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1.  Metavir 2: new tools for viral metagenome comparison and assembled virome analysis 
BMC Bioinformatics  2014;15:76.
Background
Metagenomics, based on culture-independent sequencing, is a well-fitted approach to provide insights into the composition, structure and dynamics of environmental viral communities. Following recent advances in sequencing technologies, new challenges arise for existing bioinformatic tools dedicated to viral metagenome (i.e. virome) analysis as (i) the number of viromes is rapidly growing and (ii) large genomic fragments can now be obtained by assembling the huge amount of sequence data generated for each metagenome.
Results
To face these challenges, a new version of Metavir was developed. First, all Metavir tools have been adapted to support comparative analysis of viromes in order to improve the analysis of multiple datasets. In addition to the sequence comparison previously provided, viromes can now be compared through their k-mer frequencies, their taxonomic compositions, recruitment plots and phylogenetic trees containing sequences from different datasets. Second, a new section has been specifically designed to handle assembled viromes made of thousands of large genomic fragments (i.e. contigs). This section includes an annotation pipeline for uploaded viral contigs (gene prediction, similarity search against reference viral genomes and protein domains) and an extensive comparison between contigs and reference genomes. Contigs and their annotations can be explored on the website through specifically developed dynamic genomic maps and interactive networks.
Conclusions
The new features of Metavir 2 allow users to explore and analyze viromes composed of raw reads or assembled fragments through a set of adapted tools and a user-friendly interface.
doi:10.1186/1471-2105-15-76
PMCID: PMC4002922  PMID: 24646187
Virus; Phage; Metagenomics; Web server
2.  Assessment of viral community functional potential from viral metagenomes may be hampered by contamination with cellular sequences 
Open Biology  2013;3(12):130160.
Although the importance of viruses in natural ecosystems is widely acknowledged, the functional potential of viral communities is yet to be determined. Viral genomes are traditionally believed to carry only those genes that are directly pertinent to the viral life cycle, though this view was challenged by the discovery of metabolism genes in several phage genomes. Metagenomic approaches extended these analyses to a community scale, and several studies concluded that microbial and viral communities encompass similar functional potentials. However, these conclusions could originate from the presence of cellular DNA within viral metagenomes. We developed a computational method to estimate the proportion and origin of cellular sequences in a set of 67 published viromes. A quarter of the datasets were found to contain a substantial amount of sequences originating from cellular genomes. When considering only viromes with no cellular DNA detected, the functional potential of viral and microbial communities was found to be fundamentally different—a conclusion more consistent with the actual picture drawn from known viruses. Yet a significant number of cellular metabolism genes was still retrieved in these viromes, suggesting that the presence of auxiliary genes involved in various metabolic pathways within viral genomes is a general trend in the virosphere.
doi:10.1098/rsob.130160
PMCID: PMC3877843  PMID: 24335607
phages; viruses; metagenomics; functional potential
3.  Phylogenetic Affiliation of SSU rRNA Genes Generated by Massively Parallel Sequencing: New Insights into the Freshwater Protist Diversity 
PLoS ONE  2013;8(3):e58950.
Recent advances in next-generation sequencing (NGS) technologies spur progress in determining the microbial diversity in various ecosystems by highlighting, for example, the rare biosphere. Currently, high-throughput pyrotag sequencing of PCR-amplified SSU rRNA gene regions is mainly used to characterize bacterial and archaeal communities, and rarely to characterize protist communities. In addition, although taxonomic assessment through phylogeny is considered as the most robust approach, similarity and probabilistic approaches remain the most commonly used for taxonomic affiliation. In a first part of this work, a tree-based method was compared with different approaches of taxonomic affiliation (BLAST and RDP) of 18S rRNA gene sequences and was shown to be the most accurate for near full-length sequences and for 400 bp amplicons, with the exception of amplicons covering the V5-V6 region. Secondly, the applicability of this method was tested by running a full scale test using an original pyrosequencing dataset of 18S rRNA genes of small lacustrine protists (0.2–5 µm) from eight freshwater ecosystems. Our results revealed that i) fewer than 5% of the operational taxonomic units (OTUs) identified through clustering and phylogenetic affiliation had been previously detected in lakes, based on comparison to sequence in public databases; ii) the sequencing depth provided by the NGS coupled with a phylogenetic approach allowed to shed light on clades of freshwater protists rarely or never detected with classical molecular ecology approaches; and iii) phylogenetic methods are more robust in describing the structuring of under-studied or highly divergent populations. More precisely, new putative clades belonging to Mamiellophyceae, Foraminifera, Dictyochophyceae and Euglenida were detected. Beyond the study of protists, these results illustrate that the tree-based approach for NGS based diversity characterization allows an in-depth description of microbial communities including taxonomic profiling, community structuring and the description of clades of any microorganisms (protists, Bacteria and Archaea).
