deduce the number of BAC clones required to achieve proper coverage of the genome and their respective biosynthetic pathways. The strategy has been applied to the construction of a large-insert BAC library from a marine microorganism, Hon6 (isolated from Honokohau, Maui) thought to represent a new species. The genomic library is constructed with a BAC yeast shuttle vector pClasper lacZ paving the way for the culturing of libraries in both prokaryotic and eukaryotic hosts. Flow cytometric methods are utilized to estimate the genome size of the organism and BPC implemented to assess P-coverage or percent coverage. A genetic selection strategy is illustrated, applications of which could expedite screening efforts in the identification and localization of biosynthetic pathways from marine microbial consortia, offering a powerful complement to genome sequencing and degenerate probe strategies. Implementing this approach, we report on the biotin biosynthetic pathway from the marine microorganism Hon6.

Metagenomic analysis of the microbiomes in ruminants and other herbivores

International Nuclear Information System (INIS)

Morrison, M.; Adams, S.E.; Nelson, K.E.; Attwood, G.T.

2005-01-01

Many conceptual breakthroughs in the life sciences would not have been possible without first developing techniques and instrumentation to investigate biological processes and molecules. In 1995, The Institute for Genomic Research (TIGR) completely sequenced, assembled and published the fist genome of a free-living organism, that of Haemophilus influenzae Rd. This milestone in scientific achievement has allowed microbiologists to progress from a reductionist approach of studying one gene at a time to the examination of microbial biology from an organismal perspective, using a combination of existing and newly developed (bio)chemical and computational (in silico) approaches. These fields of investigation are often defined with an 'omics' suffix. Hence, genomics refers to the holistic examination of the genetic blueprint that a microbe has acquired, at that point in evolutionary time, to support its lifestyle. Transcriptomics, proteomics and metabolomics refer to a similar level of analysis at the RNA, protein and metabolite levels, respectively. Furthermore, the latest advances in sequencing technologies and cloning vectors better enable a detailed examination of the structure and function of microbial communities, including those organisms that cannot readily be cultured, and we refer to the integrative use of the following methods as the basis of an emerging scientific discipline referred to as metagenomics: 1. Bacterial artificial chromosome and fosmid cloning technologies: Community genomic DNA is cloned in large fragments (>50-150 kilobases [kb]) to create libraries of bacterial artificial chromosomes (BACs), or smaller fragments (∼40 kb) are cloned into fosmid vectors. These libraries can then be screened by DNA- and activity-based screens for genes encoding any number of particular functions including hydrolytic and other enzymes central to schemes of carbon sequestration. 2. High throughput DNA sequencing and bioinformatics: Both BAC and fosmid libraries

Metagenomic analysis reveals presence of Treponema denticola in a tissue biopsy of the Iceman.

Directory of Open Access Journals (Sweden)

Frank Maixner

Full Text Available Ancient hominoid genome studies can be regarded by definition as metagenomic analyses since they represent a mixture of both hominoid and microbial sequences in an environment. Here, we report the molecular detection of the oral spirochete Treponema denticola in ancient human tissue biopsies of the Iceman, a 5,300-year-old Copper Age natural ice mummy. Initially, the metagenomic data of the Iceman's genomic survey was screened for bacterial ribosomal RNA (rRNA specific reads. Through ranking the reads by abundance a relatively high number of rRNA reads most similar to T. denticola was detected. Mapping of the metagenome sequences against the T. denticola genome revealed additional reads most similar to this opportunistic pathogen. The DNA damage pattern of specifically mapped reads suggests an ancient origin of these sequences. The haematogenous spread of bacteria of the oral microbiome often reported in the recent literature could already explain the presence of metagenomic reads specific for T. denticola in the Iceman's bone biopsy. We extended, however, our survey to an Iceman gingival tissue sample and a mouth swab sample and could thereby detect T. denticola and Porphyrimonas gingivalis, another important member of the human commensal oral microflora. Taken together, this study clearly underlines the opportunity to detect disease-associated microorganisms when applying metagenomics-enabled approaches on datasets of ancient human remains.

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

Science.gov (United States)

Ju, Feng; Zhang, Tong

2015-11-03

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.

MaxBin 2.0: an automated binning algorithm to recover genomes from multiple metagenomic datasets

Energy Technology Data Exchange (ETDEWEB)

Wu, Yu-Wei [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Simmons, Blake A. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Singer, Steven W. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

2015-10-29

The recovery of genomes from metagenomic datasets is a critical step to defining the functional roles of the underlying uncultivated populations. We previously developed MaxBin, an automated binning approach for high-throughput recovery of microbial genomes from metagenomes. Here, we present an expanded binning algorithm, MaxBin 2.0, which recovers genomes from co-assembly of a collection of metagenomic datasets. Tests on simulated datasets revealed that MaxBin 2.0 is highly accurate in recovering individual genomes, and the application of MaxBin 2.0 to several metagenomes from environmental samples demonstrated that it could achieve two complementary goals: recovering more bacterial genomes compared to binning a single sample as well as comparing the microbial community composition between different sampling environments. Availability and implementation: MaxBin 2.0 is freely available at http://sourceforge.net/projects/maxbin/ under BSD license. Supplementary information: Supplementary data are available at Bioinformatics online.

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.

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

Quantitative Field Testing Rotylenchulus reniformis DNA from Metagenomic Samples Isolated Directly from Soil

Science.gov (United States)

Showmaker, Kurt; Lawrence, Gary W.; Lu, Shien; Balbalian, Clarissa; Klink, Vincent P.

2011-01-01

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. PMID:22194958

Biochemical Characterization of a Family 15 Carbohydrate Esterase from a Bacterial Marine Arctic Metagenome.

Directory of Open Access Journals (Sweden)

Concetta De Santi

Full Text Available The glucuronoyl esterase enzymes of wood-degrading fungi (Carbohydrate Esterase family 15; CE15 form part of the hemicellulolytic and cellulolytic enzyme systems that break down plant biomass, and have possible applications in biotechnology. Homologous enzymes are predicted in the genomes of several bacteria, however these have been much less studied than their fungal counterparts. Here we describe the recombinant production and biochemical characterization of a bacterial CE15 enzyme denoted MZ0003, which was identified by in silico screening of a prokaryotic metagenome library derived from marine Arctic sediment. MZ0003 has high similarity to several uncharacterized gene products of polysaccharide-degrading bacterial species, and phylogenetic analysis indicates a deep evolutionary split between these CE15s and fungal homologs.MZ0003 appears to differ from previously-studied CE15s in some aspects. Some glucuronoyl esterase activity could be measured by qualitative thin-layer chromatography which confirms its assignment as a CE15, however MZ0003 can also hydrolyze a range of other esters, including p-nitrophenyl acetate, which is not acted upon by some fungal homologs. The structure of MZ0003 also appears to differ as it is predicted to have several large loop regions that are absent in previously studied CE15s, and a combination of homology-based modelling and site-directed mutagenesis indicate its catalytic residues deviate from the conserved Ser-His-Glu triad of many fungal CE15s. Taken together, these results indicate that potentially unexplored diversity exists among bacterial CE15s, and this may be accessed by investigation of the microbial metagenome. The combination of low activity on typical glucuronoyl esterase substrates, and the lack of glucuronic acid esters in the marine environment suggest that the physiological substrate of MZ0003 and its homologs is likely to be different from that of related fungal enzymes.

Abundance profiling of specific gene groups using precomputed gut metagenomes yields novel biological hypotheses.

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Konstantin Yarygin

Full Text Available The gut microbiota is essentially a multifunctional bioreactor within a human being. The exploration of its enormous metabolic potential provides insights into the mechanisms underlying microbial ecology and interactions with the host. The data obtained using "shotgun" metagenomics capture information about the whole spectrum of microbial functions. However, each new study presenting new sequencing data tends to extract only a little of the information concerning the metabolic potential and often omits specific functions. A meta-analysis of the available data with an emphasis on biomedically relevant gene groups can unveil new global trends in the gut microbiota. As a step toward the reuse of metagenomic data, we developed a method for the quantitative profiling of user-defined groups of genes in human gut metagenomes. This method is based on the quick analysis of a gene coverage matrix obtained by pre-mapping the metagenomic reads to a global gut microbial catalogue. The method was applied to profile the abundance of several gene groups related to antibiotic resistance, phages, biosynthesis clusters and carbohydrate degradation in 784 metagenomes from healthy populations worldwide and patients with inflammatory bowel diseases and obesity. We discovered country-wise functional specifics in gut resistome and virome compositions. The most distinct features of the disease microbiota were found for Crohn's disease, followed by ulcerative colitis and obesity. Profiling of the genes belonging to crAssphage showed that its abundance varied across the world populations and was not associated with clinical status. We demonstrated temporal resilience of crAssphage and the influence of the sample preparation protocol on its detected abundance. Our approach offers a convenient method to add value to accumulated "shotgun" metagenomic data by helping researchers state and assess novel biological hypotheses.

Glucose-tolerant β-glucosidase retrieved from a Kusaya gravy metagenome.

Science.gov (United States)

Uchiyama, Taku; Yaoi, Katusro; Miyazaki, Kentaro

2015-01-01

β-glucosidases (BGLs) hydrolyze cello-oligosaccharides to glucose and play a crucial role in the enzymatic saccharification of cellulosic biomass. Despite their significance for the production of glucose, most identified BGLs are commonly inhibited by low (∼mM) concentrations of glucose. Therefore, BGLs that are insensitive to glucose inhibition have great biotechnological merit. We applied a metagenomic approach to screen for such rare glucose-tolerant BGLs. A metagenomic library was created in Escherichia coli (∼10,000 colonies) and grown on LB agar plates containing 5-bromo-4-chloro-3-indolyl-β-D-glucoside, yielding 828 positive (blue) colonies. These were then arrayed in 96-well plates, grown in LB, and secondarily screened for activity in the presence of 10% (w/v) glucose. Seven glucose-tolerant clones were identified, each of which contained a single bgl gene. The genes were classified into two groups, differing by two nucleotides. The deduced amino acid sequences of these genes were identical (452 aa) and found to belong to the glycosyl hydrolase family 1. The recombinant protein (Ks5A7) was overproduced in E. coli as a C-terminal 6 × His-tagged protein and purified to apparent homogeneity. The molecular mass of the purified Ks5A7 was determined to be 54 kDa by SDS-PAGE, and 160 kDa by gel filtration analysis. The enzyme was optimally active at 45°C and pH 5.0-6.5 and retained full or 1.5-2-fold enhanced activity in the presence of 0.1-0.5 M glucose. It had a low KM (78 μM with p-nitrophenyl β-D-glucoside; 0.36 mM with cellobiose) and high V max (91 μmol min(-1) mg(-1) with p-nitrophenyl β-D-glucoside; 155 μmol min(-1) mg(-1) with cellobiose) among known glucose-tolerant BGLs and was free from substrate (0.1 M cellobiose) inhibition. The efficient use of Ks5A7 in conjunction with Trichoderma reesei cellulases in enzymatic saccharification of alkaline-treated rice straw was demonstrated by increased production of glucose.

A sampling and metagenomic sequencing-based methodology for monitoring antimicrobial resistance in swine herds

DEFF Research Database (Denmark)

Munk, Patrick; Dalhoff Andersen, Vibe; de Knegt, Leonardo

2016-01-01

Objectives Reliable methods for monitoring antimicrobial resistance (AMR) in livestock and other reservoirs are essential to understand the trends, transmission and importance of agricultural resistance. Quantification of AMR is mostly done using culture-based techniques, but metagenomic read...... mapping shows promise for quantitative resistance monitoring. Methods We evaluated the ability of: (i) MIC determination for Escherichia coli; (ii) cfu counting of E. coli; (iii) cfu counting of aerobic bacteria; and (iv) metagenomic shotgun sequencing to predict expected tetracycline resistance based...... cultivation-based techniques in terms of predicting expected tetracycline resistance based on antimicrobial consumption. Our metagenomic approach had sufficient resolution to detect antimicrobial-induced changes to individual resistance gene abundances. Pen floor manure samples were found to represent rectal...

MP3: a software tool for the prediction of pathogenic proteins in genomic and metagenomic data.

Science.gov (United States)

Gupta, Ankit; Kapil, Rohan; Dhakan, Darshan B; Sharma, Vineet K

2014-01-01

The identification of virulent proteins in any de-novo sequenced genome is useful in estimating its pathogenic ability and understanding the mechanism of pathogenesis. Similarly, the identification of such proteins could be valuable in comparing the metagenome of healthy and diseased individuals and estimating the proportion of pathogenic species. However, the common challenge in both the above tasks is the identification of virulent proteins since a significant proportion of genomic and metagenomic proteins are novel and yet unannotated. The currently available tools which carry out the identification of virulent proteins provide limited accuracy and cannot be used on large datasets. Therefore, we have developed an MP3 standalone tool and web server for the prediction of pathogenic proteins in both genomic and metagenomic datasets. MP3 is developed using an integrated Support Vector Machine (SVM) and Hidden Markov Model (HMM) approach to carry out highly fast, sensitive and accurate prediction of pathogenic proteins. It displayed Sensitivity, Specificity, MCC and accuracy values of 92%, 100%, 0.92 and 96%, respectively, on blind dataset constructed using complete proteins. On the two metagenomic blind datasets (Blind A: 51-100 amino acids and Blind B: 30-50 amino acids), it displayed Sensitivity, Specificity, MCC and accuracy values of 82.39%, 97.86%, 0.80 and 89.32% for Blind A and 71.60%, 94.48%, 0.67 and 81.86% for Blind B, respectively. In addition, the performance of MP3 was validated on selected bacterial genomic and real metagenomic datasets. To our knowledge, MP3 is the only program that specializes in fast and accurate identification of partial pathogenic proteins predicted from short (100-150 bp) metagenomic reads and also performs exceptionally well on complete protein sequences. MP3 is publicly available at http://metagenomics.iiserb.ac.in/mp3/index.php.

New Bacterial Phytase through Metagenomic Prospection

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Nathálya Farias

2018-02-01

Full Text Available Alkaline phytases from uncultured microorganisms, which hydrolyze phytate to less phosphorylated myo-inositols and inorganic phosphate, have great potential as additives in agricultural industry. The development of metagenomics has stemmed from the ineluctable evidence that as-yet-uncultured microorganisms represent the vast majority of organisms in most environments on earth. In this study, a gene encoding a phytase was cloned from red rice crop residues and castor bean cake using a metagenomics strategy. The amino acid identity between this gene and its closest published counterparts is lower than 60%. The phytase was named PhyRC001 and was biochemically characterized. This recombinant protein showed activity on sodium phytate, indicating that PhyRC001 is a hydrolase enzyme. The enzymatic activity was optimal at a pH of 7.0 and at a temperature of 35 °C. β-propeller phytases possess great potential as feed additives because they are the only type of phytase with high activity at neutral pH. Therefore, to explore and exploit the underlying mechanism for β-propeller phytase functions could be of great benefit to biotechnology.

An enrichment of CRISPR and other defense-related features in marine sponge-associated microbial metagenomes

Directory of Open Access Journals (Sweden)

Hannes Horn

2016-11-01

Full Text Available Many marine sponges are populated by dense and taxonomically diverse microbial consortia. We employed a metagenomics approach to unravel the differences in the functional gene repertoire among three Mediterranean sponge species, Petrosia ficiformis, Sarcotragus foetidus, Aplysina aerophoba and seawater. Different signatures were observed between sponge and seawater metagenomes with regard to microbial community composition, GC content, and estimated bacterial genome size. Our analysis showed further a pronounced repertoire for defense systems in sponge metagenomes. Specifically, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR, restriction modification, DNA phosphorothioation and phage growth limitation systems were enriched in sponge metagenomes. These data suggest that defense is an important functional trait for an existence within sponges that requires mechanisms to defend against foreign DNA from microorganisms and viruses. This study contributes to an understanding of the evolutionary arms race between viruses/phages and bacterial genomes and it sheds light on the bacterial defenses that have evolved in the context of the sponge holobiont.

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

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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. Keywords: Serofluid dish, Jiangshui, Fungal ITS, Cultivation-independent, Microbial diversity

Gut metagenomes of type 2 diabetic patients have characteristic single-nucleotide polymorphism distribution in Bacteroides coprocola.

Science.gov (United States)

Chen, Yaowen; Li, Zongcheng; Hu, Shuofeng; Zhang, Jian; Wu, Jiaqi; Shao, Ningsheng; Bo, Xiaochen; Ni, Ming; Ying, Xiaomin

2017-02-01

Gut microbes play a critical role in human health and disease, and researchers have begun to characterize their genomes, the so-called gut metagenome. Thus far, metagenomics studies have focused on genus- or species-level composition and microbial gene sets, while strain-level composition and single-nucleotide polymorphism (SNP) have been overlooked. The gut metagenomes of type 2 diabetes (T2D) patients have been found to be enriched with butyrate-producing bacteria and sulfate reduction functions. However, it is not known whether the gut metagenomes of T2D patients have characteristic strain patterns or SNP distributions. We downloaded public gut metagenome datasets from 170 T2D patients and 174 healthy controls and performed a systematic comparative analysis of their metagenome SNPs. We found that Bacteroides coprocola, whose relative abundance did not differ between the groups, had a characteristic distribution of SNPs in the T2D patient group. We identified 65 genes, all in B. coprocola, that had remarkably different enrichment of SNPs. The first and sixth ranked genes encode glycosyl hydrolases (GenBank accession EDU99824.1 and EDV02301.1). Interestingly, alpha-glucosidase, which is also a glycosyl hydrolase located in the intestine, is an important drug target of T2D. These results suggest that different strains of B. coprocola may have different roles in human gut and a specific set of B. coprocola strains are correlated with T2D.

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

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Alsop, Eric B; Raymond, Jason

2013-01-01

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.

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

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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.

Biofilm-Growing Bacteria Involved in the Corrosion of Concrete Wastewater Pipes: Protocols for Comparative Metagenomic Analyses

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Advances in high-throughput next-generation sequencing (NGS) technology for direct sequencing of environmental DNA (i.e. shotgun metagenomics) is transforming the field of microbiology. NGS technologies are now regularly being applied in comparative metagenomic studies, which pr...

Functional Genome Mining for Metabolites Encoded by Large Gene Clusters through Heterologous Expression of a Whole-Genome Bacterial Artificial Chromosome Library in Streptomyces spp.

Science.gov (United States)

Xu, Min; Wang, Yemin; Zhao, Zhilong; Gao, Guixi; Huang, Sheng-Xiong; Kang, Qianjin; He, Xinyi; Lin, Shuangjun; Pang, Xiuhua; Deng, Zixin

2016-01-01

ABSTRACT Genome sequencing projects in the last decade revealed numerous cryptic biosynthetic pathways for unknown secondary metabolites in microbes, revitalizing drug discovery from microbial metabolites by approaches called genome mining. In this work, we developed a heterologous expression and functional screening approach for genome mining from genomic bacterial artificial chromosome (BAC) libraries in Streptomyces spp. We demonstrate mining from a strain of Streptomyces rochei, which is known to produce streptothricins and borrelidin, by expressing its BAC library in the surrogate host Streptomyces lividans SBT5, and screening for antimicrobial activity. In addition to the successful capture of the streptothricin and borrelidin biosynthetic gene clusters, we discovered two novel linear lipopeptides and their corresponding biosynthetic gene cluster, as well as a novel cryptic gene cluster for an unknown antibiotic from S. rochei. This high-throughput functional genome mining approach can be easily applied to other streptomycetes, and it is very suitable for the large-scale screening of genomic BAC libraries for bioactive natural products and the corresponding biosynthetic pathways. IMPORTANCE Microbial genomes encode numerous cryptic biosynthetic gene clusters for unknown small metabolites with potential biological activities. Several genome mining approaches have been developed to activate and bring these cryptic metabolites to biological tests for future drug discovery. Previous sequence-guided procedures relied on bioinformatic analysis to predict potentially interesting biosynthetic gene clusters. In this study, we describe an efficient approach based on heterologous expression and functional screening of a whole-genome library for the mining of bioactive metabolites from Streptomyces. The usefulness of this function-driven approach was demonstrated by the capture of four large biosynthetic gene clusters for metabolites of various chemical types, including

Metagenomic insights into anaerobic metabolism along an Arctic peat soil profile.

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David A Lipson

Full Text Available A metagenomic analysis was performed on a soil profile from a wet tundra site in northern Alaska. The goal was to link existing biogeochemical knowledge of the system with the organisms and genes responsible for the relevant metabolic pathways. We specifically investigated how the importance of iron (Fe oxides and humic substances (HS as terminal electron acceptors in this ecosystem is expressed genetically, and how respiratory and fermentative processes varied with soil depth into the active layer and into the upper permafrost. Overall, the metagenomes reflected a microbial community enriched in a diverse range of anaerobic pathways, with a preponderance of known Fe reducing species at all depths in the profile. The abundance of sequences associated with anaerobic metabolic processes generally increased with depth, while aerobic cytochrome c oxidases decreased. Methanogenesis genes and methanogen genomes followed the pattern of CH4 fluxes: they increased steeply with depth into the active layer, but declined somewhat over the transition zone between the lower active layer and the upper permafrost. The latter was relatively enriched in fermentative and anaerobic respiratory pathways. A survey of decaheme cytochromes (MtrA, MtrC and their homologs revealed that this is a promising approach to identifying potential reducers of Fe(III or HS, and indicated a possible role for Acidobacteria as Fe reducers in these soils. Methanogens appear to coexist in the same layers, though in lower abundance, with Fe reducing bacteria and other potential competitors, including acetogens. These observations provide a rich set of hypotheses for further targeted study.

