WebCARMA: a web application for the functional and taxonomic classification of unassembled metagenomic reads

Gerlach W, Jünemann S, Tille F, Goesmann A, Stoye J. WebCARMA: a web application for the functional and taxonomic classification of unassembled metagenomic reads. BMC Bioinformatics. 2009;10(1):430.Background Metagenomics is a new field of research on natural microbial communities. High-throughput sequencing techniques like 454 or Solexa-Illumina promise new possibilities as they are able to produce huge amounts of data in much shorter time and with less efforts and costs than the traditional Sanger technique. But the data produced comes in even shorter reads (35-100 basepairs with Illumina, 100-500 basepairs with 454-sequencing). CARMA is a new software pipeline for the characterisation of species composition and the genetic potential of microbial samples using short, unassembled reads. Results In this paper, we introduce WebCARMA, a refined version of CARMA available as a web application for the taxonomic and functional classification of unassembled (ultra-)short reads from metagenomic communities. In addition, we have analysed the applicability of ultra-short reads in metagenomics. Conclusions We show that unassembled reads as short as 35 bp can be used for the taxonomic classification of a metagenome. The web application is freely available at http://webcarma.cebitec.uni-bielefeld.d

The Sorcerer II Global Ocean Sampling Expedition: Metagenomic Characterization of Viruses within Aquatic Microbial Samples

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

Evaluating the Fidelity of De Novo Short Read Metagenomic Assembly Using Simulated Data

A frequent step in metagenomic data analysis comprises the assembly of the sequenced reads. Many assembly tools have been published in the last years targeting data coming from next-generation sequencing (NGS) technologies but these assemblers have not been designed for or tested in multi-genome scenarios that characterize metagenomic studies. Here we provide a critical assessment of current de novo short reads assembly tools in multi-genome scenarios using complex simulated metagenomic data. With this approach we tested the fidelity of different assemblers in metagenomic studies demonstrating that even under the simplest compositions the number of chimeric contigs involving different species is noticeable. We further showed that the assembly process reduces the accuracy of the functional classification of the metagenomic data and that these errors can be overcome raising the coverage of the studied metagenome. The results presented here highlight the particular difficulties that de novo genome assemblers face in multi-genome scenarios demonstrating that these difficulties, that often compromise the functional classification of the analyzed data, can be overcome with a high sequencing effort

Germ Warfare in a Microbial Mat Community: CRISPRs Provide Insights into the Co-Evolution of Host and Viral Genomes

CRISPR arrays and associated cas genes are widespread in bacteria and archaea and confer acquired resistance to viruses. To examine viral immunity in the context of naturally evolving microbial populations we analyzed genomic data from two thermophilic Synechococcus isolates (Syn OS-A and Syn OS-B′) as well as a prokaryotic metagenome and viral metagenome derived from microbial mats in hotsprings at Yellowstone National Park. Two distinct CRISPR types, distinguished by the repeat sequence, are found in both the Syn OS-A and Syn OS-B′ genomes. The genome of Syn OS-A contains a third CRISPR type with a distinct repeat sequence, which is not found in Syn OS-B′, but appears to be shared with other microorganisms that inhabit the mat. The CRISPR repeats identified in the microbial metagenome are highly conserved, while the spacer sequences (hereafter referred to as “viritopes” to emphasize their critical role in viral immunity) were mostly unique and had no high identity matches when searched against GenBank. Searching the viritopes against the viral metagenome, however, yielded several matches with high similarity some of which were within a gene identified as a likely viral lysozyme/lysin protein. Analysis of viral metagenome sequences corresponding to this lysozyme/lysin protein revealed several mutations all of which translate into silent or conservative mutations which are unlikely to affect protein function, but may help the virus evade the host CRISPR resistance mechanism. These results demonstrate the varied challenges presented by a natural virus population, and support the notion that the CRISPR/viritope system must be able to adapt quickly to provide host immunity. The ability of metagenomics to track population-level variation in viritope sequences allows for a culture-independent method for evaluating the fast co-evolution of host and viral genomes and its consequence on the structuring of complex microbial communities

Population Dynamics and Diversity of Viruses, Bacteria and Phytoplankton in a Shallow Eutrophic Lake