doi:10.1371/journal.pone.0058950
PMCID: PMC3597552  PMID: 23516585
4.  Short-term responses of unicellular planktonic eukaryotes to increases in temperature and UVB radiation 
BMC Microbiology  2012;12:202.
Background
Small size eukaryotes play a fundamental role in the functioning of coastal ecosystems, however, the way in which these micro-organisms respond to combined effects of water temperature, UVB radiations (UVBR) and nutrient availability is still poorly investigated.
Results
We coupled molecular tools (18S rRNA gene sequencing and fingerprinting) with microscope-based identification and counting to experimentally investigate the short-term responses of small eukaryotes (<6 μm; from a coastal Mediterranean lagoon) to a warming treatment (+3°C) and UVB radiation increases (+20%) at two different nutrient levels. Interestingly, the increase in temperature resulted in higher pigmented eukaryotes abundances and in community structure changes clearly illustrated by molecular analyses. For most of the phylogenetic groups, some rearrangements occurred at the OTUs level even when their relative proportion (microscope counting) did not change significantly. Temperature explained almost 20% of the total variance of the small eukaryote community structure (while UVB explained only 8.4%). However, complex cumulative effects were detected. Some antagonistic or non additive effects were detected between temperature and nutrients, especially for Dinophyceae and Cryptophyceae.
Conclusions
This multifactorial experiment highlights the potential impacts, over short time scales, of changing environmental factors on the structure of various functional groups like small primary producers, parasites and saprotrophs which, in response, can modify energy flow in the planktonic food webs.
doi:10.1186/1471-2180-12-202
PMCID: PMC3478981  PMID: 22966751
Small eukaryotes; Molecular diversity; Temperature; UVB radiation; Microcosms experiment; Mediterranean lagoon
5.  Evolution and Diversity of the Microviridae Viral Family through a Collection of 81 New Complete Genomes Assembled from Virome Reads 
PLoS ONE  2012;7(7):e40418.
Recent studies suggest that members of the Microviridae (a family of ssDNA bacteriophages) might play an important role in a broad spectrum of environments, as they were found in great number among the viral fraction from seawater and human gut samples. 24 completely sequenced Microviridae have been described so far, divided into three distinct groups named Microvirus, Gokushovirinae and Alpavirinae, this last group being only composed of prophages. In this study, we present the analysis of 81 new complete Microviridae genomes, assembled from viral metagenomes originating from various ecosystems. The phylogenetic analysis of the core genes highlights the existence of four groups, confirming the three sub-families described so far and exhibiting a new group, named Pichovirinae. The genomic organizations of these viruses are strikingly coherent with their phylogeny, the Pichovirinae being the only group of this family with a different organization of the three core genes. Analysis of the structure of the major capsid protein reveals the presence of mushroom-like insertions conserved within all the groups except for the microviruses. In addition, a peptidase gene was found in 10 Microviridae and its analysis indicates a horizontal gene transfer that occurred several times between these viruses and their bacterial hosts. This is the first report of such gene transfer in Microviridae. Finally, searches against viral metagenomes revealed the presence of highly similar sequences in a variety of biomes indicating that Microviridae probably have both an important role in these ecosystems and an ancient origin.
doi:10.1371/journal.pone.0040418
PMCID: PMC3394797  PMID: 22808158
6.  Quantitative PCR Enumeration of Total/Toxic Planktothrix rubescens and Total Cyanobacteria in Preserved DNA Isolated from Lake Sediments▿† 
Applied and Environmental Microbiology  2011;77(24):8744-8753.