Comparative Metagenomics of Freshwater Microbial Communities

International Nuclear Information System (INIS)

Hemme, Chris; Deng, Ye; Tu, Qichao; Fields, Matthew; Gentry, Terry; Wu, Liyou; Tringe, Susannah; Watson, David; He, Zhili; Hazen, Terry; Tiedje, James; Rubin, Eddy; Zhou, Jizhong

2010-01-01

Previous analyses of a microbial metagenome from uranium and nitric-acid contaminated groundwater (FW106) showed significant environmental effects resulting from the rapid introduction of multiple contaminants. Effects include a massive loss of species and strain biodiversity, accumulation of toxin resistant genes in the metagenome and lateral transfer of toxin resistance genes between community members. To better understand these results in an ecological context, a second metagenome from a pristine groundwater system located along the same geological strike was sequenced and analyzed (FW301). It is hypothesized that FW301 approximates the ancestral FW106 community based on phylogenetic profiles and common geological parameters; however, even if is not the case, the datasets still permit comparisons between healthy and stressed groundwater ecosystems. Complex carbohydrate metabolism has been almost entirely lost in the stressed ecosystem. In contrast, the pristine system encodes a wide diversity of complex carbohydrate metabolism systems, suggesting that carbon turnover is very rapid and less leaky in the healthy groundwater system. FW301 encodes many (∼160+) carbon monoxide dehydrogenase genes while FW106 encodes none. This result suggests that the community is frequently exposed to oxygen from aerated rainwater percolating into the subsurface, with a resulting high rate of carbon metabolism and CO production. When oxygen levels fall, the CO then serves as a major carbon source for the community. FW301 appears to be capable of CO2 fixation via the reductive carboxylase (reverse TCA) cycle and possibly acetogenesis, activities; these activities are lacking in the heterotrophic FW106 system which relies exclusively on respiration of nitrate and/or oxygen for energy production. FW301 encodes a complete set of B12 biosynthesis pathway at high abundance suggesting the use of sodium gradients for energy production in the healthy groundwater community. Overall

A novel genome signature based on inter-nucleotide distances profiles for visualization of metagenomic data

Science.gov (United States)

Xie, Xian-Hua; Yu, Zu-Guo; Ma, Yuan-Lin; Han, Guo-Sheng; Anh, Vo

2017-09-01

There has been a growing interest in visualization of metagenomic data. The present study focuses on the visualization of metagenomic data using inter-nucleotide distances profile. We first convert the fragment sequences into inter-nucleotide distances profiles. Then we analyze these profiles by principal component analysis. Finally the principal components are used to obtain the 2-D scattered plot according to their source of species. We name our method as inter-nucleotide distances profiles (INP) method. Our method is evaluated on three benchmark data sets used in previous published papers. Our results demonstrate that the INP method is good, alternative and efficient for visualization of metagenomic data.

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.

Metagenomic and proteomic analyses to elucidate the mechanism of anaerobic benzene degradation

Energy Technology Data Exchange (ETDEWEB)

Abu Laban, Nidal [Helmholtz (Germany)

2011-07-01

This paper presents the mechanism of anaerobic benzene degradation using metagenomic and proteomic analyses. The objective of the study is to find out the microbes and biochemistry involved in benzene degradation. Hypotheses are proposed for the initial activation mechanism of benzene under anaerobic conditions. Two methods for degradation, molecular characterization and identification of benzene-degrading enzymes, are described. The physiological and molecular characteristics of iron-reducing enrichment culture are given and the process is detailed. Metagenome analysis of iron-reducing culture is presented using a pie chart. From the metagenome analysis of benzene-degrading culture, putative mobile element genes were identified in the aromatic-degrading configurations. Metaproteomic analysis of iron-reducing cultures and the anaerobic benzene degradation pathway are also elucidated. From the study, it can be concluded that gram-positive bacteria are involved in benzene degradation under iron-reducing conditions and that the catalysis mechanism of putative anaerobic benzene carboxylase needs further investigation.

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

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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.

Metagenomics for the discovery of novel biosurfactants of environmental interest from marine ecosystems.

Science.gov (United States)

Jackson, Stephen A; Borchert, Erik; O'Gara, Fergal; Dobson, Alan D W

2015-06-01

Research focused on the search for new biosurfactants aims to replace chemical surfactants, which while being cost-effective are ecologically undesirable. Metagenomics can lead to discovery of novel biosurfactants, tackling issues of low production yields. Recent successes include the heterologous production of biosurfactants. The dearth of biosurfactants discovered to date through metagenomics is puzzling given that good screening systems and heterologous host systems are available. Copyright © 2015 Elsevier Ltd. All rights reserved.

Metagenomic analysis of the airborne environment in urban spaces.

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Be, Nicholas A; Thissen, James B; Fofanov, Viacheslav Y; Allen, Jonathan E; Rojas, Mark; Golovko, George; Fofanov, Yuriy; Koshinsky, Heather; Jaing, Crystal J

2015-02-01

The organisms in aerosol microenvironments, especially densely populated urban areas, are relevant to maintenance of public health and detection of potential epidemic or biothreat agents. To examine aerosolized microorganisms in this environment, we performed sequencing on the material from an urban aerosol surveillance program. Whole metagenome sequencing was applied to DNA extracted from air filters obtained during periods from each of the four seasons. The composition of bacteria, plants, fungi, invertebrates, and viruses demonstrated distinct temporal shifts. Bacillus thuringiensis serovar kurstaki was detected in samples known to be exposed to aerosolized spores, illustrating the potential utility of this approach for identification of intentionally introduced microbial agents. Together, these data demonstrate the temporally dependent metagenomic complexity of urban aerosols and the potential of genomic analytical techniques for biosurveillance and monitoring of threats to public health.

Finding the needles in the meta-genome haystack

NARCIS (Netherlands)

Kowalchuk, G.A.; Speksnijder, A.G.C.L.; Zhang, K.; Goodman, R.M.; Veen, van J.A.

2007-01-01

In the collective genomes (the metagenome) of the microorganisms inhabiting the Earth's diverse environments is written the history of life on this planet. New molecular tools developed and used for the past 15 years by microbial ecologists are facilitating the extraction, cloning, screening, and

Metagenomic analysis of microbial consortia enriched from compost: new insights into the role of Actinobacteria in lignocellulose decomposition.

Science.gov (United States)

Wang, Cheng; Dong, Da; Wang, Haoshu; Müller, Karin; Qin, Yong; Wang, Hailong; Wu, Weixiang

2016-01-01

Compost habitats sustain a vast ensemble of microbes specializing in the degradation of lignocellulosic plant materials and are thus important both for their roles in the global carbon cycle and as potential sources of biochemical catalysts for advanced biofuels production. Studies have revealed substantial diversity in compost microbiomes, yet how this diversity relates to functions and even to the genes encoding lignocellulolytic enzymes remains obscure. Here, we used a metagenomic analysis of the rice straw-adapted (RSA) microbial consortia enriched from compost ecosystems to decipher the systematic and functional contexts within such a distinctive microbiome. Analyses of the 16S pyrotag library and 5 Gbp of metagenomic sequence showed that the phylum Actinobacteria was the predominant group among the Bacteria in the RSA consortia, followed by Proteobacteria, Firmicutes, Chloroflexi, and Bacteroidetes. The CAZymes profile revealed that CAZyme genes in the RSA consortia were also widely distributed within these bacterial phyla. Strikingly, about 46.1 % of CAZyme genes were from actinomycetal communities, which harbored a substantially expanded catalog of the cellobiohydrolase, β-glucosidase, acetyl xylan esterase, arabinofuranosidase, pectin lyase, and ligninase genes. Among these communities, a variety of previously unrecognized species was found, which reveals a greater ecological functional diversity of thermophilic Actinobacteria than previously assumed. These data underline the pivotal role of thermophilic Actinobacteria in lignocellulose biodegradation processes in the compost habitat. Besides revealing a new benchmark for microbial enzymatic deconstruction of lignocelluloses, the results suggest that actinomycetes found in compost ecosystems are potential candidates for mining efficient lignocellulosic enzymes in the biofuel industry.

Forest harvesting reduces the soil metagenomic potential for biomass decomposition.

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Cardenas, Erick; Kranabetter, J M; Hope, Graeme; Maas, Kendra R; Hallam, Steven; Mohn, William W

2015-11-01

Soil is the key resource that must be managed to ensure sustainable forest productivity. Soil microbial communities mediate numerous essential ecosystem functions, and recent studies show that forest harvesting alters soil community composition. From a long-term soil productivity study site in a temperate coniferous forest in British Columbia, 21 forest soil shotgun metagenomes were generated, totaling 187 Gb. A method to analyze unassembled metagenome reads from the complex community was optimized and validated. The subsequent metagenome analysis revealed that, 12 years after forest harvesting, there were 16% and 8% reductions in relative abundances of biomass decomposition genes in the organic and mineral soil layers, respectively. Organic and mineral soil layers differed markedly in genetic potential for biomass degradation, with the organic layer having greater potential and being more strongly affected by harvesting. Gene families were disproportionately affected, and we identified 41 gene families consistently affected by harvesting, including families involved in lignin, cellulose, hemicellulose and pectin degradation. The results strongly suggest that harvesting profoundly altered below-ground cycling of carbon and other nutrients at this site, with potentially important consequences for forest regeneration. Thus, it is important to determine whether these changes foreshadow long-term changes in forest productivity or resilience and whether these changes are broadly characteristic of harvested forests.

Enrichment allows identification of diverse, rare elements in metagenomic resistome-virulome sequencing.

Science.gov (United States)

Noyes, Noelle R; Weinroth, Maggie E; Parker, Jennifer K; Dean, Chris J; Lakin, Steven M; Raymond, Robert A; Rovira, Pablo; Doster, Enrique; Abdo, Zaid; Martin, Jennifer N; Jones, Kenneth L; Ruiz, Jaime; Boucher, Christina A; Belk, Keith E; Morley, Paul S

2017-10-17

Shotgun metagenomic sequencing is increasingly utilized as a tool to evaluate ecological-level dynamics of antimicrobial resistance and virulence, in conjunction with microbiome analysis. Interest in use of this method for environmental surveillance of antimicrobial resistance and pathogenic microorganisms is also increasing. In published metagenomic datasets, the total of all resistance- and virulence-related sequences accounts for enrichment system that incorporates unique molecular indices to count DNA molecules and correct for enrichment bias. The use of the bait-capture and enrichment system significantly increased on-target sequencing of the resistome-virulome, enabling detection of an additional 1441 gene accessions and revealing a low-abundance portion of the resistome-virulome that was more diverse and compositionally different than that detected by more traditional metagenomic assays. The low-abundance portion of the resistome-virulome also contained resistance genes with public health importance, such as extended-spectrum betalactamases, that were not detected using traditional shotgun metagenomic sequencing. In addition, the use of the bait-capture and enrichment system enabled identification of rare resistance gene haplotypes that were used to discriminate between sample origins. These results demonstrate that the rare resistome-virulome contains valuable and unique information that can be utilized for both surveillance and population genetic investigations of resistance. Access to the rare resistome-virulome using the bait-capture and enrichment system validated in this study can greatly advance our understanding of microbiome-resistome dynamics.

Preparing and Analyzing Expressed Sequence Tags (ESTs Library for the Mammary Tissue of Local Turkish Kivircik Sheep

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Nehir Ozdemir Ozgenturk

2017-01-01

Full Text Available Kivircik sheep is an important local Turkish sheep according to its meat quality and milk productivity. The aim of this study was to analyze gene expression profiles of both prenatal and postnatal stages for the Kivircik sheep. Therefore, two different cDNA libraries, which were taken from the same Kivircik sheep mammary gland tissue at prenatal and postnatal stages, were constructed. Total 3072 colonies which were randomly selected from the two libraries were sequenced for developing a sheep ESTs collection. We used Phred/Phrap computer programs for analysis of the raw EST and readable EST sequences were assembled with the CAP3 software. Putative functions of all unique sequences and statistical analysis were determined by Geneious software. Total 422 ESTs have over 80% similarity to known sequences of other organisms in NCBI classified by Panther database for the Gene Ontology (GO category. By comparing gene expression profiles, we observed some putative genes that may be relative to reproductive performance or play important roles in milk synthesis and secretion. A total of 2414 ESTs have been deposited to the NCBI GenBank database (GW996847–GW999260. EST data in this study have provided a new source of information to functional genome studies of sheep.

A Novel Prosthetic Joint Infection Pathogen, Mycoplasma salivarium, Identified by Metagenomic Shotgun Sequencing.

Science.gov (United States)

Thoendel, Matthew; Jeraldo, Patricio; Greenwood-Quaintance, Kerryl E; Chia, Nicholas; Abdel, Matthew P; Steckelberg, James M; Osmon, Douglas R; Patel, Robin

2017-07-15

Defining the microbial etiology of culture-negative prosthetic joint infection (PJI) can be challenging. Metagenomic shotgun sequencing is a new tool to identify organisms undetected by conventional methods. We present a case where metagenomics was used to identify Mycoplasma salivarium as a novel PJI pathogen in a patient with hypogammaglobulinemia. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Vikodak--A Modular Framework for Inferring Functional Potential of Microbial Communities from 16S Metagenomic Datasets.

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Sunil Nagpal

Full Text Available The overall metabolic/functional potential of any given environmental niche is a function of the sum total of genes/proteins/enzymes that are encoded and expressed by various interacting microbes residing in that niche. Consequently, prior (collated information pertaining to genes, enzymes encoded by the resident microbes can aid in indirectly (reconstructing/ inferring the metabolic/ functional potential of a given microbial community (given its taxonomic abundance profile. In this study, we present Vikodak--a multi-modular package that is based on the above assumption and automates inferring and/ or comparing the functional characteristics of an environment using taxonomic abundance generated from one or more environmental sample datasets. With the underlying assumptions of co-metabolism and independent contributions of different microbes in a community, a concerted effort has been made to accommodate microbial co-existence patterns in various modules incorporated in Vikodak.Validation experiments on over 1400 metagenomic samples have confirmed the utility of Vikodak in (a deciphering enzyme abundance profiles of any KEGG metabolic pathway, (b functional resolution of distinct metagenomic environments, (c inferring patterns of functional interaction between resident microbes, and (d automating statistical comparison of functional features of studied microbiomes. Novel features incorporated in Vikodak also facilitate automatic removal of false positives and spurious functional predictions.With novel provisions for comprehensive functional analysis, inclusion of microbial co-existence pattern based algorithms, automated inter-environment comparisons; in-depth analysis of individual metabolic pathways and greater flexibilities at the user end, Vikodak is expected to be an important value addition to the family of existing tools for 16S based function prediction.A web implementation of Vikodak can be publicly accessed at: http://metagenomics

Genomic and metagenomic challenges and opportunities for bioleaching: a mini-review.

Science.gov (United States)

Cárdenas, Juan Pablo; Quatrini, Raquel; Holmes, David S

2016-09-01

High-throughput genomic technologies are accelerating progress in understanding the diversity of microbial life in many environments. Here we highlight advances in genomics and metagenomics of microorganisms from bioleaching heaps and related acidic mining environments. Bioleaching heaps used for copper recovery provide significant opportunities to study the processes and mechanisms underlying microbial successions and the influence of community composition on ecosystem functioning. Obtaining quantitative and process-level knowledge of these dynamics is pivotal for understanding how microorganisms contribute to the solubilization of copper for industrial recovery. Advances in DNA sequencing technology provide unprecedented opportunities to obtain information about the genomes of bioleaching microorganisms, allowing predictive models of metabolic potential and ecosystem-level interactions to be constructed. These approaches are enabling predictive phenotyping of organisms many of which are recalcitrant to genetic approaches or are unculturable. This mini-review describes current bioleaching genomic and metagenomic projects and addresses the use of genome information to: (i) build metabolic models; (ii) predict microbial interactions; (iii) estimate genetic diversity; and (iv) study microbial evolution. Key challenges and perspectives of bioleaching genomics/metagenomics are addressed. Copyright © 2016 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.

Toward a Standards-Compliant Genomic and Metagenomic Publication Record

DEFF Research Database (Denmark)

Garrity, GM; Field, D; Kyrpides, N

2008-01-01

Increasingly, we are aware as a community of the growing need to manage the avalanche of genomic and metagenomic data, in addition to related data types like ribosomal RNA and barcode sequences, in a way that tightly integrates contextual data with traditional literature in a machine-readable way...... is in the midst of a publishing revolution. This revolution is marked by a growing shift away from a traditional dichotomy between "journal articles" and "database entries" and an increasing adoption of hybrid models of collecting and disseminating scientific information. With respect to genomes and metagenomes...... or communities) such as the call by the GSC for a central repository of Standard Operating Procedures describing the genomic annotation pipelines of the major sequencing centers. We argue that such an "eJournal," published under the Open Access paradigm by the GSC, could be an attractive publishing forum...

Metagenomic Classification and Characterization Marine Actinobacteria from the Gulf of Maine without Representative Genomes

Science.gov (United States)

Sachdeva, R.; Heidelberg, J.

2012-12-01

Actinobacteria represent one of the largest and most diverse bacterial phyla and unlike most marine prokaryotes are gram-positive. This phylum encompasses a broad range of physiologies, morphologies, and metabolic properties with a broad array of lifestyles. The marine actinobacterial assemblage is dominated by the orders Actinomycetales and Acidimicrobiales (also known as the marine Actinobacteria clade). The Acidimicrobiales bacteria typically outnumber the Actinomycetales bacteria and are mostly represented by the OCS155 group. Although bacteria of the order Acidimicrobiales make up ~7.6% of the 16S matches from the Global Ocean Survey shotgun metagenomic libraries; very little is known about their potential function and role in biogeochemical cycling. Samples were collected from surface seawater samples in the Gulf of Maine (GOM) from the summer and winter of 2006. Sanger sequences were generated from the 0.1-0.8 μm fractions using paired-end medium insert shotgun libraries. The resulting 2.2 Gb were assembled using the Celera Assembler package into 280 Mb of non-redundant scaffolds. Putative actinobacterial assemblies were identified using (1) ribosomal RNA genes (16S and 23S), (2) phylogenetically informative non-ribosomal core genes thought to be resistant to horizontal gene transfer (e.g. RecA and RpoB) and (3) compositional binning using oligonucleotide frequency pattern based hierarchical clustering. Binning resulted in 3.6 Mb (4.2X coverage) of actinobacterial scaffolds that were comprised of 15.1 Mb of unassembled reads. Putative actinobacterial assemblies included both summer and winter reads demonstrating that the Actinobacteria are abundant year round. Classification reveals that all of the sampled Actinobacteria are from the orders Acidimicrobiales and Actinomycetales and are similar to those found in the global ocean. The GOM Actinobacteria show a broad range of G+C % content (32-66%) indicating a high level of genomic diversity. Those assemblies

Evaluation of FTA ® paper for storage of oral meta-genomic DNA.

Science.gov (United States)

Foitzik, Magdalena; Stumpp, Sascha N; Grischke, Jasmin; Eberhard, Jörg; Stiesch, Meike

2014-10-01

The purpose of the present study was to evaluate the short-term storage of meta-genomic DNA from native oral biofilms on FTA(®) paper. Thirteen volunteers of both sexes received an acrylic splint for intraoral biofilm formation over a period of 48 hours. The biofilms were collected, resuspended in phosphate-buffered saline, and either stored on FTA(®) paper or directly processed by standard laboratory DNA extraction. The nucleic acid extraction efficiencies were evaluated by 16S rDNA targeted SSCP fingerprinting. The acquired banding pattern of FTA-derived meta-genomic DNA was compared to a standard DNA preparation protocol. Sensitivity and positive predictive values were calculated. The volunteers showed inter-individual differences in their bacterial species composition. A total of 200 bands were found for both methods and 85% of the banding patterns were equal, representing a sensitivity of 0.941 and a false-negative predictive value of 0.059. Meta-genomic DNA sampling, extraction, and adhesion using FTA(®) paper is a reliable method for storage of microbial DNA for a short period of time.

Microbial Gene Abundance and Expression Patterns across a River to Ocean Salinity Gradient.

Directory of Open Access Journals (Sweden)

Caroline S Fortunato

Full Text Available Microbial communities mediate the biogeochemical cycles that drive ecosystems, and it is important to understand how these communities are affected by changing environmental conditions, especially in complex coastal zones. As fresh and marine waters mix in estuaries and river plumes, the salinity, temperature, and nutrient gradients that are generated strongly influence bacterioplankton community structure, yet, a parallel change in functional diversity has not been described. Metagenomic and metatranscriptomic analyses were conducted on five water samples spanning the salinity gradient of the Columbia River coastal margin, including river, estuary, plume, and ocean, in August 2010. Samples were pre-filtered through 3 μm filters and collected on 0.2 μm filters, thus results were focused on changes among free-living microbial communities. Results from metagenomic 16S rRNA sequences showed taxonomically distinct bacterial communities in river, estuary, and coastal ocean. Despite the strong salinity gradient observed over sampling locations (0 to 33, the functional gene profiles in the metagenomes were very similar from river to ocean with an average similarity of 82%. The metatranscriptomes, however, had an average similarity of 31%. Although differences were few among the metagenomes, we observed a change from river to ocean in the abundance of genes encoding for catabolic pathways, osmoregulators, and metal transporters. Additionally, genes specifying both bacterial oxygenic and anoxygenic photosynthesis were abundant and expressed in the estuary and plume. Denitrification genes were found throughout the Columbia River coastal margin, and most highly expressed in the estuary. Across a river to ocean gradient, the free-living microbial community followed three different patterns of diversity: 1 the taxonomy of the community changed strongly with salinity, 2 metabolic potential was highly similar across samples, with few differences in

Microbial Gene Abundance and Expression Patterns across a River to Ocean Salinity Gradient.

Science.gov (United States)

Fortunato, Caroline S; Crump, Byron C

2015-01-01

Microbial communities mediate the biogeochemical cycles that drive ecosystems, and it is important to understand how these communities are affected by changing environmental conditions, especially in complex coastal zones. As fresh and marine waters mix in estuaries and river plumes, the salinity, temperature, and nutrient gradients that are generated strongly influence bacterioplankton community structure, yet, a parallel change in functional diversity has not been described. Metagenomic and metatranscriptomic analyses were conducted on five water samples spanning the salinity gradient of the Columbia River coastal margin, including river, estuary, plume, and ocean, in August 2010. Samples were pre-filtered through 3 μm filters and collected on 0.2 μm filters, thus results were focused on changes among free-living microbial communities. Results from metagenomic 16S rRNA sequences showed taxonomically distinct bacterial communities in river, estuary, and coastal ocean. Despite the strong salinity gradient observed over sampling locations (0 to 33), the functional gene profiles in the metagenomes were very similar from river to ocean with an average similarity of 82%. The metatranscriptomes, however, had an average similarity of 31%. Although differences were few among the metagenomes, we observed a change from river to ocean in the abundance of genes encoding for catabolic pathways, osmoregulators, and metal transporters. Additionally, genes specifying both bacterial oxygenic and anoxygenic photosynthesis were abundant and expressed in the estuary and plume. Denitrification genes were found throughout the Columbia River coastal margin, and most highly expressed in the estuary. Across a river to ocean gradient, the free-living microbial community followed three different patterns of diversity: 1) the taxonomy of the community changed strongly with salinity, 2) metabolic potential was highly similar across samples, with few differences in functional gene abundance

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

A probabilistic model to recover individual genomes from metagenomes

NARCIS (Netherlands)

J. Dröge (Johannes); A. Schönhuth (Alexander); A.C. McHardy (Alice)

2017-01-01

textabstractShotgun metagenomics of microbial communities reveal information about strains of relevance for applications in medicine, biotechnology and ecology. Recovering their genomes is a crucial but very challenging step due to the complexity of the underlying biological system and technical

An algorithm for detecting eukaryotic sequences in metagenomic ...

Indian Academy of Sciences (India)

species but also from accidental contamination from the genome of eukaryotic host cells. The latter scenario generally occurs in the case of host-associated metagenomes, e.g. microbes living in human gut. In such cases, one needs to identify and remove contaminating host DNA sequences, since the latter sequences will ...

A Utilitarian Case for Intellectual Freedom in Libraries.