We have studied the temporal variation in viral abundances and community assemblage in the eutrophic Lake Loosdrecht through epifluorescence microscopy and pulsed field gel electrophoresis (PFGE). The virioplankton community was a dynamic component of the aquatic community, with abundances ranging between 5.5 × 107 and 1.3 × 108 virus-like particles ml−1 and viral genome sizes ranging between 30 and 200 kb. Both viral abundances and community composition followed a distinct seasonal cycle, with high viral abundances observed during spring and summer. Due to the selective and parasitic nature of viral infection, it was expected that viral and host community dynamics would covary both in abundances and community composition. The temporal dynamics of the bacterial and cyanobacterial communities, as potential viral hosts, were studied in addition to a range of environmental parameters to relate these to viral community dynamics. Cyanobacterial and bacterial communities were studied applying epifluorescence microscopy, flow cytometry, and denaturing gradient gel electrophoresis (DGGE). Both bacterial and cyanobacterial communities followed a clear seasonal cycle. Contrary to expectations, viral abundances were neither correlated to abundances of the most dominant plankton groups in Lake Loosdrecht, the bacteria and the filamentous cyanobacteria, nor could we detect a correlation between the assemblage of viral and bacterial or cyanobacterial communities during the overall period. Only during short periods of strong fluctuations in microbial communities could we detect viral community assemblages to covary with cyanobacterial and bacterial communities. Methods with a higher specificity and resolution are probably needed to detect the more subtle virus–host interactions. Viral abundances did however relate to cyanobacterial community assemblage and showed a significant positive correlation to Chl-a as well as prochlorophytes, suggesting that a significant proportion of the viruses in Lake Loosdrecht may be phytoplankton and more specific cyanobacterial viruses. Temporal changes in bacterial abundances were significantly related to viral community assemblage, and vice versa, suggesting an interaction between viral and bacterial communities in Lake Loosdrecht

Effects of the social environment during adolescence on the development of social behaviour, hormones and morphology in male zebra finches (Taeniopygia guttata)

Abstract Background Individual differences in behaviour are widespread in the animal kingdom and often influenced by the size or composition of the social group during early development. In many vertebrates the effects of social interactions early in life on adult behaviour are mediated by changes in maturation and physiology. Specifically, increases in androgens and glucocorticoids in response to social stimulation seem to play a prominent role in shaping behaviour during development. In addition to the prenatal and early postnatal phase, adolescence has more recently been identified as an important period during which adult behaviour and physiology are shaped by the social environment, which so far has been studied mostly in mammals. We raised zebra finches ( Taeniopygia guttata ) under three environmental conditions differing in social complexity during adolescence\ua0-\ua0juvenile pairs, juvenile groups, and mixed-age groups - and studied males\u2019 behavioural, endocrine, and morphological maturation, and later their adult behaviour. Results As expected, group-housed males exhibited higher frequencies of social interactions. Group housing also enhanced song during adolescence, plumage development, and the frequency and intensity of adult courtship and aggression. Some traits, however, were affected more in juvenile groups and others in mixed-age groups. Furthermore, a testosterone peak during late adolescence was suppressed in groups with adults. In contrast, corticosterone concentrations did not differ between rearing environments. Unexpectedly, adult courtship in a test situation was lowest in pair-reared males and aggression depended upon the treatment of the opponent with highest rates shown by group-reared males towards pair-reared males. This contrasts with previous findings, possibly due to differences in photoperiod and the acoustic environment. Conclusion Our results support the idea that effects of the adolescent social environment on adult behaviour in vertebrates are mediated by changes in social interactions affecting behavioural and morphological maturation. We found no evidence that long-lasting differences in behaviour reflect testosterone or corticosterone levels during adolescence, although differences between juvenile and mixed-age groups suggest that testosterone and song behaviour during late adolescence may be associated