The variability of spatial distribution and the determinism of cyanobacterial blooms, as well as their impact at the lake scale, are still not understood, partly due to the lack of long-term climatic and environmental monitoring data. The paucity of these data can be alleviated by the use of proxy data from high-resolution sampling of sediments. Coupling paleolimnological and molecular tools and using biomarkers such as preserved DNA are promising approaches, although they have not been performed often enough so far. In our study, a quantitative PCR (qPCR) technique was applied to enumerate total cyanobacterial and total and toxic Planktothrix communities in preserved DNA derived from sediments of three lakes located in the French Alps (Lake Geneva, Lake Bourget, and Lake Annecy), containing a wide range of cyanobacterial species. Preserved DNA from lake sediments was analyzed to assess its quality, quantity, and integrity, with further application for qPCR. We applied the qPCR assay to enumerate the total cyanobacterial community, and multiplex qPCR assays were applied to quantify total and microcystin-producing Planktothrix populations in a single reaction tube. These methods were optimized, calibrated, and applied to sediment samples, and the specificity and reproducibility of qPCR enumeration were tested. Accurate estimation of potential inhibition within sediment samples was performed to assess the sensitivity of such enumeration by qPCR. Some precautions needed for interpreting qPCR results in the context of paleolimnological approaches are discussed. We concluded that the qPCR assay can be used successfully for the analysis of lake sediments when DNA is well preserved in order to assess the presence and dominance of cyanobacterial and Planktothrix communities.
doi:10.1128/AEM.06106-11
PMCID: PMC3233095  PMID: 21984244
7.  Assessing the Diversity and Specificity of Two Freshwater Viral Communities through Metagenomics 
PLoS ONE  2012;7(3):e33641.
Transitions between saline and fresh waters have been shown to be infrequent for microorganisms. Based on host-specific interactions, the presence of specific clades among hosts suggests the existence of freshwater-specific viral clades. Yet, little is known about the composition and diversity of the temperate freshwater viral communities, and even if freshwater lakes and marine waters harbor distinct clades for particular viral sub-families, this distinction remains to be demonstrated on a community scale.
To help identify the characteristics and potential specificities of freshwater viral communities, such communities from two lakes differing by their ecological parameters were studied through metagenomics. Both the cluster richness and the species richness of the Lake Bourget virome were significantly higher that those of the Lake Pavin, highlighting a trend similar to the one observed for microorganisms (i.e. the specie richness observed in mesotrophic lakes is greater than the one observed in oligotrophic lakes). Using 29 previously published viromes, the cluster richness was shown to vary between different environment types and appeared significantly higher in marine ecosystems than in other biomes. Furthermore, significant genetic similarity between viral communities of related environments was highlighted as freshwater, marine and hypersaline environments were separated from each other despite the vast geographical distances between sample locations within each of these biomes. An automated phylogeny procedure was then applied to marker genes of the major families of single-stranded (Microviridae, Circoviridae, Nanoviridae) and double-stranded (Caudovirales) DNA viruses. These phylogenetic analyses all spotlighted a very broad diversity and previously unknown clades undetectable by PCR analysis, clades that gathered sequences from the two lakes. Thus, the two freshwater viromes appear closely related, despite the significant ecological differences between the two lakes. Furthermore, freshwater viral communities appear genetically distinct from other aquatic ecosystems, demonstrating the specificity of freshwater viruses at a community scale for the first time.
doi:10.1371/journal.pone.0033641
PMCID: PMC3303852  PMID: 22432038
8.  Community Structure and Dynamics of Small Eukaryotes Targeted by New Oligonucleotide Probes: New Insight into the Lacustrine Microbial Food Web▿  
Applied and Environmental Microbiology  2009;75(19):6373-6381.