Science.gov (United States)

Doyle, Tony

2001-01-01

Outlines the history of censorship and intellectual and expressive freedom in American libraries; discusses the two main types of ethical theory, utilitarianism and deontology; and maintains that libraries have a special role to play in promoting unconditional intellectual freedom. (Author/LRW)

A combined meta-barcoding and shotgun metagenomic analysis of spontaneous wine fermentation.

Science.gov (United States)

Sternes, Peter R; Lee, Danna; Kutyna, Dariusz R; Borneman, Anthony R

2017-07-01

Wine is a complex beverage, comprising hundreds of metabolites produced through the action of yeasts and bacteria in fermenting grape must. Commercially, there is now a growing trend away from using wine yeast (Saccharomyces) starter cultures, toward the historic practice of uninoculated or "wild" fermentation, where the yeasts and bacteria associated with the grapes and/or winery perform the fermentation. It is the varied metabolic contributions of these numerous non-Saccharomyces species that are thought to impart complexity and desirable taste and aroma attributes to wild ferments in comparison to their inoculated counterparts. To map the microflora of spontaneous fermentation, metagenomic techniques were employed to characterize and monitor the progression of fungal species in 5 different wild fermentations. Both amplicon-based ribosomal DNA internal transcribed spacer (ITS) phylotyping and shotgun metagenomics were used to assess community structure across different stages of fermentation. While providing a sensitive and highly accurate means of characterizing the wine microbiome, the shotgun metagenomic data also uncovered a significant overabundance bias in the ITS phylotyping abundance estimations for the common non-Saccharomyces wine yeast genus Metschnikowia. By identifying biases such as that observed for Metschnikowia, abundance measurements from future ITS phylotyping datasets can be corrected to provide more accurate species representation. Ultimately, as more shotgun metagenomic and single-strain de novo assemblies for key wine species become available, the accuracy of both ITS-amplicon and shotgun studies will greatly increase, providing a powerful methodology for deciphering the influence of the microbial community on the wine flavor and aroma. © The Authors 2017. Published by Oxford University Press.

Patron Behavior Policies in the Public Library: "Kreimer v. Morristown" Revisited.

Science.gov (United States)

Geiszler, Robert W.

1998-01-01

The case of an indigent library patron recovering a judgment against a public library is used as a backdrop for discussing patron behavior policies in the public library. Highlights include First Amendment rights, the public library as an expressive forum, government rules, policy lessons from the case, and acceptable policies. (AEF)

Vinasse fertirrigation alters soil resistome dynamics: an analysis based on metagenomic profiles.

Science.gov (United States)

Braga, Lucas P P; Alves, Rafael F; Dellias, Marina T F; Navarrete, Acacio A; Basso, Thiago O; Tsai, Siu M

2017-01-01

Every year around 300 Gl of vinasse, a by-product of ethanol distillation in sugarcane mills, are flushed into more than 9 Mha of sugarcane cropland in Brazil. This practice links fermentation waste management to fertilization for plant biomass production, and it is known as fertirrigation. Here we evaluate public datasets of soil metagenomes mining for changes in antibiotic resistance genes (ARGs) of soils from sugarcane mesocosms repeatedly amended with vinasse. The metagenomes were annotated using the ResFam database. We found that the abundance of open read frames (ORFs) annotated as ARGs changed significantly across 43 different families ( p -value resistome.

A metagenomic approach to decipher the indigenous microbial communities of arsenic contaminated groundwater of Assam

Directory of Open Access Journals (Sweden)

Saurav Das

2017-06-01

Full Text Available Metagenomic approach was used to understand the structural and functional diversity present in arsenic contaminated groundwater of the Ganges Brahmaputra Delta aquifer system. A metagene dataset (coded as TTGW1 of 89,171 sequences (totaling 125,449,864 base pairs with an average length of 1406 bps was annotated. About 74,478 sequences containing 101,948 predicted protein coding regions passed the quality control. Taxonomical classification revealed abundance of bacteria that accounted for 98.3% of the microbial population of the metagenome. Eukaryota had an abundance of 1.1% followed by archea that showed 0.4% abundance. In phylum based classification, Proteobacteria was dominant (62.6% followed by Bacteroidetes (11.7%, Planctomycetes (7.7%, Verrucomicrobia (5.6%, Actinobacteria (3.7% and Firmicutes (1.9%. The Clusters of Orthologous Groups (COGs analysis indicated that the protein regulating the metabolic functions constituted a high percentage (18,199 reads; 39.3% of the whole metagenome followed by the proteins regulating the cellular processes (22.3%. About 0.07% sequences of the whole metagenome were related to genes coding for arsenic resistant mechanisms. Nearly 50% sequences of these coded for the arsenate reductase enzyme (EC. 1.20.4.1, the dominant enzyme of ars operon. Proteins associated with iron acquisition and metabolism were coded by 2% of the metagenome as revealed through SEED analysis. Our study reveals the microbial diversity and provides an insight into the functional aspect of the genes that might play crucial role in arsenic geocycle in contaminated ground water of Assam.

A Statistical Framework for the Functional Analysis of Metagenomes

Energy Technology Data Exchange (ETDEWEB)

Sharon, Itai; Pati, Amrita; Markowitz, Victor; Pinter, Ron Y.

2008-10-01

Metagenomic studies consider the genetic makeup of microbial communities as a whole, rather than their individual member organisms. The functional and metabolic potential of microbial communities can be analyzed by comparing the relative abundance of gene families in their collective genomic sequences (metagenome) under different conditions. Such comparisons require accurate estimation of gene family frequencies. They present a statistical framework for assessing these frequencies based on the Lander-Waterman theory developed originally for Whole Genome Shotgun (WGS) sequencing projects. They also provide a novel method for assessing the reliability of the estimations which can be used for removing seemingly unreliable measurements. They tested their method on a wide range of datasets, including simulated genomes and real WGS data from sequencing projects of whole genomes. Results suggest that their framework corrects inherent biases in accepted methods and provides a good approximation to the true statistics of gene families in WGS projects.

High definition for systems biology of microbial communities: metagenomics gets genome-centric and strain-resolved.

Science.gov (United States)

Turaev, Dmitrij; Rattei, Thomas

2016-06-01

The systems biology of microbial communities, organismal communities inhabiting all ecological niches on earth, has in recent years been strongly facilitated by the rapid development of experimental, sequencing and data analysis methods. Novel experimental approaches and binning methods in metagenomics render the semi-automatic reconstructions of near-complete genomes of uncultivable bacteria possible, while advances in high-resolution amplicon analysis allow for efficient and less biased taxonomic community characterization. This will also facilitate predictive modeling approaches, hitherto limited by the low resolution of metagenomic data. In this review, we pinpoint the most promising current developments in metagenomics. They facilitate microbial systems biology towards a systemic understanding of mechanisms in microbial communities with scopes of application in many areas of our daily life. Copyright © 2016 Elsevier Ltd. All rights reserved.

ORF-selector ESPRIT: a second generation library screen for soluble protein expression employing precise open reading frame selection.

Science.gov (United States)

An, Yingfeng; Yumerefendi, Hayretin; Mas, Philippe J; Chesneau, Alban; Hart, Darren J

2011-08-01

Here we present ORF-selector ESPRIT, a 9-fold enhanced version of our technology for screening incremental truncation libraries to identify soluble high yielding constructs of challenging proteins. Gene fragments are truncated at both termini to access internal domains and the resulting reading frame problem is addressed by an unbiased, intein-based open reading frame selection yielding only in-frame DNA inserts. This enriched library is then subcloned into a standard high-level expression plasmid where tens of thousands of constructs can be assayed in a two-step process using colony- and liquid-handling robots to isolate rare highly expressing clones useful for production of multi milligram quantities of purifiable proteins. The p85α protein was used to benchmark the system resulting in isolation of all known domains, either alone or in tandem. The human kinase IKK1 was then screened resulting in purification of a predicted internal domain. This strategy provides an integrated, facile route to produce soluble proteins from challenging and poorly understood target genes at quantities compatible with structural biology, screening applications and immunisation studies. The high genetic diversity that can be sampled opens the way to study more diverse systems including multisubunit complexes. Copyright © 2011 Elsevier Inc. All rights reserved.

Using herbarium-derived DNAs to assemble a large-scale DNA barcode library for the vascular plants of Canada.

Science.gov (United States)

Kuzmina, Maria L; Braukmann, Thomas W A; Fazekas, Aron J; Graham, Sean W; Dewaard, Stephanie L; Rodrigues, Anuar; Bennett, Bruce A; Dickinson, Timothy A; Saarela, Jeffery M; Catling, Paul M; Newmaster, Steven G; Percy, Diana M; Fenneman, Erin; Lauron-Moreau, Aurélien; Ford, Bruce; Gillespie, Lynn; Subramanyam, Ragupathy; Whitton, Jeannette; Jennings, Linda; Metsger, Deborah; Warne, Connor P; Brown, Allison; Sears, Elizabeth; Dewaard, Jeremy R; Zakharov, Evgeny V; Hebert, Paul D N

2017-12-01

Constructing complete, accurate plant DNA barcode reference libraries can be logistically challenging for large-scale floras. Here we demonstrate the promise and challenges of using herbarium collections for building a DNA barcode reference library for the vascular plant flora of Canada. Our study examined 20,816 specimens representing 5076 of 5190 vascular plant species in Canada (98%). For 98% of the specimens, at least one of the DNA barcode regions was recovered from the plastid loci rbcL and matK and from the nuclear ITS2 region. We used beta regression to quantify the effects of age, type of preservation, and taxonomic affiliation (family) on DNA sequence recovery. Specimen age and method of preservation had significant effects on sequence recovery for all markers, but influenced some families more (e.g., Boraginaceae) than others (e.g., Asteraceae). Our DNA barcode library represents an unparalleled resource for metagenomic and ecological genetic research working on temperate and arctic biomes. An observed decline in sequence recovery with specimen age may be associated with poor primer matches, intragenomic variation (for ITS2), or inhibitory secondary compounds in some taxa.

Variability in metagenomic samples from the Puget Sound: Relationship to temporal and anthropogenic impacts.

Directory of Open Access Journals (Sweden)

James C Wallace

Full Text Available Whole-metagenome sequencing (WMS has emerged as a powerful tool to assess potential public health risks in marine environments by measuring changes in microbial community structure and function in uncultured bacteria. In addition to monitoring public health risks such as antibiotic resistance determinants, it is essential to measure predictors of microbial variation in order to identify natural versus anthropogenic factors as well as to evaluate reproducibility of metagenomic measurements.This study expands our previous metagenomic characterization of Puget Sound by sampling new nearshore environments including the Duwamish River, an EPA superfund site, and the Hood Canal, an area characterized by highly variable oxygen levels. We also resampled a wastewater treatment plant, nearshore and open ocean sites introducing a longitudinal component measuring seasonal and locational variations and establishing metagenomics sampling reproducibility. Microbial composition from samples collected in the open sound were highly similar within the same season and location across different years, while nearshore samples revealed multi-fold seasonal variation in microbial composition and diversity. Comparisons with recently sequenced predominant marine bacterial genomes helped provide much greater species level taxonomic detail compared to our previous study. Antibiotic resistance determinants and pollution and detoxification indicators largely grouped by location showing minor seasonal differences. Metal resistance, oxidative stress and detoxification systems showed no increase in samples proximal to an EPA superfund site indicating a lack of ecosystem adaptation to anthropogenic impacts. Taxonomic analysis of common sewage influent families showed a surprising similarity between wastewater treatment plant and open sound samples suggesting a low-level but pervasive sewage influent signature in Puget Sound surface waters. Our study shows reproducibility of

Centrifuge: rapid and sensitive classification of metagenomic sequences.

Science.gov (United States)

Kim, Daehwan; Song, Li; Breitwieser, Florian P; Salzberg, Steven L

2016-12-01

Centrifuge is a novel microbial classification engine that enables rapid, accurate, and sensitive labeling of reads and quantification of species on desktop computers. The system uses an indexing scheme based on the Burrows-Wheeler transform (BWT) and the Ferragina-Manzini (FM) index, optimized specifically for the metagenomic classification problem. Centrifuge requires a relatively small index (4.2 GB for 4078 bacterial and 200 archaeal genomes) and classifies sequences at very high speed, allowing it to process the millions of reads from a typical high-throughput DNA sequencing run within a few minutes. Together, these advances enable timely and accurate analysis of large metagenomics data sets on conventional desktop computers. Because of its space-optimized indexing schemes, Centrifuge also makes it possible to index the entire NCBI nonredundant nucleotide sequence database (a total of 109 billion bases) with an index size of 69 GB, in contrast to k-mer-based indexing schemes, which require far more extensive space. © 2016 Kim et al.; Published by Cold Spring Harbor Laboratory Press.

Bacterial tag encoded FLX titanium amplicon pyrosequencing (bTEFAP based assessment of prokaryotic diversity in metagenome of Lonar soda lake, India

Directory of Open Access Journals (Sweden)

Pravin Dudhagara

2015-06-01

Full Text Available Bacterial diversity and archaeal diversity in metagenome of the Lonar soda lake sediment were assessed by bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP. Metagenome comprised 5093 sequences with 2,531,282 bp and 53 ± 2% G + C content. Metagenome sequence data are available at NCBI under the Bioproject database with accession no. PRJNA218849. Metagenome sequence represented the presence of 83.1% bacterial and 10.5% archaeal origin. A total of 14 different bacteria demonstrating 57 species were recorded with dominating species like Coxiella burnetii (17%, Fibrobacter intestinalis (12% and Candidatus Cloacamonas acidaminovorans (11%. Occurrence of two archaeal phyla representing 24 species, among them Methanosaeta harundinacea (35%, Methanoculleus chikugoensis (12% and Methanolinea tarda (11% were dominating species. Significant presence of 11% sequences as an unclassified indicated the possibilities for unknown novel prokaryotes from the metagenome.

Re-Analysis of Metagenomic Sequences from Acute Flaccidmyelitis Patients Reveals Alternatives to Enterovirus D68 Infection

Science.gov (United States)

2015-07-13

caused in some cases by infection with enterovirus D68. We found that among the patients whose symptoms were previously attributed to enterovirus D68...distribution is unlimited. Re-analysis of metagenomic sequences from acute flaccidmyelitis patients reveals alternatives to enterovirus D68...Street Baltimore, MD 21218 -2685 ABSTRACT Re-analysis of metagenomic sequences from acute flaccidmyelitis patients reveals alternatives to enterovirus

The Construction of cDNA Libraries from Human Single Preimplantation Embryos and Their Use in the Study of Gene Expression During Development

OpenAIRE

Adjaye, James; Daniels, Rob; Monk, Marilyn

1998-01-01

Purpose:The construction and application of polymerase chain reaction (PCR)-based cDNA libraries from unfertilized human oocytes and single preimplantation-stage embryos are described. The purpose of these studies is to provide a readily available resource for the study of gene expression during human preimplantation development.

GenomePeek—an online tool for prokaryotic genome and metagenome analysis

Directory of Open Access Journals (Sweden)

Katelyn McNair

2015-06-01

Full Text Available As more and more prokaryotic sequencing takes place, a method to quickly and accurately analyze this data is needed. Previous tools are mainly designed for metagenomic analysis and have limitations; such as long runtimes and significant false positive error rates. The online tool GenomePeek (edwards.sdsu.edu/GenomePeek was developed to analyze both single genome and metagenome sequencing files, quickly and with low error rates. GenomePeek uses a sequence assembly approach where reads to a set of conserved genes are extracted, assembled and then aligned against the highly specific reference database. GenomePeek was found to be faster than traditional approaches while still keeping error rates low, as well as offering unique data visualization options.

Statistical comparison of two or more SAGE libraries: one tag at a time

NARCIS (Netherlands)

Schaaf, Gerben J.; van Ruissen, Fred; van Kampen, Antoine; Kool, Marcel; Ruijter, Jan M.

2008-01-01

Several statistical tests have been introduced for the comparison of serial analysis of gene expression (SAGE) libraries to quantitatively analyze the differential expression of genes. As each SAGE library is only one measurement, the necessary information on biological variation or experimental

Using herbarium-derived DNAs to assemble a large-scale DNA barcode library for the vascular plants of Canada1

Science.gov (United States)

Kuzmina, Maria L.; Braukmann, Thomas W. A.; Fazekas, Aron J.; Graham, Sean W.; Dewaard, Stephanie L.; Rodrigues, Anuar; Bennett, Bruce A.; Dickinson, Timothy A.; Saarela, Jeffery M.; Catling, Paul M.; Newmaster, Steven G.; Percy, Diana M.; Fenneman, Erin; Lauron-Moreau, Aurélien; Ford, Bruce; Gillespie, Lynn; Subramanyam, Ragupathy; Whitton, Jeannette; Jennings, Linda; Metsger, Deborah; Warne, Connor P.; Brown, Allison; Sears, Elizabeth; Dewaard, Jeremy R.; Zakharov, Evgeny V.; Hebert, Paul D. N.

2017-01-01

Premise of the study: Constructing complete, accurate plant DNA barcode reference libraries can be logistically challenging for large-scale floras. Here we demonstrate the promise and challenges of using herbarium collections for building a DNA barcode reference library for the vascular plant flora of Canada. Methods: Our study examined 20,816 specimens representing 5076 of 5190 vascular plant species in Canada (98%). For 98% of the specimens, at least one of the DNA barcode regions was recovered from the plastid loci rbcL and matK and from the nuclear ITS2 region. We used beta regression to quantify the effects of age, type of preservation, and taxonomic affiliation (family) on DNA sequence recovery. Results: Specimen age and method of preservation had significant effects on sequence recovery for all markers, but influenced some families more (e.g., Boraginaceae) than others (e.g., Asteraceae). Discussion: Our DNA barcode library represents an unparalleled resource for metagenomic and ecological genetic research working on temperate and arctic biomes. An observed decline in sequence recovery with specimen age may be associated with poor primer matches, intragenomic variation (for ITS2), or inhibitory secondary compounds in some taxa. PMID:29299394

An Improved Methodology to Overcome Key Issues in Human Fecal Metagenomic DNA Extraction

Directory of Open Access Journals (Sweden)

Jitendra Kumar

2016-12-01

Full Text Available Microbes are ubiquitously distributed in nature, and recent culture-independent studies have highlighted the significance of gut microbiota in human health and disease. Fecal DNA is the primary source for the majority of human gut microbiome studies. However, further improvement is needed to obtain fecal metagenomic DNA with sufficient amount and good quality but low host genomic DNA contamination. In the current study, we demonstrate a quick, robust, unbiased, and cost-effective method for the isolation of high molecular weight (>23 kb metagenomic DNA (260/280 ratio >1.8 with a good yield (55.8 ± 3.8 ng/mg of feces. We also confirm that there is very low human genomic DNA contamination (eubacterial: human genomic DNA marker genes = 227.9:1 in the human feces. The newly-developed method robustly performs for fresh as well as stored fecal samples as demonstrated by 16S rRNA gene sequencing using 454 FLX+. Moreover, 16S rRNA gene analysis indicated that compared to other DNA extraction methods tested, the fecal metagenomic DNA isolated with current methodology retains species richness and does not show microbial diversity biases, which is further confirmed by qPCR with a known quantity of spike-in genomes. Overall, our data highlight a protocol with a balance between quality, amount, user-friendliness, and cost effectiveness for its suitability toward usage for culture-independent analysis of the human gut microbiome, which provides a robust solution to overcome key issues associated with fecal metagenomic DNA isolation in human gut microbiome studies.

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.

Unravelling core microbial metabolisms in the hypersaline microbial mats of Shark Bay using high-throughput metagenomics

Energy Technology Data Exchange (ETDEWEB)

Ruvindy, Rendy; White III, Richard Allen; Neilan, Brett Anthony; Burns, Brendan Paul

2015-05-29

Modern microbial mats are potential analogues of some of Earth’s earliest ecosystems. Excellent examples can be found in Shark Bay, Australia, with mats of various morphologies. To further our understanding of the functional genetic potential of these complex microbial ecosystems, we conducted for the first time shotgun metagenomic analyses. We assembled metagenomic nextgeneration sequencing data to classify the taxonomic and metabolic potential across diverse morphologies of marine mats in Shark Bay. The microbial community across taxonomic classifications using protein-coding and small subunit rRNA genes directly extracted from the metagenomes suggests that three phyla Proteobacteria, Cyanobacteria and Bacteriodetes dominate all marine mats. However, the microbial community structure between Shark Bay and Highbourne Cay (Bahamas) marine systems appears to be distinct from each other. The metabolic potential (based on SEED subsystem classifications) of the Shark Bay and Highbourne Cay microbial communities were also distinct. Shark Bay metagenomes have a metabolic pathway profile consisting of both heterotrophic and photosynthetic pathways, whereas Highbourne Cay appears to be dominated almost exclusively by photosynthetic pathways. Alternative non-rubisco-based carbon metabolism including reductive TCA cycle and 3-hydroxypropionate/4-hydroxybutyrate pathways is highly represented in Shark Bay metagenomes while not represented in Highbourne Cay microbial mats or any other mat forming ecosystems investigated to date. Potentially novel aspects of nitrogen cycling were also observed, as well as putative heavy metal cycling (arsenic, mercury, copper and cadmium). Finally, archaea are highly represented in Shark Bay and may have critical roles in overall ecosystem function in these modern microbial mats.

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.

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

Directory of Open Access Journals (Sweden)

Alexander L Greninger

2010-10-01

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.

Complementation of radiation-sensitive Ataxia telangiectasia cells after transfection of cDNA expression libraries and cosmid clones from wildtype cells

International Nuclear Information System (INIS)

Fritz, E.