Bioinformatics for the human microbiome project

Microbes inhabit virtually all sites of the human body, yet we know very little about the role they play in our health. In recent years, there has been increasing interest in studying human-associated microbial communities, particularly since microbial dysbioses have now been implicated in a number of human diseases [1]–[3]. Dysbiosis, the disruption of the normal microbial community structure, however, is impossible to define without first establishing what “normal microbial community structure” means within the healthy human microbiome. Recent advances in sequencing technologies have made it feasible to perform large-scale studies of microbial communities, providing the tools necessary to begin to address this question [4], [5]. This led to the implementation of the Human Microbiome Project (HMP) in 2007, an initiative funded by the National Institutes of Health Roadmap for Biomedical Research and constructed as a large, genome-scale community research project [6]. Any such project must plan for data analysis, computational methods development, and the public availability of tools and data; here, we provide an overview of the corresponding bioinformatics organization, history, and results from the HMP (Figure 1).National Institutes of Health (U.S.) (NIH U54HG004969)National Institutes of Health (U.S.) (grant R01HG004885)National Institutes of Health (U.S.) (grant R01HG005975)National Institutes of Health (U.S.) (grant R01HG005969

Metagenomics - a guide from sampling to data analysis

Metagenomics applies a suite of genomic technologies and bioinformatics tools to directly access the genetic content of entire communities of organisms. The field of metagenomics has been responsible for substantial advances in microbial ecology, evolution, and diversity over the past 5 to 10 years, and many research laboratories are actively engaged in it now. With the growing numbers of activities also comes a plethora of methodological knowledge and expertise that should guide future developments in the field. This review summarizes the current opinions in metagenomics, and provides practical guidance and advice on sample processing, sequencing technology, assembly, binning, annotation, experimental design, statistical analysis, data storage, and data sharing. As more metagenomic datasets are generated, the availability of standardized procedures and shared data storage and analysis becomes increasingly important to ensure that output of individual projects can be assessed and compared

Planktonic Microbes in the Gulf of Maine Area

In the Gulf of Maine area (GoMA), as elsewhere in the ocean, the organisms of greatest numerical abundance are microbes. Viruses in GoMA are largely cyanophages and bacteriophages, including podoviruses which lack tails. There is also evidence of Mimivirus and Chlorovirus in the metagenome. Bacteria in GoMA comprise the dominant SAR11 phylotype cluster, and other abundant phylotypes such as SAR86-like cluster, SAR116-like cluster, Roseobacter, Rhodospirillaceae, Acidomicrobidae, Flavobacteriales, Cytophaga, and unclassified Alphaproteobacteria and Gammaproteobacteria clusters. Bacterial epibionts of the dinoflagellate Alexandrium fundyense include Rhodobacteraceae, Flavobacteriaceae, Cytophaga spp., Sulfitobacter spp., Sphingomonas spp., and unclassified Bacteroidetes. Phototrophic prokaryotes in GoMA include cyanobacteria that contain chlorophyll (mainly Synechococcus), aerobic anoxygenic phototrophs that contain bacteriochlorophyll, and bacteria that contain proteorhodopsin. Eukaryotic microalgae in GoMA include Bacillariophyceae, Dinophyceae, Prymnesiophyceae, Prasinophyceae, Trebouxiophyceae, Cryptophyceae, Dictyochophyceae, Chrysophyceae, Eustigmatophyceae, Pelagophyceae, Synurophyceae, and Xanthophyceae. There are no records of Bolidophyceae, Aurearenophyceae, Raphidophyceae, and Synchromophyceae in GoMA. In total, there are records for 665 names and 229 genera of microalgae. Heterotrophic eukaryotic protists in GoMA include Dinophyceae, Alveolata, Apicomplexa, amoeboid organisms, Labrynthulida, and heterotrophic marine stramenopiles (MAST). Ciliates include Strombidium, Lohmaniella, Tontonia, Strobilidium, Strombidinopsis and the mixotrophs Laboea strobila and Myrionecta rubrum (ex Mesodinium rubra). An inventory of selected microbial groups in each of 14 physiographic regions in GoMA is made by combining information on the depth-dependent variation of cell density and the depth-dependent variation of water volume. Across the entire GoMA, an estimate for the minimum abundance of cell-based microbes is 1.7×1025 organisms. By one account, this number of microbes implies a richness of 105 to 106 taxa in the entire water volume of GoMA. Morphological diversity in microplankton is well-described but the true extent of taxonomic diversity, especially in the femtoplankton, picoplankton and nanoplankton – whether autotrophic, heterotrophic, or mixotrophic, is unknown