The seasonal dynamics of the small eukaryotic fraction (cell diameter, 0.2 to 5 μm) was investigated in a mesotrophic lake by tyramide signal amplification-fluorescence in situ hybridization targeting seven different phylogenetic groups: Chlorophyceae, Chrysophyceae, Cryptophyceae, Cercozoa, LKM11, Perkinsozoa (two clades), and Fungi. The abundance of small eukaryotes ranged from 1,692 to 10,782 cells ml−1. The dominant groups were the Chrysophyceae and the Chlorophyceae, which represented 19.6% and 17.9% of small eukaryotes, respectively. The results also confirmed the quantitative importance of putative parasites, Fungi and Perkinsozoa, in the small heterotrophic eukaryotic assemblage. The relative abundances recorded for the Perkinsozoa group reached as much as 31.6% of total targeted eukaryotes during the summer. The dynamics of Perkinsozoa clade 1 coincided with abundance variations in Peridinium and Ceratium spp. (Dinoflagellates), while the dynamics of Perkinsozoa clade 2 was linked to the presence of Dinobryon spp. (Chrysophyceae). Fungi, represented by chytrids, reached maximal abundance in December (569 cells ml−1) and were mainly correlated with the dynamics of diatoms, especially Melosira varians. A further new finding of this study is the recurrent presence of Cercozoa (6.2%) and LKM11 (4.5%) cells. This quantitative approach based on newly designed probes offers a promising means of in-depth analysis of microbial food webs in lakes, especially by revealing the phylogenetic composition of the small heterotrophic flagellate assemblage, for which an important fraction of cells are generally unidentified by classical microscopy (on average, 96.8% of the small heterotrophic flagellates were identified by the specific probes we used in this study).
doi:10.1128/AEM.00607-09
PMCID: PMC2753085  PMID: 19666727
9.  Unexpected Importance of Potential Parasites in the Composition of the Freshwater Small-Eukaryote Community▿  
Applied and Environmental Microbiology  2008;74(10):2940-2949.
The diversity of small eukaryotes (0.2 to 5 μm) in a mesotrophic lake (Lake Bourget) was investigated using 18S rRNA gene library construction and fluorescent in situ hybridization coupled with tyramide signal amplification (TSA-FISH). Samples collected from the epilimnion on two dates were used to extend a data set previously obtained using similar approaches for lakes with a range of trophic types. A high level of diversity was recorded for this system with intermediate trophic status, and the main sequences from Lake Bourget were affiliated with ciliates (maximum, 19% of the operational taxonomic units [OTUs]), cryptophytes (33%), stramenopiles (13.2%), and cercozoa (9%). Although the comparison of TSA-FISH results and clone libraries suggested that the level of Chlorophyceae may have been underestimated using PCR with 18S rRNA primers, heterotrophic organisms dominated the small-eukaryote assemblage. We found that a large fraction of the sequences belonged to potential parasites of freshwater phytoplankton, including sequences affiliated with fungi and Perkinsozoa. On average, these sequences represented 30% of the OTUs (40% of the clones) obtained for each of two dates for Lake Bourget. Our results provide information on lacustrine small-eukaryote diversity and structure, adding to the phylogenetic data available for lakes with various trophic types.
doi:10.1128/AEM.01156-07
PMCID: PMC2394938  PMID: 18359836
10.  Succession and Regulation Factors of Small Eukaryote Community Composition in a Lacustrine Ecosystem (Lake Pavin) 
The structure and dynamics of small eukaryotes (cells with a diameter less than 5 μm) were studied over two consecutive years in an oligomesotrophic lake (Lake Pavin in France). Water samples were collected at 5 and 30 m below the surface; when the lake was stratified, these depths corresponded to the epilimnion and hypolimnion. Changes in small-eukaryote structure were analyzed using terminal restriction fragment length polymorphism (T-RFLP) and cloning and sequencing of the 18S rRNA genes. Terminal restriction fragments from clones were used to reveal the dominant taxa in T-RFLP profiles of the environmental samples. Spumella-like cells (Chrysophyceae) did not dominate the small eukaryote community identified by molecular techniques in lacustrine ecosystems. Small eukaryotes appeared to be dominated by heterotrophic cells, particularly Cercozoa, which represented nearly half of the identified phylotypes, followed by the Fungi-LKM11 group (25%), choanoflagellates (10.3%) and Chrysophyceae (8.9%). Bicosoecida, Cryptophyta, and ciliates represented less than 9% of the community studied. No seasonal reproducibility in temporal evolution of the small-eukaryote community was observed from 1 year to the next. The T-RFLP patterns were related to bottom-up (resources) and top-down (grazing) variables using canonical correspondence analysis. The results showed a strong top-down regulation of small eukaryotes by zooplankton, more exactly, by cladocerans at 5 m and copepods at 30 m. Among bottom-up factors, temperature had a significant effect at both depths. The concentrations of nitrogenous nutrients and total phosphorus also had an effect on small-eukaryote dynamics at 5 m, whereas bacterial abundance and dissolved oxygen played a more important structuring role in the deeper zone.