1994-06-01

In this Ph.D.-thesis, phenotypic complementation of AT-cells (AT5BIVA) by transfection of cDNA-expression-libraries was adressed: After stable transfection of cDNA-expression-libraries G418 resistant clones were selected for enhanced radioresistance by a fractionated X-ray selection. One surviving transfectant clone (clone 514) exhibited enhanced radiation resistance in dose-response experiments and further X-ray selections. Cell cycle analysis revealed complementation of untreated and irradiated 514-cells in cell cycle progression. The rate of DNA synthesis, however, is not diminished after irradiation but shows the reverse effect. A transfected cDNA-fragment (AT500-cDNA) was isolated from the genomic DNA of 514-cells and proved to be an unknown DNA sequence. A homologous sequence could be detected in genomic DNA from human cell lines, but not in DNA from other species. The cDNA-sequence could be localized to human chromosome 11. In human cells the cDNA sequence is part of two large mRNAs. 4 different cosmid clones containing high molecular genomic DNA from normal human cells could be isolated from a library, each hybridizing to the AT500-cDNA. After stable transfection into AT-cells, one cosmid-clone was able to confer enhanced radiation resistance both in X-ray selections and dose-response experiments. The results indicate that the cloned cDNA-fragment is based on an unknown gene from human chromosome 11 which partially complements the radiosensitivity and the defective cell cycle progression in AT5BIVA cells. (orig.) [de

Metagenomic investigation of the microbial diversity in a chrysotile asbestos mine pit pond, Lowell, Vermont, USA

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Heather E. Driscoll

2016-12-01

Full Text Available Here we report on a metagenomics investigation of the microbial diversity in a serpentine-hosted aquatic habitat created by chrysotile asbestos mining activity at the Vermont Asbestos Group (VAG Mine in northern Vermont, USA. The now-abandoned VAG Mine on Belvidere Mountain in the towns of Eden and Lowell includes three open-pit quarries, a flooded pit, mill buildings, roads, and >26 million metric tons of eroding mine waste that contribute alkaline mine drainage to the surrounding watershed. Metagenomes and water chemistry originated from aquatic samples taken at three depths (0.5 m, 3.5 m, and 25 m along the water column at three distinct, offshore sites within the mine's flooded pit (near 44°46′00.7673″, −72°31′36.2699″; UTM NAD 83 Zone 18 T 0695720 E, 4960030 N. Whole metagenome shotgun Illumina paired-end sequences were quality trimmed and analyzed based on a translated nucleotide search of NCBI-NR protein database and lowest common ancestor taxonomic assignments. Our results show strata within the pit pond water column can be distinguished by taxonomic composition and distribution, pH, temperature, conductivity, light intensity, and concentrations of dissolved oxygen. At the phylum level, metagenomes from 0.5 m and 3.5 m contained a similar distribution of taxa and were dominated by Actinobacteria (46% and 53% of reads, respectively, Proteobacteria (45% and 38%, respectively, and Bacteroidetes (7% in both. The metagenomes from 25 m showed a greater diversity of phyla and a different distribution of reads than the two upper strata: Proteobacteria (60%, Actinobacteria (18%, Planctomycetes, (10%, Bacteroidetes (5% and Cyanobacteria (2.5%, Armatimonadetes (<1%, Verrucomicrobia (<1%, Firmicutes (<1%, and Nitrospirae (<1%. Raw metagenome sequence data from each sample reside in NCBI's Short Read Archive (SRA ID: SRP056095 and are accessible through NCBI BioProject PRJNA277916.

Binning sequences using very sparse labels within a metagenome

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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

Metagenome-derived haloalkane dehalogenases with novel catalytic properties

Czech Academy of Sciences Publication Activity Database

Kotík, Michael; Vaňáček, P.; Kuňka, A.; Prokop, Z.; Dambrovský, J.

2017-01-01

Roč. 101, č. 16 (2017), s. 6385-6397 ISSN 0175-7598 R&D Projects: GA ČR GAP504/10/0137; GA MŠk(CZ) LM2015047; GA MŠk(CZ) LM2015055 Institutional support: RVO:61388971 Keywords : Haloalkane dehalogenase * Metagenomic DNA * Heterologous production Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 3.420, year: 2016

The GAAS metagenomic tool and its estimations of viral and microbial average genome size in four major biomes.

Science.gov (United States)

Angly, Florent E; Willner, Dana; Prieto-Davó, Alejandra; Edwards, Robert A; Schmieder, Robert; Vega-Thurber, Rebecca; Antonopoulos, Dionysios A; Barott, Katie; Cottrell, Matthew T; Desnues, Christelle; Dinsdale, Elizabeth A; Furlan, Mike; Haynes, Matthew; Henn, Matthew R; Hu, Yongfei; Kirchman, David L; McDole, Tracey; McPherson, John D; Meyer, Folker; Miller, R Michael; Mundt, Egbert; Naviaux, Robert K; Rodriguez-Mueller, Beltran; Stevens, Rick; Wegley, Linda; Zhang, Lixin; Zhu, Baoli; Rohwer, Forest

2009-12-01

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 environmental conditions.

Fast and accurate taxonomic assignments of metagenomic sequences using MetaBin.

Directory of Open Access Journals (Sweden)

Vineet K Sharma

Full Text Available Taxonomic assignment of sequence reads is a challenging task in metagenomic data analysis, for which the present methods mainly use either composition- or homology-based approaches. Though the homology-based methods are more sensitive and accurate, they suffer primarily due to the time needed to generate the Blast alignments. We developed the MetaBin program and web server for better homology-based taxonomic assignments using an ORF-based approach. By implementing Blat as the faster alignment method in place of Blastx, the analysis time has been reduced by severalfold. It is benchmarked using both simulated and real metagenomic datasets, and can be used for both single and paired-end sequence reads of varying lengths (≥45 bp. To our knowledge, MetaBin is the only available program that can be used for the taxonomic binning of short reads (<100 bp with high accuracy and high sensitivity using a homology-based approach. The MetaBin web server can be used to carry out the taxonomic analysis, by either submitting reads or Blastx output. It provides several options including construction of taxonomic trees, creation of a composition chart, functional analysis using COGs, and comparative analysis of multiple metagenomic datasets. MetaBin web server and a standalone version for high-throughput analysis are available freely at http://metabin.riken.jp/.

Metagenome Sequence Analysis of Filamentous Microbial Communities Obtained from Geochemically Distinct Geothermal Channels Reveals Specialization of Three Aquificales Lineages

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Cristina eTakacs-vesbach

2013-05-01

Full Text Available The Aquificales are thermophilic microorganisms that inhabit hydrothermal systems worldwide and are considered one of the earliest lineages of the domain Bacteria. We analyzed metagenome sequence obtained from six thermal ‘filamentous streamer’ communities (~40 Mbp per site, which targeted three different groups of Aquificales found in Yellowstone National Park (YNP. Unassembled metagenome sequence and PCR-amplified 16S rRNA gene libraries revealed that acidic, sulfidic sites were dominated by Hydrogenobaculum (Aquificaceae populations, whereas the circumneutral pH (6.5 - 7.8 sites containing dissolved sulfide were dominated by Sulfurihydrogenibium spp. (Hydrogenothermaceae. Thermocrinis (Aquificaceae populations were found primarily in the circumneutral sites with undetectable sulfide, and to a lesser extent in one sulfidic system at pH 8. Phylogenetic analysis of assembled sequence containing 16S rRNA genes as well as conserved protein-encoding genes revealed that the composition and function of these communities varied across geochemical conditions. Each Aquificales lineage contained genes for CO2 fixation by the reverse TCA cycle, but only the Sulfurihydrogenibium populations perform citrate cleavage using ATP citrate lyase (Acl. The Aquificaceae populations use an alternative pathway catalyzed by two separate enzymes, citryl CoA synthetase (Ccs and citryl CoA lyase (Ccl. All three Aquificales lineages contained evidence of aerobic respiration, albeit due to completely different types of heme Cu oxidases (subunit I involved in oxygen reduction. The distribution of Aquificales populations and differences among functional genes involved in energy generation and electron transport is consistent with the hypothesis that geochemical parameters (e.g., pH, sulfide, H2, O2 have resulted in niche specialization among members of the Aquificales.

Shotgun pyrosequencing metagenomic analyses of dusts from swine confinement and grain facilities.

Science.gov (United States)

Boissy, Robert J; Romberger, Debra J; Roughead, William A; Weissenburger-Moser, Lisa; Poole, Jill A; LeVan, Tricia D

2014-01-01

Inhalation of agricultural dusts causes inflammatory reactions and symptoms such as headache, fever, and malaise, which can progress to chronic airway inflammation and associated diseases, e.g. asthma, chronic bronchitis, chronic obstructive pulmonary disease, and hypersensitivity pneumonitis. Although in many agricultural environments feed particles are the major constituent of these dusts, the inflammatory responses that they provoke are likely attributable to particle-associated bacteria, archaebacteria, fungi, and viruses. In this study, we performed shotgun pyrosequencing metagenomic analyses of DNA from dusts from swine confinement facilities or grain elevators, with comparisons to dusts from pet-free households. DNA sequence alignment showed that 19% or 62% of shotgun pyrosequencing metagenomic DNA sequence reads from swine facility or household dusts, respectively, were of swine or human origin, respectively. In contrast only 2% of such reads from grain elevator dust were of mammalian origin. These metagenomic shotgun reads of mammalian origin were excluded from our analyses of agricultural dust microbiota. The ten most prevalent bacterial taxa identified in swine facility compared to grain elevator or household dust were comprised of 75%, 16%, and 42% gram-positive organisms, respectively. Four of the top five swine facility dust genera were assignable (Clostridium, Lactobacillus, Ruminococcus, and Eubacterium, ranging from 4% to 19% relative abundance). The relative abundances of these four genera were lower in dust from grain elevators or pet-free households. These analyses also highlighted the predominance in swine facility dust of Firmicutes (70%) at the phylum level, Clostridia (44%) at the Class level, and Clostridiales at the Order level (41%). In summary, shotgun pyrosequencing metagenomic analyses of agricultural dusts show that they differ qualitatively and quantitatively at the level of microbial taxa present, and that the bioinformatic analyses

Shotgun pyrosequencing metagenomic analyses of dusts from swine confinement and grain facilities.

Directory of Open Access Journals (Sweden)

Robert J Boissy

Full Text Available Inhalation of agricultural dusts causes inflammatory reactions and symptoms such as headache, fever, and malaise, which can progress to chronic airway inflammation and associated diseases, e.g. asthma, chronic bronchitis, chronic obstructive pulmonary disease, and hypersensitivity pneumonitis. Although in many agricultural environments feed particles are the major constituent of these dusts, the inflammatory responses that they provoke are likely attributable to particle-associated bacteria, archaebacteria, fungi, and viruses. In this study, we performed shotgun pyrosequencing metagenomic analyses of DNA from dusts from swine confinement facilities or grain elevators, with comparisons to dusts from pet-free households. DNA sequence alignment showed that 19% or 62% of shotgun pyrosequencing metagenomic DNA sequence reads from swine facility or household dusts, respectively, were of swine or human origin, respectively. In contrast only 2% of such reads from grain elevator dust were of mammalian origin. These metagenomic shotgun reads of mammalian origin were excluded from our analyses of agricultural dust microbiota. The ten most prevalent bacterial taxa identified in swine facility compared to grain elevator or household dust were comprised of 75%, 16%, and 42% gram-positive organisms, respectively. Four of the top five swine facility dust genera were assignable (Clostridium, Lactobacillus, Ruminococcus, and Eubacterium, ranging from 4% to 19% relative abundance. The relative abundances of these four genera were lower in dust from grain elevators or pet-free households. These analyses also highlighted the predominance in swine facility dust of Firmicutes (70% at the phylum level, Clostridia (44% at the Class level, and Clostridiales at the Order level (41%. In summary, shotgun pyrosequencing metagenomic analyses of agricultural dusts show that they differ qualitatively and quantitatively at the level of microbial taxa present, and that the

Insights into resistome and stress responses genes in Bubalus bubalis rumen through metagenomic analysis.

Science.gov (United States)

Reddy, Bhaskar; Singh, Krishna M; Patel, Amrutlal K; Antony, Ancy; Panchasara, Harshad J; Joshi, Chaitanya G

2014-10-01

Buffalo rumen microbiota experience variety of diets and represents a huge reservoir of mobilome, resistome and stress responses. However, knowledge of metagenomic responses to such conditions is still rudimentary. We analyzed the metagenomes of buffalo rumen in the liquid and solid phase of the rumen biomaterial from river buffalo adapted to varying proportion of concentrate to green or dry roughages, using high-throughput sequencing to know the occurrence of antibiotics resistance genes, genetic exchange between bacterial population and environmental reservoirs. A total of 3914.94 MB data were generated from all three treatments group. The data were analysed with Metagenome rapid annotation system tools. At phyla level, Bacteroidetes were dominant in all the treatments followed by Firmicutes. Genes coding for functional responses to stress (oxidative stress and heat shock proteins) and resistome genes (resistance to antibiotics and toxic compounds, phages, transposable elements and pathogenicity islands) were prevalent in similar proportion in liquid and solid fraction of rumen metagenomes. The fluoroquinolone resistance, MDR efflux pumps and Methicillin resistance genes were broadly distributed across 11, 9, and 14 bacterial classes, respectively. Bacteria responsible for phages replication and prophages and phage packaging and rlt-like streptococcal phage genes were mostly assigned to phyla Bacteroides, Firmicutes and proteaobacteria. Also, more reads matching the sigma B genes were identified in the buffalo rumen. This study underscores the presence of diverse mechanisms of adaptation to different diet, antibiotics and other stresses in buffalo rumen, reflecting the proportional representation of major bacterial groups.

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...

A transposase strategy for creating libraries of circularly permuted proteins.

Science.gov (United States)

Mehta, Manan M; Liu, Shirley; Silberg, Jonathan J

2012-05-01

A simple approach for creating libraries of circularly permuted proteins is described that is called PERMutation Using Transposase Engineering (PERMUTE). In PERMUTE, the transposase MuA is used to randomly insert a minitransposon that can function as a protein expression vector into a plasmid that contains the open reading frame (ORF) being permuted. A library of vectors that express different permuted variants of the ORF-encoded protein is created by: (i) using bacteria to select for target vectors that acquire an integrated minitransposon; (ii) excising the ensemble of ORFs that contain an integrated minitransposon from the selected vectors; and (iii) circularizing the ensemble of ORFs containing integrated minitransposons using intramolecular ligation. Construction of a Thermotoga neapolitana adenylate kinase (AK) library using PERMUTE revealed that this approach produces vectors that express circularly permuted proteins with distinct sequence diversity from existing methods. In addition, selection of this library for variants that complement the growth of Escherichia coli with a temperature-sensitive AK identified functional proteins with novel architectures, suggesting that PERMUTE will be useful for the directed evolution of proteins with new functions.

Metagenomic analyses of bacteria on human hairs: a qualitative assessment for applications in forensic science.

Science.gov (United States)

Tridico, Silvana R; Murray, Dáithí C; Addison, Jayne; Kirkbride, Kenneth P; Bunce, Michael

2014-01-01

Mammalian hairs are one of the most ubiquitous types of trace evidence collected in the course of forensic investigations. However, hairs that are naturally shed or that lack roots are problematic substrates for DNA profiling; these hair types often contain insufficient nuclear DNA to yield short tandem repeat (STR) profiles. Whilst there have been a number of initial investigations evaluating the value of metagenomics analyses for forensic applications (e.g. examination of computer keyboards), there have been no metagenomic evaluations of human hairs-a substrate commonly encountered during forensic practice. This present study attempts to address this forensic capability gap, by conducting a qualitative assessment into the applicability of metagenomic analyses of human scalp and pubic hair. Forty-two DNA extracts obtained from human scalp and pubic hairs generated a total of 79,766 reads, yielding 39,814 reads post control and abundance filtering. The results revealed the presence of unique combinations of microbial taxa that can enable discrimination between individuals and signature taxa indigenous to female pubic hairs. Microbial data from a single co-habiting couple added an extra dimension to the study by suggesting that metagenomic analyses might be of evidentiary value in sexual assault cases when other associative evidence is not present. Of all the data generated in this study, the next-generation sequencing (NGS) data generated from pubic hair held the most potential for forensic applications. Metagenomic analyses of human hairs may provide independent data to augment other forensic results and possibly provide association between victims of sexual assault and offender when other associative evidence is absent. Based on results garnered in the present study, we believe that with further development, bacterial profiling of hair will become a valuable addition to the forensic toolkit.

Metagenome of a Versatile Chemolithoautotroph from Expanding Oceanic Dead Zones

Energy Technology Data Exchange (ETDEWEB)

Walsh, David A.; Zaikova, Elena; Howes, Charles L.; Song, Young; Wright, Jody; Tringe, Susannah G.; Tortell, Philippe D.; Hallam, Steven J.

2009-07-15

Oxygen minimum zones (OMZs), also known as oceanic"dead zones", are widespread oceanographic features currently expanding due to global warming and coastal eutrophication. Although inhospitable to metazoan life, OMZs support a thriving but cryptic microbiota whose combined metabolic activity is intimately connected to nutrient and trace gas cycling within the global ocean. Here we report time-resolved metagenomic analyses of a ubiquitous and abundant but uncultivated OMZ microbe (SUP05) closely related to chemoautotrophic gill symbionts of deep-sea clams and mussels. The SUP05 metagenome harbors a versatile repertoire of genes mediating autotrophic carbon assimilation, sulfur-oxidation and nitrate respiration responsive to a wide range of water column redox states. Thus, SUP05 plays integral roles in shaping nutrient and energy flow within oxygen-deficient oceanic waters via carbon sequestration, sulfide detoxification and biological nitrogen loss with important implications for marine productivity and atmospheric greenhouse control.

A metagenomic framework for the study of airborne microbial communities.

Science.gov (United States)

Yooseph, Shibu; Andrews-Pfannkoch, Cynthia; Tenney, Aaron; McQuaid, Jeff; Williamson, Shannon; Thiagarajan, Mathangi; Brami, Daniel; Zeigler-Allen, Lisa; Hoffman, Jeff; Goll, Johannes B; Fadrosh, Douglas; Glass, John; Adams, Mark D; Friedman, Robert; Venter, J Craig

2013-01-01

Understanding the microbial content of the air has important scientific, health, and economic implications. While studies have primarily characterized the taxonomic content of air samples by sequencing the 16S or 18S ribosomal RNA gene, direct analysis of the genomic content of airborne microorganisms has not been possible due to the extremely low density of biological material in airborne environments. We developed sampling and amplification methods to enable adequate DNA recovery to allow metagenomic profiling of air samples collected from indoor and outdoor environments. Air samples were collected from a large urban building, a medical center, a house, and a pier. Analyses of metagenomic data generated from these samples reveal airborne communities with a high degree of diversity and different genera abundance profiles. The identities of many of the taxonomic groups and protein families also allows for the identification of the likely sources of the sampled airborne bacteria.

Structural and Functional Insights from the Metagenome of an Acidic Hot Spring Microbial Planktonic Community in the Colombian Andes

NARCIS (Netherlands)

Jiménez Avella, Diego; Dini Andreote, Fernando; Chaves, Diego; Montaña, José Salvador; Osorio-Forero, Cesar; Junca, Howard; Zambrano, María Mercedes; Baena, Sandra

2012-01-01

A taxonomic and annotated functional description of microbial life was deduced from 53 Mb of metagenomic sequence retrieved from a planktonic fraction of the Neotropical high Andean (3,973 meters above sea level) acidic hot spring El Coquito (EC). A classification of unassembled metagenomic reads

Analysis of expressed sequence tags generated from full-length enriched cDNA libraries of melon

Directory of Open Access Journals (Sweden)

Bendahmane Abdelhafid

2011-05-01

Full Text Available Abstract Background Melon (Cucumis melo, an economically important vegetable crop, belongs to the Cucurbitaceae family which includes several other important crops such as watermelon, cucumber, and pumpkin. It has served as a model system for sex determination and vascular biology studies. However, genomic resources currently available for melon are limited. Result We constructed eleven full-length enriched and four standard cDNA libraries from fruits, flowers, leaves, roots, cotyledons, and calluses of four different melon genotypes, and generated 71,577 and 22,179 ESTs from full-length enriched and standard cDNA libraries, respectively. These ESTs, together with ~35,000 ESTs available in public domains, were assembled into 24,444 unigenes, which were extensively annotated by comparing their sequences to different protein and functional domain databases, assigning them Gene Ontology (GO terms, and mapping them onto metabolic pathways. Comparative analysis of melon unigenes and other plant genomes revealed that 75% to 85% of melon unigenes had homologs in other dicot plants, while approximately 70% had homologs in monocot plants. The analysis also identified 6,972 gene families that were conserved across dicot and monocot plants, and 181, 1,192, and 220 gene families specific to fleshy fruit-bearing plants, the Cucurbitaceae family, and melon, respectively. Digital expression analysis identified a total of 175 tissue-specific genes, which provides a valuable gene sequence resource for future genomics and functional studies. Furthermore, we identified 4,068 simple sequence repeats (SSRs and 3,073 single nucleotide polymorphisms (SNPs in the melon EST collection. Finally, we obtained a total of 1,382 melon full-length transcripts through the analysis of full-length enriched cDNA clones that were sequenced from both ends. Analysis of these full-length transcripts indicated that sizes of melon 5' and 3' UTRs were similar to those of tomato, but

High throughtput comparisons and profiling of metagenomes for industrially relevant enzymes

KAUST Repository

Alam, Intikhab

2016-01-01

.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

IDENTIFICATION OF AVIAN-SPECIFIC FECAL METAGENOMIC SEQUENCES USING GENOME FRAGMENT ENRICHMENTS

Science.gov (United States)

Sequence analysis of microbial genomes has provided biologists the opportunity to compare genetic differences between closely related microorganisms. While random sequencing has also been used to study natural microbial communities, metagenomic comparisons via sequencing analysis...

The new science of metagenomics: revealing the secrets of our microbial planet

National Research Council Canada - National Science Library

Committee on Metagenomics: Challenges and Functional Applications, National Research Council

2007-01-01

.... The emerging field of metagenomics offers a new way of exploring the microbial world that will transform modern microbiology and lead to practical applications in medicine, agriculture, alternative...

Construction of a cDNA library for sea cucumber Acaudina leucoprocta and differential expression of ferritin peptide

Science.gov (United States)

Zhou, Jun; Hou, Fujing; Li, Ye; Su, Xiurong; Li, Taiwu; Jin, Chunhua

2016-07-01

Acaudina leucoprocta is an edible sea cucumber of economic interest that is widely distributed in China. Little information is available concerning the molecular genetics of this species although such knowledge would contribute to a better understanding of the optimal conditions for its aquaculture and its mechanisms of defense against disease. Therefore, we constructed a cDNA library and, based on bioinformatics analysis of the sequences, the functions of 75% of the cDNAs were identified, including those involved in cell structure, energy metabolism, mitochondrial function, and signal transduction pathways. Approximately 25% of genes in the library were unmatched. The gene for A. leucoprocta ferritin was also cloned. The predicted amino-acid sequence of ferritin displayed significant homology with other sea-cucumber counterparts but indicated that it was a new member of the ferritin family. Semiquantitative real-time RT-PCR indicated the highest levels of ferritin mRNA expression in the intestine. A polyclonal antibody of ferritin was also produced. These data provide a set of molecular tools essential for further studies of the functions of ferritin protein in A. leucoprocta.

The microbiome of Brazilian mangrove sediments as revealed by metagenomics

NARCIS (Netherlands)

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

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

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

Combining gene prediction methods to improve metagenomic gene annotation

Directory of Open Access Journals (Sweden)

Rosen Gail L

2011-01-01

Full Text Available Abstract Background Traditional gene annotation methods rely on characteristics that may not be available in short reads generated from next generation technology, resulting in suboptimal performance for metagenomic (environmental samples. Therefore, in recent years, new programs have been developed that optimize performance on short reads. In this work, we benchmark three metagenomic gene prediction programs and combine their predictions to improve metagenomic read gene annotation. Results We not only analyze the programs' performance at different read-lengths like similar studies, but also separate different types of reads, including intra- and intergenic regions, for analysis. The main deficiencies are in the algorithms' ability to predict non-coding regions and gene edges, resulting in more false-positives and false-negatives than desired. In fact, the specificities of the algorithms are notably worse than the sensitivities. By combining the programs' predictions, we show significant improvement in specificity at minimal cost to sensitivity, resulting in 4% improvement in accuracy for 100 bp reads with ~1% improvement in accuracy for 200 bp reads and above. To correctly annotate the start and stop of the genes, we find that a consensus of all the predictors performs best for shorter read lengths while a unanimous agreement is better for longer read lengths, boosting annotation accuracy by 1-8%. We also demonstrate use of the classifier combinations on a real dataset. Conclusions To optimize the performance for both prediction and annotation accuracies, we conclude that the consensus of all methods (or a majority vote is the best for reads 400 bp and shorter, while using the intersection of GeneMark and Orphelia predictions is the best for reads 500 bp and longer. We demonstrate that most methods predict over 80% coding (including partially coding reads on a real human gut sample sequenced by Illumina technology.

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

Information-seeking behavior of basic science researchers: implications for library services.

Science.gov (United States)

Haines, Laura L; Light, Jeanene; O'Malley, Donna; Delwiche, Frances A

2010-01-01

This study examined the information-seeking behaviors of basic science researchers to inform the development of customized library services. A qualitative study using semi-structured interviews was conducted on a sample of basic science researchers employed at a university medical school. The basic science researchers used a variety of information resources ranging from popular Internet search engines to highly technical databases. They generally relied on basic keyword searching, using the simplest interface of a database or search engine. They were highly collegial, interacting primarily with coworkers in their laboratories and colleagues employed at other institutions. They made little use of traditional library services and instead performed many traditional library functions internally. Although the basic science researchers expressed a positive attitude toward the library, they did not view its resources or services as integral to their work. To maximize their use by researchers, library resources must be accessible via departmental websites. Use of library services may be increased by cultivating relationships with key departmental administrative personnel. Despite their self-sufficiency, subjects expressed a desire for centralized information about ongoing research on campus and shared resources, suggesting a role for the library in creating and managing an institutional repository.

Recaf: Java dialects as libraries

NARCIS (Netherlands)

Biboudis, A. (Aggelos); P.A. Inostroza Valdera (Pablo); T. van der Storm (Tijs)

2016-01-01

textabstractMainstream programming languages like Java have limited support for language extensibility. Without mechanisms for syntactic abstraction, new programming styles can only be embedded in the form of libraries, limiting expressiveness. In this paper, we present Recaf, a lightweight tool for

Library associations and social responsibilities: the cases of ALA ...

African Journals Online (AJOL)

Library associations should not only promote traditional library activities such as the promotion of reading and literacy, but also serve as progressive forces for change. They can serve to protect and enhance civil liberties and freedom of expression, and can promote community organization for enhancing the quality of life.

A human gut microbial gene catalogue established by metagenomic sequencing

DEFF Research Database (Denmark)

dos Santos, Marcelo Bertalan Quintanilha; Sicheritz-Pontén, Thomas; Nielsen, Henrik Bjørn

2010-01-01

To understand the impact of gut microbes on human health and well-being it is crucial to assess their genetic potential. Here we describe the Illumina-based metagenomic sequencing, assembly and characterization of 3.3 million non-redundant microbial genes, derived from 576.7 gigabases of sequence...

High frequency of phylogenetically diverse reductive dehalogenase-homologous genes in deep subseafloor sedimentary metagenomes

Directory of Open Access Journals (Sweden)

Mikihiko eKawai

2014-03-01

Full Text Available Marine subsurface sediments on the Pacific margin harbor diverse microbial communities even at depths of several hundreds meters below the seafloor (mbsf or more. Previous PCR-based molecular analysis showed the presence of diverse reductive dehalogenase gene (rdhA homologs in marine subsurface sediment, suggesting that anaerobic respiration of organohalides is one of the possible energy-yielding pathways in the organic-rich sedimentary habitat. However, primer-independent molecular characterization of rdhA has remained to be demonstrated. Here, we studied the diversity and frequency of rdhA homologs by metagenomic analysis of five different depth horizons (0.8, 5.1, 18.6, 48.5 and 107.0 mbsf at Site C9001 off the Shimokita Peninsula of Japan. From all metagenomic pools, remarkably diverse rdhA-homologous sequences, some of which are affiliated with novel clusters, were observed with high frequency. As a comparison, we also examined frequency of dissimilatory sulfite reductase genes (dsrAB, key functional genes for microbial sulfate reduction. The dsrAB were also widely observed in the metagenomic pools whereas the frequency of dsrAB genes was generally smaller than that of rdhA-homologous genes. The phylogenetic composition of rdhA-homologous genes was similar among the five depth horizons. Our metagenomic data revealed that subseafloor rdhA homologs are more diverse than previously identified from PCR-based molecular studies. Spatial distribution of similar rdhA homologs across wide depositional ages indicates that the heterotrophic metabolic processes mediated by the genes can be ecologically important, functioning in the organic-rich subseafloor sedimentary biosphere.

Exploring nucleo-cytoplasmic large DNA viruses in Tara Oceans microbial metagenomes.

Science.gov (United States)

Hingamp, Pascal; Grimsley, Nigel; Acinas, Silvia G; Clerissi, Camille; Subirana, Lucie; Poulain, Julie; Ferrera, Isabel; Sarmento, Hugo; Villar, Emilie; Lima-Mendez, Gipsi; Faust, Karoline; Sunagawa, Shinichi; Claverie, Jean-Michel; Moreau, Hervé; Desdevises, Yves; Bork, Peer; Raes, Jeroen; de Vargas, Colomban; Karsenti, Eric; Kandels-Lewis, Stefanie; Jaillon, Olivier; Not, Fabrice; Pesant, Stéphane; Wincker, Patrick; Ogata, Hiroyuki

2013-09-01

Nucleo-cytoplasmic large DNA viruses (NCLDVs) constitute a group of eukaryotic viruses that can have crucial ecological roles in the sea by accelerating the turnover of their unicellular hosts or by causing diseases in animals. To better characterize the diversity, abundance and biogeography of marine NCLDVs, we analyzed 17 metagenomes derived from microbial samples (0.2-1.6 μm size range) collected during the Tara Oceans Expedition. The sample set includes ecosystems under-represented in previous studies, such as the Arabian Sea oxygen minimum zone (OMZ) and Indian Ocean lagoons. By combining computationally derived relative abundance and direct prokaryote cell counts, the abundance of NCLDVs was found to be in the order of 10(4)-10(5) genomes ml(-1) for the samples from the photic zone and 10(2)-10(3) genomes ml(-1) for the OMZ. The Megaviridae and Phycodnaviridae dominated the NCLDV populations in the metagenomes, although most of the reads classified in these families showed large divergence from known viral genomes. Our taxon co-occurrence analysis revealed a potential association between viruses of the Megaviridae family and eukaryotes related to oomycetes. In support of this predicted association, we identified six cases of lateral gene transfer between Megaviridae and oomycetes. Our results suggest that marine NCLDVs probably outnumber eukaryotic organisms in the photic layer (per given water mass) and that metagenomic sequence analyses promise to shed new light on the biodiversity of marine viruses and their interactions with potential hosts.

Novel resistance functions uncovered using functional metagenomic investigations of resistance reservoirs

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Erica C. Pehrsson

2013-06-01

Full Text Available Rates of infection with antibiotic-resistant bacteria have increased precipitously over the past several decades, with far-reaching healthcare and societal costs. Recent evidence has established a link between antibiotic resistance genes in human pathogens and those found in non-pathogenic, commensal, and environmental organisms, prompting deeper investigation of natural and human-associated reservoirs of antibiotic resistance. Functional metagenomic selections, in which shotgun-cloned DNA fragments are selected for their ability to confer survival to an indicator host, have been increasingly applied to the characterization of many antibiotic resistance reservoirs. These experiments have demonstrated that antibiotic resistance genes are highly diverse and widely distributed, many times bearing little to no similarity to known sequences. Through unbiased selections for survival to antibiotic exposure, functional metagenomics can improve annotations by reducing the discovery of false-positive resistance and by allowing for the identification of previously unrecognizable resistance genes. In this review, we summarize the novel resistance functions uncovered using functional metagenomic investigations of natural and human-impacted resistance reservoirs. Examples of novel antibiotic resistance genes include those highly divergent from known sequences, those for which sequence is entirely unable to predict resistance function, bifunctional resistance genes, and those with unconventional, atypical resistance mechanisms. Overcoming antibiotic resistance in the clinic will require a better understanding of existing resistance reservoirs and the dissemination networks that govern horizontal gene exchange, informing best practices to limit the spread of resistance-conferring genes to human pathogens.

The Projects of Two Different Universtiy Libraries in England

Directory of Open Access Journals (Sweden)

Çev.: Muhittin Gürbüz

2017-03-01

Full Text Available To respond to the changing needs of the 21st century, libraries are changing and must continue to change. In a competitive area, libraries either have to be brave and innovative or have to demonstrate their relevance, value and impact to make their community difference. In this study, information was given about the projects of two different universtiy libraries in England. Firstly, Study Happy project of Warwick University Library was mentioned. With the project, besides the information service which is traditional task of library, by organizing various activities aimed at providing a comfortable learning area for its users. Secondly, Reads and Rights project of Bath Spa University library was expressed. The main aim of the project is to stimulate thinking and conversations about equality and diversity and at the same time about books, reading and libraries.

Improved glycerol to ethanol conversion by E. coli using a metagenomic fragment isolated from an anaerobic reactor.

Science.gov (United States)

Loaces, Inés; Rodríguez, Cecilia; Amarelle, Vanesa; Fabiano, Elena; Noya, Francisco

2016-10-01

Crude glycerol obtained as a by-product of biodiesel production is a reliable feedstock with the potential to be converted into reduced chemicals with high yields. It has been previously shown that ethanol is the primary product of glycerol fermentation by Escherichia coli. However, few efforts were made to enhance this conversion by means of the expression of heterologous genes with the potential to improve glycerol transport or metabolism. In this study, a fosmid-based metagenomic library constructed from an anaerobic reactor purge sludge was screened for genetic elements that promote the use and fermentation of crude glycerol by E. coli. One clone was selected based on its improved growth rate on this feedstock. The corresponding fosmid, named G1, was fully sequenced (41 kbp long) and the gene responsible for the observed phenotype was pinpointed by in vitro insertion mutagenesis. Ethanol production from both pure and crude glycerol was evaluated using the parental G1 clone harboring the ethanologenic plasmid pLOI297 or the industrial strain LY180 complemented with G1. In mineral salts media containing 50 % (v/v) pure glycerol, ethanol concentrations increased two-fold on average when G1 was present in the cells reaching up to 20 g/L after 24 h fermentation. Similar fermentation experiments were done using crude instead of pure glycerol. With an initial OD620 of 8.0, final ethanol concentrations after 24 h were much higher reaching 67 and 75 g/L with LY180 cells carrying the control fosmid or the G1 fosmid, respectively. This translates into a specific ethanol production rate of 0.39 g h(-1) OD(-1) L(-1).

MetaGenSense: A web-application for analysis and exploration of high throughput sequencing metagenomic data [version 3; referees: 1 approved, 2 approved with reservations

Directory of Open Access Journals (Sweden)

Damien Correia

2016-12-01

Full Text Available The detection and characterization of emerging infectious agents has been a continuing public health concern. High Throughput Sequencing (HTS or Next-Generation Sequencing (NGS technologies have proven to be promising approaches for efficient and unbiased detection of pathogens in complex biological samples, providing access to comprehensive analyses. As NGS approaches typically yield millions of putatively representative reads per sample, efficient data management and visualization resources have become mandatory. Most usually, those resources are implemented through a dedicated Laboratory Information Management System (LIMS, solely to provide perspective regarding the available information. We developed an easily deployable web-interface, facilitating management and bioinformatics analysis of metagenomics data-samples. It was engineered to run associated and dedicated Galaxy workflows for the detection and eventually classification of pathogens. The web application allows easy interaction with existing Galaxy metagenomic workflows, facilitates the organization, exploration and aggregation of the most relevant sample-specific sequences among millions of genomic sequences, allowing them to determine their relative abundance, and associate them to the most closely related organism or pathogen. The user-friendly Django-Based interface, associates the users’ input data and its metadata through a bio-IT provided set of resources (a Galaxy instance, and both sufficient storage and grid computing power. Galaxy is used to handle and analyze the user’s input data from loading, indexing, mapping, assembly and DB-searches. Interaction between our application and Galaxy is ensured by the BioBlend library, which gives API-based access to Galaxy’s main features. Metadata about samples, runs, as well as the workflow results are stored in the LIMS. For metagenomic classification and exploration purposes, we show, as a proof of concept, that integration

Metagenomic profiling reveals lignocellulose degrading system in a microbial community associated with a wood-feeding beetle.

Directory of Open Access Journals (Sweden)

Erin D Scully

Full Text Available The Asian longhorned beetle (Anoplophoraglabripennis is an invasive, wood-boring pest that thrives in the heartwood of deciduous tree species. A large impediment faced by A. glabripennis as it feeds on woody tissue is lignin, a highly recalcitrant biopolymer that reduces access to sugars and other nutrients locked in cellulose and hemicellulose. We previously demonstrated that lignin, cellulose, and hemicellulose are actively deconstructed in the beetle gut and that the gut harbors an assemblage of microbes hypothesized to make significant contributions to these processes. While lignin degrading mechanisms have been well characterized in pure cultures of white rot basidiomycetes, little is known about such processes in microbial communities associated with wood-feeding insects. The goals of this study were to develop a taxonomic and functional profile of a gut community derived from an invasive population of larval A. glabripennis collected from infested host trees and to identify genes that could be relevant for the digestion of woody tissue and nutrient acquisition. To accomplish this goal, we taxonomically and functionally characterized the A. glabripennis midgut microbiota through amplicon and shotgun metagenome sequencing and conducted a large-scale comparison with the metagenomes from a variety of other herbivore-associated communities. This analysis distinguished the A. glabripennis larval gut metagenome from the gut communities of other herbivores, including previously sequenced termite hindgut metagenomes. Genes encoding enzymes were identified in the A. glabripennis gut metagenome that could have key roles in woody tissue digestion including candidate lignin degrading genes (laccases, dye-decolorizing peroxidases, novel peroxidases and β-etherases, 36 families of glycoside hydrolases (such as cellulases and xylanases, and genes that could facilitate nutrient recovery, essential nutrient synthesis, and detoxification. This community

ELIXIR pilot action: Marine metagenomics – towards a domain specific set of sustainable services [version 1; referees: 1 approved, 2 approved with reservations

Directory of Open Access Journals (Sweden)

Espen Mikal Robertsen

2017-01-01

Full Text Available Metagenomics, the study of genetic material recovered directly from environmental samples, has the potential to provide insight into the structure and function of heterogeneous microbial communities.  There has been an increased use of metagenomics to discover and understand the diverse biosynthetic capacities of marine microbes, thereby allowing them to be exploited for industrial, food, and health care products. This ELIXIR pilot action was motivated by the need to establish dedicated data resources and harmonized metagenomics pipelines for the marine domain, in order to enhance the exploration and exploitation of marine genetic resources. In this paper, we summarize some of the results from the ELIXIR pilot action “Marine metagenomics – towards user centric services”.

Diversity Indices as Measures of Functional Annotation Methods in Metagenomics Studies

KAUST Repository

Jankovic, Boris R.

2016-01-01

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

Metagenomics of Bacterial Diversity in Villa Luz Caves with Sulfur Water Springs

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Giuseppe D’Auria

2018-01-01

Full Text Available New biotechnology applications require in-depth preliminary studies of biodiversity. The methods of massive sequencing using metagenomics and bioinformatics tools offer us sufficient and reliable knowledge to understand environmental diversity, to know new microorganisms, and to take advantage of their functional genes. Villa Luz caves, in the southern Mexican state of Tabasco, are fed by at least 26 groundwater inlets, containing 300–500 mg L-1 H2S and <0.1 mg L-1 O2. We extracted environmental DNA for metagenomic analysis of collected samples in five selected Villa Luz caves sites, with pH values from 2.5 to 7. Foreign organisms found in this underground ecosystem can oxidize H2S to H2SO4. These include: biovermiculites, a bacterial association that can grow on the rock walls; snottites, that are whitish, viscous biofilms hanging from the rock walls, and sacks or bags of phlegm, which live within the aquatic environment of the springs. Through the emergency food assistance program (TEFAP pyrosequencing, a total of 20,901 readings of amplification products from hypervariable regions V1 and V3 of 16S rRNA bacterial gene in whole and pure metagenomic DNA samples were generated. Seven bacterial phyla were identified. As a result, Proteobacteria was more frequent than Acidobacteria. Finally, acidophilic Proteobacteria was detected in UJAT5 sample

Bounds on the distribution of the number of gaps when circles and lines are covered by fragments: Theory and practical application to genomic and metagenomic projects

Directory of Open Access Journals (Sweden)

Marchesi Julian R

2007-03-01

Full Text Available Abstract Background The question of how a circle or line segment becomes covered when random arcs are marked off has arisen repeatedly in bioinformatics. The number of uncovered gaps is of particular interest. Approximate distributions for the number of gaps have been given in the literature, one motivation being ease of computation. Error bounds for these approximate distributions have not been given. Results We give bounds on the probability distribution of the number of gaps when a circle is covered by fragments of fixed size. The absolute error in the approximation is typically on the order of 0.1% at 10× coverage depth. The method can be applied to coverage problems on the interval, including edge effects, and applications are given to metagenomic libraries and shotgun sequencing.

ESPRIT: an automated, library-based method for mapping and soluble expression of protein domains from challenging targets.

Science.gov (United States)

Yumerefendi, Hayretin; Tarendeau, Franck; Mas, Philippe J; Hart, Darren J

2010-10-01

Expression of sufficient quantities of soluble protein for structural biology and other applications is often a very difficult task, especially when multimilligram quantities are required. In order to improve yield, solubility or crystallisability of a protein, it is common to subclone shorter genetic constructs corresponding to single- or multi-domain fragments. However, it is not always clear where domain boundaries are located, especially when working on novel targets with little or no sequence similarity to other proteins. Several methods have been described employing aspects of directed evolution to the recombinant expression of challenging proteins. These combine the construction of a random library of genetic constructs of a target with a screening or selection process to identify solubly expressing protein fragments. Here we review several datasets from the ESPRIT (Expression of Soluble Proteins by Random Incremental Truncation) technology to provide a view on its capabilities. Firstly, we demonstrate how it functions using the well-characterised NF-kappaB p50 transcription factor as a model system. Secondly, application of ESPRIT to the challenging PB2 subunit of influenza polymerase has led to several novel atomic resolution structures; here we present an overview of the screening phase of that project. Thirdly, analysis of the human kinase TBK1 is presented to show how the ESPRIT technology rapidly addresses the compatibility of challenging targets with the Escherichia coli expression system.

Metagenomic Evidence for H2 Oxidation and H2 Production by Serpentinite-Hosted Subsurface Microbial Communities

Science.gov (United States)

Brazelton, William J.; Nelson, Bridget; Schrenk, Matthew O.

2012-01-01

Ultramafic rocks in the Earth’s mantle represent a tremendous reservoir of carbon and reducing power. Upon tectonic uplift and exposure to fluid flow, serpentinization of these materials generates copious energy, sustains abiogenic synthesis of organic molecules, and releases hydrogen gas (H2). In order to assess the potential for microbial H2 utilization fueled by serpentinization, we conducted metagenomic surveys of a marine serpentinite-hosted hydrothermal chimney (at the Lost City hydrothermal field) and two continental serpentinite-hosted alkaline seeps (at the Tablelands Ophiolite, Newfoundland). Novel [NiFe]-hydrogenase sequences were identified at both the marine and continental sites, and in both cases, phylogenetic analyses indicated aerobic, potentially autotrophic Betaproteobacteria belonging to order Burkholderiales as the most likely H2-oxidizers. Both sites also yielded metagenomic evidence for microbial H2 production catalyzed by [FeFe]-hydrogenases in anaerobic Gram-positive bacteria belonging to order Clostridiales. In addition, we present metagenomic evidence at both sites for aerobic carbon monoxide utilization and anaerobic carbon fixation via the Wood–Ljungdahl pathway. In general, our results point to H2-oxidizing Betaproteobacteria thriving in shallow, oxic–anoxic transition zones and the anaerobic Clostridia thriving in anoxic, deep subsurface habitats. These data demonstrate the feasibility of metagenomic investigations into novel subsurface habitats via surface-exposed seeps and indicate the potential for H2-powered primary production in serpentinite-hosted subsurface habitats. PMID:22232619

Metagenomic evidence for h(2) oxidation and h(2) production by serpentinite-hosted subsurface microbial communities.

Science.gov (United States)

Brazelton, William J; Nelson, Bridget; Schrenk, Matthew O

2012-01-01

Ultramafic rocks in the Earth's mantle represent a tremendous reservoir of carbon and reducing power. Upon tectonic uplift and exposure to fluid flow, serpentinization of these materials generates copious energy, sustains abiogenic synthesis of organic molecules, and releases hydrogen gas (H(2)). In order to assess the potential for microbial H(2) utilization fueled by serpentinization, we conducted metagenomic surveys of a marine serpentinite-hosted hydrothermal chimney (at the Lost City hydrothermal field) and two continental serpentinite-hosted alkaline seeps (at the Tablelands Ophiolite, Newfoundland). Novel [NiFe]-hydrogenase sequences were identified at both the marine and continental sites, and in both cases, phylogenetic analyses indicated aerobic, potentially autotrophic Betaproteobacteria belonging to order Burkholderiales as the most likely H(2)-oxidizers. Both sites also yielded metagenomic evidence for microbial H(2) production catalyzed by [FeFe]-hydrogenases in anaerobic Gram-positive bacteria belonging to order Clostridiales. In addition, we present metagenomic evidence at both sites for aerobic carbon monoxide utilization and anaerobic carbon fixation via the Wood-Ljungdahl pathway. In general, our results point to H(2)-oxidizing Betaproteobacteria thriving in shallow, oxic-anoxic transition zones and the anaerobic Clostridia thriving in anoxic, deep subsurface habitats. These data demonstrate the feasibility of metagenomic investigations into novel subsurface habitats via surface-exposed seeps and indicate the potential for H(2)-powered primary production in serpentinite-hosted subsurface habitats.