doi:10.1128/AEM.72.4.2971-2981.2006
PMCID: PMC1449018  PMID: 16598004
11.  Anaerobic Microbial Communities in Lake Pavin, a Unique Meromictic Lake in France†  
Applied and Environmental Microbiology  2005;71(11):7389-7400.
The Bacteria and Archaea from the meromictic Lake Pavin were analyzed in samples collected along a vertical profile in the anoxic monimolimnion and were compared to those in samples from the oxic mixolimnion. Nine targeted 16S rRNA oligonucleotide probes were used to assess the distribution of Bacteria and Archaea and to investigate the in situ occurrence of sulfate-reducing bacteria and methane-producing Archaea involved in the terminal steps of the anaerobic degradation of organic material. The diversity of the complex microbial communities was assessed from the 16S rRNA polymorphisms present in terminal restriction fragment (TRF) depth patterns. The densities of the microbial community increased in the anoxic layer, and Archaea detected with probe ARCH915 represented the largest microbial group in the water column, with a mean Archaea/Eubacteria ratio of 1.5. Terminal restriction fragment length polymorphism (TRFLP) analysis revealed an elevated archaeal and bacterial phylotype richness in anoxic bottom-water samples. The structure of the Archaea community remained rather homogeneous, while TRFLP patterns for the eubacterial community revealed a heterogeneous distribution of eubacterial TRFs.
doi:10.1128/AEM.71.11.7389-7400.2005
PMCID: PMC1287608  PMID: 16269781
12.  Genetic Diversity of Small Eukaryotes in Lakes Differing by Their Trophic Status 
Applied and Environmental Microbiology  2005;71(10):5935-5942.
Small eukaryotes, cells with a diameter of less than 5 μm, are fundamental components of lacustrine planktonic systems. In this study, small-eukaryote diversity was determined by sequencing cloned 18S rRNA genes in three libraries from lakes of differing trophic status in the Massif Central, France: the oligotrophic Lake Godivelle, the oligomesotrophic Lake Pavin, and the eutrophic Lake Aydat. This analysis shows that the least diversified library was in the eutrophic lake (12 operational taxonomic units [OTUs]) and the most diversified was in the oligomesotrophic lake (26 OTUs). Certain groups were present in at least two ecosystems, while the others were specific to one lake on the sampling date. Cryptophyta, Chrysophyceae, and the strictly heterotrophic eukaryotes, Ciliophora and fungi, were identified in the three libraries. Among the small eukaryotes found only in two lakes, Choanoflagellida and environmental sequences (LKM11) were not detected in the eutrophic system whereas Cercozoa were confined to the oligomesotrophic and eutrophic lakes. Three OTUs, linked to the Perkinsozoa, were detected only in the Aydat library, where they represented 60% of the clones of the library. Chlorophyta and Haptophyta lineages were represented by a single clone and were present only in Godivelle and Pavin, respectively. Of the 127 clones studied, classical pigmented organisms (autotrophs and mixotrophs) represented only a low proportion regardless of the library's origin. This study shows that the small-eukaryote community composition may differ as a function of trophic status; certain lineages could be detected only in a single ecosystem.
doi:10.1128/AEM.71.10.5935-5942.2005
PMCID: PMC1266003  PMID: 16204507

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