Metagenomic evidence for H2 oxidation and H2 production by serpentinite-hosted subsurface microbial communities

Directory of Open Access Journals (Sweden)

William J Brazelton

2012-01-01

Full Text Available Ultramafic rocks in the Earth’s mantle represent a tremendous reservoir of carbon and reducing power. Upon tectonic uplift and exposure to fluid flow, serpentinization of these materials generates copious energy, sustains abiogenic synthesis of organic molecules, and releases hydrogen gas (H2. In order to assess the potential for microbial H2 utilization fueled by serpentinization, we conducted metagenomic surveys of a marine serpentinite-hosted hydrothermal chimney (at the Lost City hydrothermal field and two continental serpentinite- hosted alkaline seeps (at the Tablelands Ophiolite, Newfoundland. Novel [NiFe]-hydrogenase sequences were identified at both the marine and continental sites, and in both cases, phylogenetic analyses indicated aerobic, potentially autotrophic Betaproteobacteria belonging to order Burkholderiales as the most likely H2-oxidizers. Both sites also yielded metagenomic evidence for microbial H2 production catalyzed by [FeFe]-hydrogenases in anaerobic Gram- positive bacteria belonging to order Clostridiales. In addition, we present metagenomic evidence at both sites for aerobic carbon monoxide utilization and anaerobic carbon fixation via the Wood-Ljungdahl pathway. In general, our results point to H2-oxidizing Betaproteobacteria thriving in shallow, oxic-anoxic transition zones and the anaerobic Clostridia thriving in anoxic, deep subsurface habitats. These data demonstrate the feasibility of metagenomic investigations into novel subsurface habitats via surface-exposed seeps and indicate the potential for H2- powered primary production in serpentinite-hosted subsurface habitats.

Glycoside Hydrolases from a targeted Compost Metagenome, activity-screening and functional characterization

Directory of Open Access Journals (Sweden)

Dougherty Michael J

2012-07-01

Full Text Available Abstract Background Metagenomics approaches provide access to environmental genetic diversity for biotechnology applications, enabling the discovery of new enzymes and pathways for numerous catalytic processes. Discovery of new glycoside hydrolases with improved biocatalytic properties for the efficient conversion of lignocellulosic material to biofuels is a critical challenge in the development of economically viable routes from biomass to fuels and chemicals. Results Twenty-two putative ORFs (open reading frames were identified from a switchgrass-adapted compost community based on sequence homology to related gene families. These ORFs were expressed in E. coli and assayed for predicted activities. Seven of the ORFs were demonstrated to encode active enzymes, encompassing five classes of hemicellulases. Four enzymes were over expressed in vivo, purified to homogeneity and subjected to detailed biochemical characterization. Their pH optima ranged between 5.5 - 7.5 and they exhibit moderate thermostability up to ~60-70°C. Conclusions Seven active enzymes were identified from this set of ORFs comprising five different hemicellulose activities. These enzymes have been shown to have useful properties, such as moderate thermal stability and broad pH optima, and may serve as the starting points for future protein engineering towards the goal of developing efficient enzyme cocktails for biomass degradation under diverse process conditions.

About the Library - Betty Petersen Memorial Library

Science.gov (United States)

branch library of the NOAA Central Library. The library serves the NOAA Science Center in Camp Springs , Maryland. History and Mission: Betty Petersen Memorial Library began as a reading room in the NOAA Science Science Center staff and advises the library on all aspects of the library program. Library Newsletters

Display of a Maize cDNA library on baculovirus infected insect cells

Directory of Open Access Journals (Sweden)

Jones Ian M

2008-08-01

Full Text Available Abstract Background Maize is a good model system for cereal crop genetics and development because of its rich genetic heritage and well-characterized morphology. The sequencing of its genome is well advanced, and new technologies for efficient proteomic analysis are needed. Baculovirus expression systems have been used for the last twenty years to express in insect cells a wide variety of eukaryotic proteins that require complex folding or extensive posttranslational modification. More recently, baculovirus display technologies based on the expression of foreign sequences on the surface of Autographa californica (AcMNPV have been developed. We investigated the potential of a display methodology for a cDNA library of maize young seedlings. Results We constructed a full-length cDNA library of young maize etiolated seedlings in the transfer vector pAcTMVSVG. The library contained a total of 2.5 × 105 independent clones. Expression of two known maize proteins, calreticulin and auxin binding protein (ABP1, was shown by western blot analysis of protein extracts from insect cells infected with the cDNA library. Display of the two proteins in infected insect cells was shown by selective biopanning using magnetic cell sorting and demonstrated proof of concept that the baculovirus maize cDNA display library could be used to identify and isolate proteins. Conclusion The maize cDNA library constructed in this study relies on the novel technology of baculovirus display and is unique in currently published cDNA libraries. Produced to demonstrate proof of principle, it opens the way for the development of a eukaryotic in vivo display tool which would be ideally suited for rapid screening of the maize proteome for binding partners, such as proteins involved in hormone regulation or defence.

Stable isotope probing in the metagenomics era: a bridge towards improved bioremediation

Science.gov (United States)

Uhlik, Ondrej; Leewis, Mary-Cathrine; Strejcek, Michal; Musilova, Lucie; Mackova, Martina; Leigh, Mary Beth; Macek, Tomas

2012-01-01

Microbial biodegradation and biotransformation reactions are essential to most bioremediation processes, yet the specific organisms, genes, and mechanisms involved are often not well understood. Stable isotope probing (SIP) enables researchers to directly link microbial metabolic capability to phylogenetic and metagenomic information within a community context by tracking isotopically labeled substances into phylogenetically and functionally informative biomarkers. SIP is thus applicable as a tool for the identification of active members of the microbial community and associated genes integral to the community functional potential, such as biodegradative processes. The rapid evolution of SIP over the last decade and integration with metagenomics provides researchers with a much deeper insight into potential biodegradative genes, processes, and applications, thereby enabling an improved mechanistic understanding that can facilitate advances in the field of bioremediation. PMID:23022353

Comparing and Evaluating Metagenome Assembly Tools from a Microbiologist's Perspective - Not Only Size Matters!

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John Vollmers

Full Text Available With the constant improvement in cost-efficiency and quality of Next Generation Sequencing technologies, shotgun-sequencing approaches -such as metagenomics- have nowadays become the methods of choice for studying and classifying microorganisms from various habitats. The production of data has dramatically increased over the past years and processing and analysis steps are becoming more and more of a bottleneck. Limiting factors are partly the availability of computational resources, but mainly the bioinformatics expertise in establishing and applying appropriate processing and analysis pipelines. Fortunately, a large diversity of specialized software tools is nowadays available. Nevertheless, choosing the most appropriate methods for answering specific biological questions can be rather challenging, especially for non-bioinformaticians. In order to provide a comprehensive overview and guide for the microbiological scientific community, we assessed the most common and freely available metagenome assembly tools with respect to their output statistics, their sensitivity for low abundant community members and variability in resulting community profiles as well as their ease-of-use. In contrast to the highly anticipated "Critical Assessment of Metagenomic Interpretation" (CAMI challenge, which uses general mock community-based assembler comparison we here tested assemblers on real Illumina metagenome sequencing data from natural communities of varying complexity sampled from forest soil and algal biofilms. Our observations clearly demonstrate that different assembly tools can prove optimal, depending on the sample type, available computational resources and, most importantly, the specific research goal. In addition, we present detailed descriptions of the underlying principles and pitfalls of publically available assembly tools from a microbiologist's perspective, and provide guidance regarding the user-friendliness, sensitivity and reliability of

America's Star Libraries, 2010: Top-Rated Libraries

Science.gov (United States)

Lyons, Ray; Lance, Keith Curry

2010-01-01

The "LJ" Index of Public Library Service 2010, "Library Journal"'s national rating of public libraries, identifies 258 "star" libraries. Created by Ray Lyons and Keith Curry Lance, and based on 2008 data from the IMLS, it rates 7,407 public libraries. The top libraries in each group get five, four, or three stars. All included libraries, stars or…

Synthetic promoter libraries- tuning of gene expression

DEFF Research Database (Denmark)

Hammer, Karin; Mijakovic, Ivan; Jensen, Peter Ruhdal

2006-01-01

knockout and strong overexpression. However, applications such as metabolic optimization and control analysis necessitate a continuous set of expression levels with only slight increments in strength to cover a specific window around the wildtype expression level of the studied gene; this requirement can......The study of gene function often requires changing the expression of a gene and evaluating the consequences. In principle, the expression of any given gene can be modulated in a quasi-continuum of discrete expression levels but the traditional approaches are usually limited to two extremes: gene...

The Metagenome of Utricularia gibba's Traps: Into the Microbial Input to a Carnivorous Plant

Science.gov (United States)

Alcaraz, Luis David; Martínez-Sánchez, Shamayim; Torres, Ignacio; Ibarra-Laclette, Enrique; Herrera-Estrella, Luis

2016-01-01

The genome and transcriptome sequences of the aquatic, rootless, and carnivorous plant Utricularia gibba L. (Lentibulariaceae), were recently determined. Traps are necessary for U. gibba because they help the plant to survive in nutrient-deprived environments. The U. gibba's traps (Ugt) are specialized structures that have been proposed to selectively filter microbial inhabitants. To determine whether the traps indeed have a microbiome that differs, in composition or abundance, from the microbiome in the surrounding environment, we used whole-genome shotgun (WGS) metagenomics to describe both the taxonomic and functional diversity of the Ugt microbiome. We collected U. gibba plants from their natural habitat and directly sequenced the metagenome of the Ugt microbiome and its surrounding water. The total predicted number of species in the Ugt was more than 1,100. Using pan-genome fragment recruitment analysis, we were able to identify to the species level of some key Ugt players, such as Pseudomonas monteilii. Functional analysis of the Ugt metagenome suggests that the trap microbiome plays an important role in nutrient scavenging and assimilation while complementing the hydrolytic functions of the plant. PMID:26859489

Construction of a cDNA library from female adult of Toxocara canis, and analysis of EST and immune-related genes expressions.

Science.gov (United States)

Zhou, Rongqiong; Xia, Qingyou; Huang, Hancheng; Lai, Min; Wang, Zhenxin

2011-10-01

Toxocara canis is a widespread intestinal nematode parasite of dogs, which can also cause disease in humans. We employed an expressed sequence tag (EST) strategy in order to study gene-expression including development, digestion and reproduction of T. canis. ESTs provided a rapid way to identify genes, particularly in organisms for which we have very little molecular information. In this study, a cDNA library was constructed from a female adult of T. canis and 215 high-quality ESTs from 5'-ends of the cDNA clones representing 79 unigenes were obtained. The titer of the primary cDNA library was 1.83×10(6)pfu/mL with a recombination rate of 99.33%. Most of the sequences ranged from 300 to 900bp with an average length of 656bp. Cluster analysis of these ESTs allowed identification of 79 unique sequences containing 28 contigs and 51 singletons. BLASTX searches revealed that 18 unigenes (22.78% of the total) or 70 ESTs (32.56% of the total) were novel genes that had no significant matches to any protein sequences in the public databases. The rest of the 61 unigenes (77.22% of the total) or 145 ESTs (67.44% of the total) were closely matched to the known genes or sequences deposited in the public databases. These genes were classified into seven groups based on their known or putative biological functions. We also confirmed the gene expression patterns of several immune-related genes using RT-PCR examination. This work will provide a valuable resource for the further investigations in the stage-, sex- and tissue-specific gene transcription or expression. Copyright © 2011. Published by Elsevier Inc.

Identification of genes and pathways related to phenol degradation in metagenomic libraries from petroleum refinery wastewater.

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Cynthia C Silva

Full Text Available Two fosmid libraries, totaling 13,200 clones, were obtained from bioreactor sludge of petroleum refinery wastewater treatment system. The library screening based on PCR and biological activity assays revealed more than 400 positive clones for phenol degradation. From these, 100 clones were randomly selected for pyrosequencing in order to evaluate the genetic potential of the microorganisms present in wastewater treatment plant for biodegradation, focusing mainly on novel genes and pathways of phenol and aromatic compound degradation. The sequence analysis of selected clones yielded 129,635 reads at an estimated 17-fold coverage. The phylogenetic analysis showed Burkholderiales and Rhodocyclales as the most abundant orders among the selected fosmid clones. The MG-RAST analysis revealed a broad metabolic profile with important functions for wastewater treatment, including metabolism of aromatic compounds, nitrogen, sulphur and phosphorus. The predicted 2,276 proteins included phenol hydroxylases and cathecol 2,3- dioxygenases, involved in the catabolism of aromatic compounds, such as phenol, byphenol, benzoate and phenylpropanoid. The sequencing of one fosmid insert of 33 kb unraveled the gene that permitted the host, Escherichia coli EPI300, to grow in the presence of aromatic compounds. Additionally, the comparison of the whole fosmid sequence against bacterial genomes deposited in GenBank showed that about 90% of sequence showed no identity to known sequences of Proteobacteria deposited in the NCBI database. This study surveyed the functional potential of fosmid clones for aromatic compound degradation and contributed to our knowledge of the biodegradative capacity and pathways of microbial assemblages present in refinery wastewater treatment system.

Featured Library: Parrish Library

OpenAIRE

Kirkwood, Hal P, Jr

2015-01-01

The Roland G. Parrish Library of Management & Economics is located within the Krannert School of Management at Purdue University. Between 2005 - 2007 work was completed on a white paper that focused on a student-centered vision for the Management & Economics Library. The next step was a massive collection reduction and a re-envisioning of both the services and space of the library. Thus began a 3 phase renovation from a 2 floor standard, collection-focused library into a single floor, 18,000s...

Meta4: a web application for sharing and annotating metagenomic gene predictions using web services.

Science.gov (United States)

Richardson, Emily J; Escalettes, Franck; Fotheringham, Ian; Wallace, Robert J; Watson, Mick

2013-01-01

Whole-genome shotgun metagenomics experiments produce DNA sequence data from entire ecosystems, and provide a huge amount of novel information. Gene discovery projects require up-to-date information about sequence homology and domain structure for millions of predicted proteins to be presented in a simple, easy-to-use system. There is a lack of simple, open, flexible tools that allow the rapid sharing of metagenomics datasets with collaborators in a format they can easily interrogate. We present Meta4, a flexible and extensible web application that can be used to share and annotate metagenomic gene predictions. Proteins and predicted domains are stored in a simple relational database, with a dynamic front-end which displays the results in an internet browser. Web services are used to provide up-to-date information about the proteins from homology searches against public databases. Information about Meta4 can be found on the project website, code is available on Github, a cloud image is available, and an example implementation can be seen at.

Microbial survival strategies in ancient permafrost: insights from metagenomics.

Science.gov (United States)

Mackelprang, Rachel; Burkert, Alexander; Haw, Monica; Mahendrarajah, Tara; Conaway, Christopher H; Douglas, Thomas A; Waldrop, Mark P

2017-10-01

In permafrost (perennially frozen ground) microbes survive oligotrophic conditions, sub-zero temperatures, low water availability and high salinity over millennia. Viable life exists in permafrost tens of thousands of years old but we know little about the metabolic and physiological adaptations to the challenges presented by life in frozen ground over geologic time. In this study we asked whether increasing age and the associated stressors drive adaptive changes in community composition and function. We conducted deep metagenomic and 16 S rRNA gene sequencing across a Pleistocene permafrost chronosequence from 19 000 to 33 000 years before present (kyr). We found that age markedly affected community composition and reduced diversity. Reconstruction of paleovegetation from metagenomic sequence suggests vegetation differences in the paleo record are not responsible for shifts in community composition and function. Rather, we observed shifts consistent with long-term survival strategies in extreme cryogenic environments. These include increased reliance on scavenging detrital biomass, horizontal gene transfer, chemotaxis, dormancy, environmental sensing and stress response. Our results identify traits that may enable survival in ancient cryoenvironments with no influx of energy or new materials.

Functional metagenomic profiling of intestinal microbiome in extreme ageing

Science.gov (United States)

Rampelli, Simone; Candela, Marco; Turroni, Silvia; Biagi, Elena; Collino, Sebastiano; Franceschi, Claudio; O'Toole, Paul W; Brigidi, Patrizia

2013-01-01

Age-related alterations in human gut microbiota composition have been thoroughly described, but a detailed functional description of the intestinal bacterial coding capacity is still missing. In order to elucidate the contribution of the gut metagenome to the complex mosaic of human longevity, we applied shotgun sequencing to total fecal bacterial DNA in a selection of samples belonging to a well-characterized human ageing cohort. The age-related trajectory of the human gut microbiome was characterized by loss of genes for shortchain fatty acid production and an overall decrease in the saccharolytic potential, while proteolytic functions were more abundant than in the intestinal metagenome of younger adults. This altered functional profile was associated with a relevant enrichment in “pathobionts”, i.e. opportunistic pro-inflammatory bacteria generally present in the adult gut ecosystem in low numbers. Finally, as a signature for long life we identified 116 microbial genes that significantly correlated with ageing. Collectively, our data emphasize the relationship between intestinal bacteria and human metabolism, by detailing the modifications in the gut microbiota as a consequence of and/or promoter of the physiological changes occurring in the human host upon ageing. PMID:24334635

Functional metagenomic profiling of intestinal microbiome in extreme ageing.

Science.gov (United States)

Rampelli, Simone; Candela, Marco; Turroni, Silvia; Biagi, Elena; Collino, Sebastiano; Franceschi, Claudio; O'Toole, Paul W; Brigidi, Patrizia

2013-12-01

Age-related alterations in human gut microbiota composition have been thoroughly described, but a detailed functional description of the intestinal bacterial coding capacity is still missing. In order to elucidate the contribution of the gut metagenome to the complex mosaic of human longevity, we applied shotgun sequencing to total fecal bacterial DNA in a selection of samples belonging to a well-characterized human ageing cohort. The age-related trajectory of the human gut microbiome was characterized by loss of genes for shortchain fatty acid production and an overall decrease in the saccharolytic potential, while proteolytic functions were more abundant than in the intestinal metagenome of younger adults. This altered functional profile was associated with a relevant enrichment in "pathobionts", i.e. opportunistic pro-inflammatory bacteria generally present in the adult gut ecosystem in low numbers. Finally, as a signature for long life we identified 116 microbial genes that significantly correlated with ageing. Collectively, our data emphasize the relationship between intestinal bacteria and human metabolism, by detailing the modifications in the gut microbiota as a consequence of and/or promoter of the physiological changes occurring in the human host upon ageing.

IDENTIFICATION OF CHICKEN-SPECIFIC FECAL MICROBIAL SEQUENCES USING A METAGENOMIC APPROACH

Science.gov (United States)

In this study, we applied a genome fragment enrichment (GFE) method to select for genomic regions that differ between different fecal metagenomes. Competitive DNA hybridizations were performed between chicken fecal DNA and pig fecal DNA (C-P) and between chicken fecal DNA and an ...

Metagenomic analysis of faecal microbiome as a tool towards targeted non-invasive biomarkers for colorectal cancer

DEFF Research Database (Denmark)

Yu, Jun; Feng, Qiang; Wong, Sunny Hei

2017-01-01

known associations of Fusobacterium nucleatum and Peptostreptococcus stomatis with CRC, we found significant associations with several species, including Parvimonas micra and Solobacterium moorei. We identified 20 microbial gene markers that differentiated CRC and control microbiomes, and validated 4...... in the independent Chinese cohort with AUC=0.84 and OR of 23. These genes were enriched in early-stage (I-II) patient microbiomes, highlighting the potential for using faecal metagenomic biomarkers for early diagnosis of CRC. CONCLUSIONS: We present the first metagenomic profiling study of CRC faecal microbiomes...

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.

Multigroup cross section library; WIMS library

International Nuclear Information System (INIS)

Kannan, Umasankari

2000-01-01

The WIMS library has been extensively used in thermal reactor calculations. This multigroup constants library was originally developed from the UKNDL in the late 60's and has been updated in 1986. This library has been distributed with the WIMS-D code by NEA data bank. The references to WIMS library in literature are the 'old' which is the original as developed by the AEA Winfrith and the 'new' which is the current 1986 WIMS library. IAEA has organised a CRP where a new and fully updated WIMS library will soon be available. This paper gives an overview of the definitions of the group constants that go into any basic nuclear data library used for reactor calculations. This paper also outlines the contents of the WIMS library and some of its shortcomings

Expression of maize prolamins in Escherichia Coli

International Nuclear Information System (INIS)

Wang, Szu-zhen; Esen, Asim

1985-01-01

We have constructed a cDNA expression library of developing corn (Zea manys L.) endosperm using plasmid pUC8 as vector and Escherichia coli strain DH1 as host. The expression library was screened with non-radioactive immunological probes to detect the expression of gamma-zein and alpha-zein. When anti-gamma-zein antibody was used as the probe, 23 colonies gave positive reactions. The lengths of cDNA inserts of the 23 colonies were found to be 250-900 base pairs. When anti-alpha zein antibody was used, however, fewer colonies gave positive reactions. The library was also screened by colony-hybridization with 32 P-labeled DNA probes. Based on immunological and hybridization screening of the library and other evidence, we conclude that alpha-zein was either toxic to E. coli cells or rapidly degraded whereas gamma-zein and its fragments were readily expressed. (author)

Microcystin-LR nanobody screening from an alpaca phage display nanobody library and its expression and application.

Science.gov (United States)

Xu, Chongxin; Yang, Ying; Liu, Liwen; Li, Jianhong; Liu, Xiaoqin; Zhang, Xiao; Liu, Yuan; Zhang, Cunzheng; Liu, Xianjin

2018-04-30

Microcystin-LR (MC-LR) is a type of biotoxin that pollutes the ecological environment and food. The study aimed to obtain new nanobodies from phage nanobody library for determination of MC-LR. The toxin was conjugated to keyhole limpet haemocyanin (KLH) and bovine serum albumin (BSA), respectively, then the conjugates were used as coated antigens for enrichment (coated MC-LR-KLH) and screening (coated MC-LR-BSA) of MC-LR phage nanobodies from an alpaca phage display nanobody library. The antigen-specific phage particles were enriched effectively with four rounds of biopanning. At the last round of enrichment, total 20 positive monoclonal phage nanobodies were obtained from the library, which were analyzed after monoclonal phage enzyme linked immunosorbent assay (ELISA), colony PCR and DNA sequencing. The most three positive nanobody genes, ANAb12, ANAb9 and ANAb7 were cloned into pET26b vector, then the nanobodies were expressed in Escherichia coli BL21 respectively. After being purified, the molecular weight (M.W.) of all nanobodies were approximate 15kDa with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified nanobodies, ANAb12, ANAb9 and ANAb7 were used to establish the indirect competitive ELISA (IC-ELISA) for MC-LR, and their half-maximum inhibition concentrations (IC 50 ) were 0.87, 1.17 and 1.47μg/L, their detection limits (IC 10 ) were 0.06, 0.08 and 0.12μg/L, respectively. All of them showed strong cross-reactivity (CRs) of 82.7-116.9% for MC-RR, MC-YR and MC-WR, and weak CRs of less than 4.56% for MC-LW, less than 0.1% for MC-LY and MC-LF. It was found that all the IC-ELISAs for MC-LR spiked in tap water samples detection were with good accuracy, stability and repeatability, their recoveries were 84.0-106.5%, coefficient of variations (CVs) were 3.4-10.6%. These results showed that IC-ELISA based on the nanobodies from the alpaca phage display antibody library were promising for high sensitive determination of multiple

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...... provincialism). Molecular functional richness and diversity show a distinct latitudinal gradient peaking at 20° N and correlate with primary production. The latter can also be predicted from the molecular functional composition of an environmental sample. Together, our results show that the functional community...

HORSE SPECIES SYMPOSIUM: Canine intestinal microbiology and metagenomics: From phylogeny to function.

Science.gov (United States)

Guard, B C; Suchodolski, J S

2016-06-01

Recent molecular studies have revealed a complex microbiota in the dog intestine. Convincing evidence has been reported linking changes in microbial communities to acute and chronic gastrointestinal inflammation, especially in canine inflammatory bowel disease (IBD). The most common microbial changes observed in intestinal inflammation are decreases in the bacterial phyla Firmicutes (i.e., Lachnospiraceae, Ruminococcaceae, and ) and Bacteroidetes, with concurrent increases in Proteobacteria (i.e., ). Due to the important role of microbial-derived metabolites for host health, it is important to elucidate the metabolic consequences of gastrointestinal dysbiosis and physiological pathways implicated in specific disease phenotypes. Metagenomic studies have used shotgun sequencing of DNA as well as phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) to characterize functional changes in the bacterial metagenome in gastrointestinal disease. Furthermore, wide-scale and untargeted measurements of metabolic products derived by the host and the microbiota in intestinal samples allow a better understanding of the functional alterations that occur in gastrointestinal disease. For example, changes in bile acid metabolism and tryptophan catabolism recently have been reported in humans and dogs. Also, metabolites associated with the pentose phosphate pathway were significantly altered in chronic gastrointestinal inflammation and indicate the presence of oxidative stress in dogs with IBD. This review focuses on the advancements made in canine metagenomics and metabolomics and their implications in understanding gastrointestinal disease as well as the development of better treatment approaches.

Educational Needs of Public Library Users

Directory of Open Access Journals (Sweden)

Simona Šinko

2014-09-01

Full Text Available AbstractPurpose: The article explains the role of user education in public libraries based on the assessment of users’ educational needs in a selected public library. We present a part of the results of the needs assessment study conducted on users of the Ljubljana City Library (LCL. In this presentation, we focus on the thematic areas of users’ interest, the most appropriate time of day and the most appropriate period of the year for participating in education. The aim of presenting this information is to support the development of an appropriate adult education provision and an effective use of human, financial and material resources in public libraries.Methodology/approach: The basic research method used was descriptive and causal non- experimental. The study involved 943 adults LCL users who have filled in an online or a printed form of the questionnaire.Results: Results of data analysis showed that users express a greater degree of interest in the following course topics: travel, foreign languages, literature, public speaking and health. They express the lowest degree of interest in the topics: policy, motoring and mathematics. Afternoons and evenings suit them best, whereas the autumn and winter season are in their view the best time of the year for participating in education. The analysis also showed some statistically significant differences in relation to sex, age, status and educational level of the users.Research limitation: By the practical application of results the characteristics of the sample of users and the environment from which they come, as well as the shortcomings of the questionnaire as an instrument for data collection, have to be considered.Originality/practical implications: The data obtained are useful in preparing the basis of educational activities in LCL, as well as for the design of educational programs in other comparable public libraries and other providers of adult education. The survey results can also

The Human Gut Antibiotic Resistome in the Metagenomic Era: Progress and Perspectives

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Yongfei Hu

2016-04-01

Full Text Available The human gut is populated by a vast number of bacteria, which play a critical role in human health. In recent years, attention has focused on the gut bacteria as a reservoir of antibiotic resistance genes (ARGs. Both culture-dependent and culture-independent methods have been applied to investigate numerous ARGs, collectively called the antibiotic resistome, harbored by gut bacteria. This has led to an increased understanding of the overall profile of the gut antibiotic resistome, although it remains incompletely understood. In this review, we summarize the recent research findings on the human gut antibiotic resistome, with an emphasis on progress achieved using the culture-independent metagenomic strategy. We also describe the features of different available ARG databases used for annotation in metagenomic analysis, discuss the potential problems and limitations in current research, and suggest several directions for future investigation.

Library 3.0 intelligent libraries and apomediation

CERN Document Server

Kwanya, Tom; Underwood, Peter

2015-01-01

The emerging generation of research and academic library users expect the delivery of user-centered information services. 'Apomediation' refers to the supporting role librarians can give users by stepping in when users need help. Library 3.0 explores the ongoing debates on the "point oh” phenomenon and its impact on service delivery in libraries. This title analyses Library 3.0 and its potential in creating intelligent libraries capable of meeting contemporary needs, and the growing role of librarians as apomediators. Library 3.0 is divided into four chapters. The first chapter introduces and places the topic in context. The second chapter considers "point oh” libraries. The third chapter covers library 3.0 librarianship, while the final chapter explores ways libraries can move towards '3.0'.

The mzqLibrary--An open source Java library supporting the HUPO-PSI quantitative proteomics standard.

Science.gov (United States)

Qi, Da; Zhang, Huaizhong; Fan, Jun; Perkins, Simon; Pisconti, Addolorata; Simpson, Deborah M; Bessant, Conrad; Hubbard, Simon; Jones, Andrew R

2015-09-01

The mzQuantML standard has been developed by the Proteomics Standards Initiative for capturing, archiving and exchanging quantitative proteomic data, derived from mass spectrometry. It is a rich XML-based format, capable of representing data about two-dimensional features from LC-MS data, and peptides, proteins or groups of proteins that have been quantified from multiple samples. In this article we report the development of an open source Java-based library of routines for mzQuantML, called the mzqLibrary, and associated software for visualising data called the mzqViewer. The mzqLibrary contains routines for mapping (peptide) identifications on quantified features, inference of protein (group)-level quantification values from peptide-level values, normalisation and basic statistics for differential expression. These routines can be accessed via the command line, via a Java programming interface access or a basic graphical user interface. The mzqLibrary also contains several file format converters, including import converters (to mzQuantML) from OpenMS, Progenesis LC-MS and MaxQuant, and exporters (from mzQuantML) to other standards or useful formats (mzTab, HTML, csv). The mzqViewer contains in-built routines for viewing the tables of data (about features, peptides or proteins), and connects to the R statistical library for more advanced plotting options. The mzqLibrary and mzqViewer packages are available from https://code.google.com/p/mzq-lib/. © 2015 The Authors. PROTEOMICS Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Statistical evaluation of SAGE libraries: consequences for experimental design

NARCIS (Netherlands)

Ruijter, Jan M.; van Kampen, Antoine H. C.; Baas, Frank

2002-01-01

Since the introduction of serial analysis of gene expression (SAGE) as a method to quantitatively analyze the differential expression of genes, several statistical tests have been published for the pairwise comparison of SAGE libraries. Testing the difference between the number of specific tags

Strain-Level Discrimination of Shiga Toxin-Producing Escherichia coli in Spinach Using Metagenomic Sequencing.

Directory of Open Access Journals (Sweden)

Susan R Leonard

Full Text Available Consumption of fresh bagged spinach contaminated with Shiga toxin-producing Escherichia coli (STEC has led to severe illness and death; however current culture-based methods to detect foodborne STEC are time consuming. Since not all STEC strains are considered pathogenic to humans, it is crucial to incorporate virulence characterization of STEC in the detection method. In this study, we assess the comprehensiveness of utilizing a shotgun metagenomics approach for detection and strain-level identification by spiking spinach with a variety of genomically disparate STEC strains at a low contamination level of 0.1 CFU/g. Molecular serotyping, virulence gene characterization, microbial community analysis, and E. coli core gene single nucleotide polymorphism (SNP analysis were performed on metagenomic sequence data from enriched samples. It was determined from bacterial community analysis that E. coli, which was classified at the phylogroup level, was a major component of the population in most samples. However, in over half the samples, molecular serotyping revealed the presence of indigenous E. coli which also contributed to the percent abundance of E. coli. Despite the presence of additional E. coli strains, the serotype and virulence genes of the spiked STEC, including correct Shiga toxin subtype, were detected in 94% of the samples with a total number of reads per sample averaging 2.4 million. Variation in STEC abundance and/or detection was observed in replicate spiked samples, indicating an effect from the indigenous microbiota during enrichment. SNP analysis of the metagenomic data correctly placed the spiked STEC in a phylogeny of related strains in cases where the indigenous E. coli did not predominate in the enriched sample. Also, for these samples, our analysis demonstrates that strain-level phylogenetic resolution is possible using shotgun metagenomic data for determining the genomic relatedness of a contaminating STEC strain to other

Placing the public library – a comparative analysis of political perceptions

DEFF Research Database (Denmark)

Evjen, Sunniva

2012-01-01

This thesis explores politicians’ perceptions of the public library and public library development. While many call for a redefined vision for public libraries, eye-catching libraries are built in major cities around the world. What library visions are expressed through such projects? I attempt...... to discern how local politicians view the role of the public library, and how do they want to develop it in their local context, using concepts from institutional theory in the analysis. The research questions include issues concerning norms politicians connect with public libraries compared with those...... and document analysis of local and national policy documents. One important premise for this study has been to find cases where there are on-going developments; in the shape of main library construction. The findings show that politicians have extensive knowledge about the norms and values found...

Croatian library leaders’ views on (their library quality

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Kornelija Petr Balog

2014-04-01

Full Text Available The purpose of this paper is to determine and describe the library culture in Croatian public libraries. Semi-structured interviews with 14 library directors (ten public and four academic were conducted. The tentative discussion topics were: definition of quality, responsibility for quality, satisfaction with library services, familiarization with user perspective of library and librarians, monitoring of user expectations and opinions. These interviews incorporate some of the findings of the project Evaluation of library and information services: public and academic libraries. The project investigates library culture in Croatian public and academic libraries and their preparedness for activities of performance measurement. The interviews reveal that library culture has changed positively in the past few years and that library leaders have positive attitude towards quality and evaluation activities. Library culture in Croatian libraries is a relatively new concept and as such was not actively developed and/or created. This article looks into the library culture of Croatian libraries, but at the same time investigates whether there is any trace of culture of assessment in them. Also, this article brings the latest update on views, opinions and atmosphere in Croatian public and academic libraries.

Snapshot of the eukaryotic gene expression in muskoxen rumen--a metatranscriptomic approach.

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Meng Qi

Full Text Available BACKGROUND: Herbivores rely on digestive tract lignocellulolytic microorganisms, including bacteria, fungi and protozoa, to derive energy and carbon from plant cell wall polysaccharides. Culture independent metagenomic studies have been used to reveal the genetic content of the bacterial species within gut microbiomes. However, the nature of the genes encoded by eukaryotic protozoa and fungi within these environments has not been explored using metagenomic or metatranscriptomic approaches. METHODOLOGY/PRINCIPAL FINDINGS: In this study, a metatranscriptomic approach was used to investigate the functional diversity of the eukaryotic microorganisms within the rumen of muskoxen (Ovibos moschatus, with a focus on plant cell wall degrading enzymes. Polyadenylated RNA (mRNA was sequenced on the Illumina Genome Analyzer II system and 2.8 gigabases of sequences were obtained and 59129 contigs assembled. Plant cell wall degrading enzyme modules including glycoside hydrolases, carbohydrate esterases and polysaccharide lyases were identified from over 2500 contigs. These included a number of glycoside hydrolase family 6 (GH6, GH48 and swollenin modules, which have rarely been described in previous gut metagenomic studies. CONCLUSIONS/SIGNIFICANCE: The muskoxen rumen metatranscriptome demonstrates a much higher percentage of cellulase enzyme discovery and an 8.7x higher rate of total carbohydrate active enzyme discovery per gigabase of sequence than previous rumen metagenomes. This study provides a snapshot of eukaryotic gene expression in the muskoxen rumen, and identifies a number of candidate genes coding for potentially valuable lignocellulolytic enzymes.

Snapshot of the Eukaryotic Gene Expression in Muskoxen Rumen—A Metatranscriptomic Approach

Science.gov (United States)

O'Toole, Nicholas; Barboza, Perry S.; Ungerfeld, Emilio; Leigh, Mary Beth; Selinger, L. Brent; Butler, Greg; Tsang, Adrian; McAllister, Tim A.; Forster, Robert J.

2011-01-01

Background Herbivores rely on digestive tract lignocellulolytic microorganisms, including bacteria, fungi and protozoa, to derive energy and carbon from plant cell wall polysaccharides. Culture independent metagenomic studies have been used to reveal the genetic content of the bacterial species within gut microbiomes. However, the nature of the genes encoded by eukaryotic protozoa and fungi within these environments has not been explored using metagenomic or metatranscriptomic approaches. Methodology/Principal Findings In this study, a metatranscriptomic approach was used to investigate the functional diversity of the eukaryotic microorganisms within the rumen of muskoxen (Ovibos moschatus), with a focus on plant cell wall degrading enzymes. Polyadenylated RNA (mRNA) was sequenced on the Illumina Genome Analyzer II system and 2.8 gigabases of sequences were obtained and 59129 contigs assembled. Plant cell wall degrading enzyme modules including glycoside hydrolases, carbohydrate esterases and polysaccharide lyases were identified from over 2500 contigs. These included a number of glycoside hydrolase family 6 (GH6), GH48 and swollenin modules, which have rarely been described in previous gut metagenomic studies. Conclusions/Significance The muskoxen rumen metatranscriptome demonstrates a much higher percentage of cellulase enzyme discovery and an 8.7x higher rate of total carbohydrate active enzyme discovery per gigabase of sequence than previous rumen metagenomes. This study provides a snapshot of eukaryotic gene expression in the muskoxen rumen, and identifies a number of candidate genes coding for potentially valuable lignocellulolytic enzymes. PMID:21655220

Metagenome and Metatranscriptome Analyses Using Protein Family Profiles.

Directory of Open Access Journals (Sweden)

Cuncong Zhong

2016-07-01

Full Text Available Analyses of metagenome data (MG and metatranscriptome data (MT are often challenged by a paucity of complete reference genome sequences and the uneven/low sequencing depth of the constituent organisms in the microbial community, which respectively limit the power of reference-based alignment and de novo sequence assembly. These limitations make accurate protein family classification and abundance estimation challenging, which in turn hamper downstream analyses such as abundance profiling of metabolic pathways, identification of differentially encoded/expressed genes, and de novo reconstruction of complete gene and protein sequences from the protein family of interest. The profile hidden Markov model (HMM framework enables the construction of very useful probabilistic models for protein families that allow for accurate modeling of position specific matches, insertions, and deletions. We present a novel homology detection algorithm that integrates banded Viterbi algorithm for profile HMM parsing with an iterative simultaneous alignment and assembly computational framework. The algorithm searches a given profile HMM of a protein family against a database of fragmentary MG/MT sequencing data and simultaneously assembles complete or near-complete gene and protein sequences of the protein family. The resulting program, HMM-GRASPx, demonstrates superior performance in aligning and assembling homologs when benchmarked on both simulated marine MG and real human saliva MG datasets. On real supragingival plaque and stool MG datasets that were generated from healthy individuals, HMM-GRASPx accurately estimates the abundances of the antimicrobial resistance (AMR gene families and enables accurate characterization of the resistome profiles of these microbial communities. For real human oral microbiome MT datasets, using the HMM-GRASPx estimated transcript abundances significantly improves detection of differentially expressed (DE genes. Finally, HMM

Genometa--a fast and accurate classifier for short metagenomic shotgun reads.

Science.gov (United States)

Davenport, Colin F; Neugebauer, Jens; Beckmann, Nils; Friedrich, Benedikt; Kameri, Burim; Kokott, Svea; Paetow, Malte; Siekmann, Björn; Wieding-Drewes, Matthias; Wienhöfer, Markus; Wolf, Stefan; Tümmler, Burkhard; Ahlers, Volker; Sprengel, Frauke

2012-01-01

Metagenomic studies use high-throughput sequence data to investigate microbial communities in situ. However, considerable challenges remain in the analysis of these data, particularly with regard to speed and reliable analysis of microbial species as opposed to higher level taxa such as phyla. We here present Genometa, a computationally undemanding graphical user interface program that enables identification of bacterial species and gene content from datasets generated by inexpensive high-throughput short read sequencing technologies. Our approach was first verified on two simulated metagenomic short read datasets, detecting 100% and 94% of the bacterial species included with few false positives or false negatives. Subsequent comparative benchmarking analysis against three popular metagenomic algorithms on an Illumina human gut dataset revealed Genometa to attribute the most reads to bacteria at species level (i.e. including all strains of that species) and demonstrate similar or better accuracy than the other programs. Lastly, speed was demonstrated to be many times that of BLAST due to the use of modern short read aligners. Our method is highly accurate if bacteria in the sample are represented by genomes in the reference sequence but cannot find species absent from the reference. This method is one of the most user-friendly and resource efficient approaches and is thus feasible for rapidly analysing millions of short reads on a personal computer. The Genometa program, a step by step tutorial and Java source code are freely available from http://genomics1.mh-hannover.de/genometa/ and on http://code.google.com/p/genometa/. This program has been tested on Ubuntu Linux and Windows XP/7.

Genometa--a fast and accurate classifier for short metagenomic shotgun reads.

Directory of Open Access Journals (Sweden)

Colin F Davenport

Full Text Available Metagenomic studies use high-throughput sequence data to investigate microbial communities in situ. However, considerable challenges remain in the analysis of these data, particularly with regard to speed and reliable analysis of microbial species as opposed to higher level taxa such as phyla. We here present Genometa, a computationally undemanding graphical user interface program that enables identification of bacterial species and gene content from datasets generated by inexpensive high-throughput short read sequencing technologies. Our approach was first verified on two simulated metagenomic short read datasets, detecting 100% and 94% of the bacterial species included with few false positives or false negatives. Subsequent comparative benchmarking analysis against three popular metagenomic algorithms on an Illumina human gut dataset revealed Genometa to attribute the most reads to bacteria at species level (i.e. including all strains of that species and demonstrate similar or better accuracy than the other programs. Lastly, speed was demonstrated to be many times that of BLAST due to the use of modern short read aligners. Our method is highly accurate if bacteria in the sample are represented by genomes in the reference sequence but cannot find species absent from the reference. This method is one of the most user-friendly and resource efficient approaches and is thus feasible for rapidly analysing millions of short reads on a personal computer.The Genometa program, a step by step tutorial and Java source code are freely available from http://genomics1.mh-hannover.de/genometa/ and on http://code.google.com/p/genometa/. This program has been tested on Ubuntu Linux and Windows XP/7.

Construction of CRISPR Libraries for Functional Screening.

Science.gov (United States)

Carstens, Carsten P; Felts, Katherine A; Johns, Sarah E

2018-01-01

Identification of gene function has been aided by the ability to generate targeted gene knockouts or transcriptional repression using the CRISPR/CAS9 system. Using pooled libraries of guide RNA expression vectors that direct CAS9 to a specific genomic site allows identification of genes that are either enriched or depleted in response to a selection scheme, thus linking the affected gene to the chosen phenotype. The quality of the data generated by the screening is dependent on the quality of the guide RNA delivery library with regards to error rates and especially evenness of distribution of the guides. Here, we describe a method for constructing complex plasmid libraries based on pooled designed oligomers with high representation and tight distributions. The procedure allows construction of plasmid libraries of >60,000 members with a 95th/5th percentile ratio of less than 3.5.

Ecological and genetic interactions between cyanobacteria and viruses in a low-oxygen mat community inferred through metagenomics and metatranscriptomics.

Science.gov (United States)

Voorhies, Alexander A; Eisenlord, Sarah D; Marcus, Daniel N; Duhaime, Melissa B; Biddanda, Bopaiah A; Cavalcoli, James D; Dick, Gregory J

2016-02-01

Metagenomic and metatranscriptomic sequencing was conducted on cyanobacterial mats of the Middle Island Sinkhole (MIS), Lake Huron. Metagenomic data from 14 samples collected over 5 years were used to reconstruct genomes of two genotypes of a novel virus, designated PhV1 type A and PhV1 type B. Both viral genotypes encode and express nblA, a gene involved in degrading phycobilisomes, which are complexes of pigmented proteins that harvest light for photosynthesis. Phylogenetic analysis indicated that the viral-encoded nblA is derived from the host cyanobacterium, Phormidium MIS-PhA. The cyanobacterial host also has two complete CRISPR (clustered regularly interspaced short palindromic repeats) systems that serve as defence mechanisms for bacteria and archaea against viruses and plasmids. One 45 bp CRISPR spacer from Phormidium had 100% nucleotide identity to PhV1 type B, but this region was absent from PhV1 type A. Transcripts from PhV1 and the Phormidium CRISPR loci were detected in all six metatranscriptomic data sets (three during the day and three at night), indicating that both are transcriptionally active in the environment. These results reveal ecological and genetic interactions between viruses and cyanobacteria at MIS, highlighting the value of parallel analysis of viruses and hosts in understanding ecological interactions in natural communities. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

[Construction of fetal mesenchymal stem cell cDNA subtractive library].

Science.gov (United States)

Yang, Li; Wang, Dong-Mei; Li, Liang; Bai, Ci-Xian; Cao, Hua; Li, Ting-Yu; Pei, Xue-Tao

2002-04-01

To identify differentially expressed genes between fetal mesenchymal stem cell (MSC) and adult MSC, especially specified genes expressed in fetal MSC, a cDNA subtractive library of fetal MSC was constructed using suppression subtractive hybridization (SSH) technique. At first, total RNA was isolated from fetal and adult MSC. Using SMART PCR synthesis method, single-strand and double-strand cDNAs were synthesized. After Rsa I digestion, fetal MSC cDNAs were divided into two groups and ligated to adaptor 1 and adaptor 2 respectively. Results showed that the amplified library contains 890 clones. Analysis of 890 clones with PCR demonstrated that 768 clones were positive. The positive rate is 86.3%. The size of inserted fragments in these positive clones was between 0.2 - 1 kb, with an average of 400 - 600 bp. SSH is a convenient and effective method for screening differentially expressed genes. The constructed cDNA subtractive library of fetal MSC cDNA lays solid foundation for screening and cloning new and specific function related genes of fetal MSC.

Metagenomic exploration reveals a marked change in the river resistome and mobilome after treated wastewater discharges.

Science.gov (United States)

Lekunberri, Itziar; Balcázar, José Luis; Borrego, Carles M

2018-03-01

Mobile genetic elements (MGEs) are key agents in the spread of antibiotic resistance genes (ARGs) across environments. Here we used metagenomics to compare the river resistome (collection of all ARGs) and mobilome (e.g., integrases, transposases, integron integrases and insertion sequence common region "ISCR" elements) between samples collected upstream (n = 6) and downstream (n = 6) of an urban wastewater treatment plant (UWWTP). In comparison to upstream metagenomes, downstream metagenomes showed a drastic increase in the abundance of ARGs, as well as markers of MGEs, particularly integron integrases and ISCR elements. These changes were accompanied by a concomitant prevalence of 16S rRNA gene signatures of bacteria affiliated to families encompassing well-known human and animal pathogens. Our results confirm that chronic discharges of treated wastewater severely impact the river resistome affecting not only the abundance and diversity of ARGs but also their potential spread by enriching the river mobilome in a wide variety of MGEs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Libraries of Synthetic TALE-Activated Promoters: Methods and Applications.

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Schreiber, T; Tissier, A

2016-01-01

The discovery of proteins with programmable DNA-binding specificities triggered a whole array of applications in synthetic biology, including genome editing, regulation of transcription, and epigenetic modifications. Among those, transcription activator-like effectors (TALEs) due to their natural function as transcription regulators, are especially well-suited for the development of orthogonal systems for the control of gene expression. We describe here the construction and testing of libraries of synthetic TALE-activated promoters which are under the control of a single TALE with a given DNA-binding specificity. These libraries consist of a fixed DNA-binding element for the TALE, a TATA box, and variable sequences of 19 bases upstream and 43 bases downstream of the DNA-binding element. These libraries were cloned using a Golden Gate cloning strategy making them usable as standard parts in a modular cloning system. The broad range of promoter activities detected and the versatility of these promoter libraries make them valuable tools for applications in the fine-tuning of expression in metabolic engineering projects or in the design and implementation of regulatory circuits. © 2016 Elsevier Inc. All rights reserved.

Metagenomic and Metatranscriptomic Analyses of Diverse Watermelon Cultivars Reveal the Role of Fruit Associated Microbiome in Carbohydrate Metabolism and Ripening of Mature Fruits

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Thangasamy Saminathan

2018-01-01

Full Text Available The plant microbiome is a key determinant of plant health and productivity, and changes in the plant microbiome can alter the tolerance to biotic and abiotic stresses and the quality of end produce. Little is known about the microbial diversity and its effect on carbohydrate metabolism in ripe fruits. In this study, we aimed to understand the diversity and function of microorganisms in relation to carbohydrate metabolism of ripe watermelon fruits. We used 16S metagenomics and RNAseq metatranscriptomics for analysis of red (PI459074, Congo, and SDRose and yellow fruit-flesh cultivars (PI227202, PI435990, and JBush of geographically and metabolically diverse watermelon cultivars. Metagenomics data showed that Proteobacteria were abundant in SDRose and PI227202, whereas Cyanobacteria were most abundant in Congo and PI4559074. In the case of metatranscriptome data, Proteobacteria was the most abundant in all cultivars. High expression of genes linked to infectious diseases and the expression of peptidoglycan hydrolases associated to pathogenicity of eukaryotic hosts was observed in SDRose, which could have resulted in low microbial diversity in this cultivar. The presence of GH28, associated with polygalacturonase activity in JBush and SDRose could be related to cell wall modifications including de-esterification and depolymerization, and consequent loss of galacturonic acid and neutral sugars. Moreover, based on the KEGG annotation of the expressed genes, nine α-galactosidase genes involved in key processes of galactosyl oligosaccharide metabolism, such as raffinose family were identified and galactose metabolism pathway was reconstructed. Results of this study underline the links between the host and fruit-associated microbiome in carbohydrate metabolism of the ripe fruits. The cultivar difference in watermelon reflects the quantum and diversity of the microbiome, which would benefit watermelon and other plant breeders aiming at the holobiont

Secretory Overexpression of Bacillus thermocatenulatus Lipase in Saccharomyces cerevisiae Using Combinatorial Library Strategy.

Science.gov (United States)

Kajiwara, Shota; Yamada, Ryosuke; Ogino, Hiroyasu

2018-04-10

Simple and cost-effective lipase expression host microorganisms are highly desirable. A combinatorial library strategy is used to improve the secretory expression of lipase from Bacillus thermocatenulatus (BTL2) in the culture supernatant of Saccharomyces cerevisiae. A plasmid library including expression cassettes composed of sequences encoding one of each 15 promoters, 15 secretion signals, and 15 terminators derived from yeast species, S. cerevisiae, Pichia pastoris, and Hansenula polymorpha, is constructed. The S. cerevisiae transformant YPH499/D4, comprising H. polymorpha GAP promoter, S. cerevisiae SAG1 secretion signal, and P. pastoris AOX1 terminator, is selected by high-throughput screening. This transformant expresses BTL2 extra-cellularly with a 130-fold higher than the control strain, comprising S. cerevisiae PGK1 promoter, S. cerevisiae α-factor secretion signal, and S. cerevisiae PGK1 terminator, after cultivation for 72 h. This combinatorial library strategy holds promising potential for application in the optimization of the secretory expression of proteins in yeast. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Seasonal patterns in Arctic prasinophytes and inferred ecology of Bathycoccus unveiled in an Arctic winter metagenome.

Science.gov (United States)

Joli, Nathalie; Monier, Adam; Logares, Ramiro; Lovejoy, Connie

2017-06-01

Prasinophytes occur in all oceans but rarely dominate phytoplankton populations. In contrast, a single ecotype of the prasinophyte Micromonas is frequently the most abundant photosynthetic taxon reported in the Arctic from summer through autumn. However, seasonal dynamics of prasinophytes outside of this period are little known. To address this, we analyzed high-throughput V4 18S rRNA amplicon data collected from November to July in the Amundsen Gulf Region, Beaufort Sea, Arctic. Surprisingly during polar sunset in November and December, we found a high proportion of reads from both DNA and RNA belonging to another prasinophyte, Bathycoccus. We then analyzed a metagenome from a December sample and the resulting Bathycoccus metagenome assembled genome (MAG) covered ~90% of the Bathycoccus Ban7 reference genome. In contrast, only ~20% of a reference Micromonas genome was found in the metagenome. Our phylogenetic analysis of marker genes placed the Arctic Bathycoccus in the B1 coastal clade. In addition, substitution rates of 129 coding DNA sequences were ~1.6% divergent between the Arctic MAG and coastal Chilean upwelling MAGs and 17.3% between it and a South East Atlantic open ocean MAG in the B2 Clade. The metagenomic analysis also revealed a winter viral community highly skewed toward viruses targeting Micromonas, with a much lower diversity of viruses targeting Bathycoccus. Overall a combination of Micromonas being relatively less able to maintain activity under dark winter conditions and viral suppression of Micromonas may have contributed to the success of Bathycoccus in the Amundsen Gulf during winter.

BrAD-seq: Breath Adapter Directional sequencing: a streamlined, ultra-simple and fast library preparation protocol for strand specific mRNA library construction.

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Brad Thomas Townsley

2015-05-01

Full Text Available Next Generation Sequencing (NGS is driving rapid advancement in biological understanding and RNA-sequencing (RNA-seq has become an indispensable tool for biology and medicine. There is a growing need for access to these technologies although preparation of NGS libraries remains a bottleneck to wider adoption. Here we report a novel method for the production of strand specific RNA-seq libraries utilizing inherent properties of double-stranded cDNA to capture and incorporate a sequencing adapter. Breath Adapter Directional sequencing (BrAD-seq reduces sample handling and requires far fewer enzymatic steps than most available methods to produce high quality strand-specific RNA-seq libraries. The method we present is optimized for 3-prime Digital Gene Expression (DGE libraries and can easily extend to full transcript coverage shotgun (SHO type strand-specific libraries and is modularized to accommodate a diversity of RNA and DNA input materials. BrAD-seq offers a highly streamlined and inexpensive option for RNA-seq libraries.

The Genomes OnLine Database (GOLD) v.4: status of genomic and metagenomic projects and their associated metadata

Science.gov (United States)

Pagani, Ioanna; Liolios, Konstantinos; Jansson, Jakob; Chen, I-Min A.; Smirnova, Tatyana; Nosrat, Bahador; Markowitz, Victor M.; Kyrpides, Nikos C.

2012-01-01

The Genomes OnLine Database (GOLD, http://www.genomesonline.org/) is a comprehensive resource for centralized monitoring of genome and metagenome projects worldwide. Both complete and ongoing projects, along with their associated metadata, can be accessed in GOLD through precomputed tables and a search page. As of September 2011, GOLD, now on version 4.0, contains information for 11 472 sequencing projects, of which 2907 have been completed and their sequence data has been deposited in a public repository. Out of these complete projects, 1918 are finished and 989 are permanent drafts. Moreover, GOLD contains information for 340 metagenome studies associated with 1927 metagenome samples. GOLD continues to expand, moving toward the goal of providing the most comprehensive repository of metadata information related to the projects and their organisms/environments in accordance with the Minimum Information about any (x) Sequence specification and beyond. PMID:22135293

Metagenomic Sequencing of Marine Periphyton: Taxonomic and Functional Insights into Biofilm Communities

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Kemal eSanli

2015-10-01

Full Text Available Periphyton communities are complex phototrophic, multispecies biofilms that develop on surfaces in aquatic environments. These communities harbor a large diversity of organisms comprising viruses, bacteria, algae, fungi, protozoans and metazoans. However, thus far the total biodiversity of periphyton has not been described. In this study, we use metagenomics to characterize periphyton communities from the marine environment of the Swedish west coast. Although we found approximately ten times more eukaryotic rRNA marker gene sequences compared to prokaryotic, the whole metagenome-based similarity searches showed that bacteria constitute the most abundant phyla in these biofilms. We show that marine periphyton encompass a range of heterotrophic and phototrophic organisms. Heterotrophic bacteria, including the majority of proteobacterial clades and Bacteroidetes, and eukaryotic macro-invertebrates were found to dominate periphyton. The phototrophic groups comprise Cyanobacteria and the alpha-proteobacterial genus Roseobacter, followed by different micro- and macro-algae. We also assess the metabolic pathways that predispose these communities to an attached lifestyle. Functional indicators of the biofilm form of life in periphyton involve genes coding for enzymes that catalyze the production and degradation of extracellular polymeric substances, mainly in the form of complex sugars such as starch and glycogen-like meshes together with chitin. Genes for 278 different transporter proteins were detected in the metagenome, constituting the most abundant protein complexes. Finally, genes encoding enzymes that participate in anaerobic pathways, such as denitrification and methanogenesis, were detected suggesting the presence of anaerobic or low-oxygen micro-zones within the biofilms.

Metagenomic detection of viruses in aerosol samples from workers in animal slaughterhouses.

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Richard J Hall

Full Text Available Published studies have shown that workers in animal slaughterhouses are at a higher risk of lung cancers as compared to the general population. No specific causal agents have been identified, and exposures to several chemicals have been examined and found to be unrelated. Evidence suggests a biological aetiology as the risk is highest for workers who are exposed to live animals or to biological material containing animal faeces, urine or blood. To investigate possible biological exposures in animal slaughterhouses, we used a metagenomic approach to characterise the profile of organisms present within an aerosol sample. An assessment of aerosol exposures for individual workers was achieved by the collection of personal samples that represent the inhalable fraction of dust/bioaerosol in workplace air in both cattle and sheep slaughterhouses. Two sets of nine personal aerosol samples were pooled for the cattle processing and sheep processing areas respectively, with a total of 332,677,346 sequence reads and 250,144,492 sequence reads of 85 bp in length produced for each. Eukaryotic genome sequence was found in both sampling locations, and bovine, ovine and human sequences were common. Sequences from WU polyomavirus and human papillomavirus 120 were detected in the metagenomic dataset from the cattle processing area, and these sequences were confirmed as being present in the original personal aerosol samples. This study presents the first metagenomic description of personal aerosol exposure and this methodology could be applied to a variety of environments. Also, the detection of two candidate viruses warrants further investigation in the setting of occupational exposures in animal slaughterhouses.

Tuning the performance of a natural treatment process using metagenomics for improved trace organic chemical attenuation

KAUST Repository

Drewes, Jorg

2014-02-01

By utilizing high-throughput sequencing and metagenomics, this study revealed how the microbial community characteristics including composition, diversity, as well as functional genes in managed aquifer recharge (MAR) systems can be tuned to enhance removal of trace organic chemicals of emerging concern (CECs). Increasing the humic content of the primary substrate resulted in higher microbial diversity. Lower concentrations and a higher humic content of the primary substrate promoted the attenuation of biodegradable CECs in laboratory and field MAR systems. Metagenomic results indicated that the metabolic capabilities of xenobiotic biodegradation were significantly promoted for the microbiome under carbon-starving conditions. © IWA Publishing 2014.

From Libraries to ‘Libratories’

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Leo Waaijers

2006-04-01

Full Text Available While the eighties of the last century were a time of local automation for libraries and the nineties the decade in which libraries embraced the internet and the WWW, now is the age in which the big search engines and institutional repositories are gaining a firm footing. This heralds a new era in both the evolution of scholarly communication and its agencies themselves, i.e. the libraries. Until now libraries and publishers have developed a digital variant of existing processes and products, i.e. catalogues posted on the Web, scanned copies of articles, e-mail notification about acquisitions or expired lending periods, or traditional journals in a digital jacket. However, the new OAI repositories and services based upon them have given rise to entirely new processes and products, libraries transforming themselves into partners in setting up virtual learning environments, building an institution’s digital showcase, maintaining academics’ personal websites, designing refereed portals and – further into the future – taking part in organising virtual research environments or collaboratories. Libraries are set to metamorphose into ‘libratories’, an imaginary word to express their combined functions of library, repository and collaboratory. In such environments scholarly communication will be liberated from its current copyright bridle while its coverage will be both broader - including primary data, audiovisuals and dynamic models - and deeper, with cross-disciplinary analyses of methodologies and applications of instruments. Universities will make it compulsory to store in their institutional repositories the results of research conducted within their walls for purposes of academic reporting, review committees, and other modes of clarification and explanation. Big search engines will provide access to this profusion of information and organise its mass customisation.

Libraries for users services in academic libraries

CERN Document Server

Alvite, Luisa

2010-01-01

This book reviews the quality and evolution of academic library services. It revises service trends offered by academic libraries and the challenge of enhancing traditional ones such as: catalogues, repositories and digital collections, learning resources centres, virtual reference services, information literacy and 2.0 tools.studies the role of the university library in the new educational environment of higher educationrethinks libraries in academic contextredefines roles for academic libraries

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

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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

Construction of cDNA library and preliminary analysis of expressed sequence tags from Siberian tiger

Science.gov (United States)

Liu, Chang-Qing; Lu, Tao-Feng; Feng, Bao-Gang; Liu, Dan; Guan, Wei-Jun; Ma, Yue-Hui

2010-01-01

In this study we successfully constructed a full-length cDNA library from Siberian tiger, Panthera tigris altaica, the most well-known wild Animal. Total RNA was extracted from cultured Siberian tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.30×106 pfu/ml and 1.62×109 pfu/ml respectively. The proportion of recombinants from unamplified library was 90.5% and average length of exogenous inserts was 1.13 kb. A total of 282 individual ESTs with sizes ranging from 328 to 1,142bps were then analyzed the BLASTX score revealed that 53.9% of the sequences were classified as strong match, 38.6% as nominal and 7.4% as weak match. 28.0% of them were found to be related to enzyme/catalytic protein, 20.9% ESTs to metabolism, 13.1% ESTs to transport, 12.1% ESTs to signal transducer/cell communication, 9.9% ESTs to structure protein, 3.9% ESTs to immunity protein/defense metabolism, 3.2% ESTs to cell cycle, and 8.9 ESTs classified as novel genes. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genomic research of Siberian tigers. PMID:20941376

Metagenomic and satellite analyses of red snow in the Russian Arctic

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Nao Hisakawa

2015-12-01

Full Text Available Cryophilic algae thrive in liquid water within snow and ice in alpine and polar regions worldwide. Blooms of these algae lower albedo (reflection of sunlight, thereby altering melting patterns (Kohshima, Seko & Yoshimura, 1993; Lutz et al., 2014; Thomas & Duval, 1995. Here metagenomic DNA analysis and satellite imaging were used to investigate red snow in Franz Josef Land in the Russian Arctic. Franz Josef Land red snow metagenomes confirmed that the communities are composed of the autotroph Chlamydomonas nivalis that is supporting a complex viral and heterotrophic bacterial community. Comparisons with white snow communities from other sites suggest that white snow and ice are initially colonized by fungal-dominated communities and then succeeded by the more complex C. nivalis-heterotroph red snow. Satellite image analysis showed that red snow covers up to 80% of the surface of snow and ice fields in Franz Josef Land and globally. Together these results show that C. nivalis supports a local food web that is on the rise as temperatures warm, with potential widespread impacts on alpine and polar environments worldwide.

Metagenomic analysis of microbial communities yields insight into impacts of nanoparticle design

Science.gov (United States)

Metch, Jacob W.; Burrows, Nathan D.; Murphy, Catherine J.; Pruden, Amy; Vikesland, Peter J.

2018-01-01

Next-generation DNA sequencing and metagenomic analysis provide powerful tools for the environmentally friendly design of nanoparticles. Herein we demonstrate this approach using a model community of environmental microbes (that is, wastewater-activated sludge) dosed with gold nanoparticles of varying surface coatings and morphologies. Metagenomic analysis was highly sensitive in detecting the microbial community response to gold nanospheres and nanorods with either cetyltrimethylammonium bromide or polyacrylic acid surface coatings. We observed that the gold-nanoparticle morphology imposes a stronger force in shaping the microbial community structure than does the surface coating. Trends were consistent in terms of the compositions of both taxonomic and functional genes, which include antibiotic resistance genes, metal resistance genes and gene-transfer elements associated with cell stress that are relevant to public health. Given that nanoparticle morphology remained constant, the potential influence of gold dissolution was minimal. Surface coating governed the nanoparticle partitioning between the bioparticulate and aqueous phases.

Characterization of Bacterial Hydrocarbon Degradation Potential in the Red Sea Through Metagenomic and Cultivation Methods

KAUST Repository

Bianchi, Patrick

2018-01-01

The focus of this thesis is on the characterization at the metagenomic level of the water column of the Red Sea and on the isolation and characterization of novel hydrocarbon-degrading species and genomes adapted to the unique environmental characteristics of the basin. The presence of metabolic genes responsible of both linear and aromatic hydrocarbon degradation has been evaluated from a metagenomic survey and a meta-analysis of already available datasets. In parallel, water column-based microcosms have been established with crude oil as the sole carbon source, with aim to isolate potential novel bacterial species and provide new genome-based insights on the hydrocarbon degradation potential available in the Red Sea.

SCHOOL COMMUNITY PERCEPTION OF LIBRARY APPS AGAINTS LIBRARY EMPOWERMENT

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Achmad Riyadi Alberto

2017-07-01

Full Text Available Abstract. This research is motivated by the development of information and communication technology (ICT in the library world so rapidly that allows libraries in the present to develop its services into digital-based services. This study aims to find out the school community’s perception of library apps developed by Riche Cynthia Johan, Hana Silvana, and Holin Sulistyo and its influence on library empowerment at the library of SD Laboratorium Percontohan UPI Bandung. Library apps in this research belong to the context of m-libraries, which is a library that meets the needs of its users by using mobile platforms such as smartphones,computers, and other mobile devices. Empowerment of library is the utilization of all aspects of the implementation of libraries to the best in order to achieve the expected goals. An analysis of the schoolcommunity’s perception of library apps using the Technology Acceptance Model (TAM includes: ease of use, usefulness, usability, usage trends, and real-use conditions. While the empowerment of the library includes aspects: information empowerment, empowerment of learning resources, empowerment of human resources, empowerment of library facilities, and library promotion. The research method used in this research is descriptive method with quantitative approach. Population and sample in this research is school community at SD Laboratorium Percontohan UPI Bandung. Determination of sample criteria by using disproportionate stratified random sampling with the number of samples of 83 respondents. Data analysis using simple linear regression to measure the influence of school community perception about library apps to library empowerment. The result of data analysis shows that there is influence between school community perception about library apps to library empowerment at library of SD Laboratorium Percontohan UPI Bandung which is proved by library acceptance level and library empowerment improvement.

A robust and accurate binning algorithm for metagenomic sequences with arbitrary species abundance ratio.

Science.gov (United States)

Leung, Henry C M; Yiu, S M; Yang, Bin; Peng, Yu; Wang, Yi; Liu, Zhihua; Chen, Jingchi; Qin, Junjie; Li, Ruiqiang; Chin, Francis Y L

2011-06-01

With the rapid development of next-generation sequencing techniques, metagenomics, also known as environmental genomics, has emerged as an exciting research area that enables us to analyze the microbial environment in which we live. An important step for metagenomic data analysis is the identification and taxonomic characterization of DNA fragments (reads or contigs) resulting from sequencing a sample of mixed species. This step is referred to as 'binning'. Binning algorithms that are based on sequence similarity and sequence composition markers rely heavily on the reference genomes of known microorganisms or phylogenetic markers. Due to the limited availability of reference genomes and the bias and low availability of markers, these algorithms may not be applicable in all cases. Unsupervised binning algorithms which can handle fragments from unknown species provide an alternative approach. However, existing unsupervised binning algorithms only work on datasets either with balanced species abundance ratios or rather different abundance ratios, but not both. In this article, we present MetaCluster 3.0, an integrated binning method based on the unsupervised top--down separation and bottom--up merging strategy, which can bin metagenomic fragments of species with very balanced abundance ratios (say 1:1) to very different abundance ratios (e.g. 1:24) with consistently higher accuracy than existing methods. MetaCluster 3.0 can be downloaded at http://i.cs.hku.hk/~alse/MetaCluster/.

A highly optimized grid deployment: the metagenomic analysis example.

Science.gov (United States)

Aparicio, Gabriel; Blanquer, Ignacio; Hernández, Vicente

2008-01-01

Computational resources and computationally expensive processes are two topics that are not growing at the same ratio. The availability of large amounts of computing resources in Grid infrastructures does not mean that efficiency is not an important issue. It is necessary to analyze the whole process to improve partitioning and submission schemas, especially in the most critical experiments. This is the case of metagenomic analysis, and this text shows the work done in order to optimize a Grid deployment, which has led to a reduction of the response time and the failure rates. Metagenomic studies aim at processing samples of multiple specimens to extract the genes and proteins that belong to the different species. In many cases, the sequencing of the DNA of many microorganisms is hindered by the impossibility of growing significant samples of isolated specimens. Many bacteria cannot survive alone, and require the interaction with other organisms. In such cases, the information of the DNA available belongs to different kinds of organisms. One important stage in Metagenomic analysis consists on the extraction of fragments followed by the comparison and analysis of their function stage. By the comparison to existing chains, whose function is well known, fragments can be classified. This process is computationally intensive and requires of several iterations of alignment and phylogeny classification steps. Source samples reach several millions of sequences, which could reach up to thousands of nucleotides each. These sequences are compared to a selected part of the "Non-redundant" database which only implies the information from eukaryotic species. From this first analysis, a refining process is performed and alignment analysis is restarted from the results. This process implies several CPU years. The article describes and analyzes the difficulties to fragment, automate and check the above operations in current Grid production environments. This environment has been

Rock Music's Place in the Library.

Science.gov (United States)

Politis, John

1983-01-01

Discussion of the importance of rock music as an expression of aural culture includes its history, rock music today, and the development of a rock music collection in the library (placement of collection and books which aid in developing a collection of permanent value). Three references are included. (EJS)