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Malausa T.,French National Institute for Agricultural Research | Gilles A.,Aix - Marseille University | Meglecz E.,Aix - Marseille University | Blanquart H.,Campus Of Linstitut Pasteur | And 16 more authors.
Molecular Ecology Resources | Year: 2011

Microsatellites (or SSRs: simple sequence repeats) are among the most frequently used DNA markers in many areas of research. The use of microsatellite markers is limited by the difficulties involved in their de novo isolation from species for which no genomic resources are available. We describe here a high-throughput method for isolating microsatellite markers based on coupling multiplex microsatellite enrichment and next-generation sequencing on 454 GS-FLX Titanium platforms. The procedure was calibrated on a model species (Apis mellifera) and validated on 13 other species from various taxonomic groups (animals, plants and fungi), including taxa for which severe difficulties were previously encountered using traditional methods. We obtained from 11497 to 34483 sequences depending on the species and the number of detected microsatellite loci ranged from 199 to 5791. We thus demonstrated that this procedure can be readily and successfully applied to a large variety of taxonomic groups, at much lower cost than would have been possible with traditional protocols. This method is expected to speed up the acquisition of high-quality genetic markers for nonmodel organisms. © 2011 Blackwell Publishing Ltd.


Gilles A.,Aix - Marseille University | Meglecz E.,Aix - Marseille University | Pech N.,Aix - Marseille University | Ferreira S.,Campus Of Linstitut Pasteur | And 2 more authors.
BMC Genomics | Year: 2011

Background: The rapid evolution of 454 GS-FLX sequencing technology has not been accompanied by a reassessment of the quality and accuracy of the sequences obtained. Current strategies for decision-making and error-correction are based on an initial analysis by Huse et al. in 2007, for the older GS20 system based on experimental sequences. We analyze here the quality of 454 sequencing data and identify factors playing a role in sequencing error, through the use of an extensive dataset for Roche control DNA fragments.Results: We obtained a mean error rate for 454 sequences of 1.07%. More importantly, the error rate is not randomly distributed; it occasionally rose to more than 50% in certain positions, and its distribution was linked to several experimental variables. The main factors related to error are the presence of homopolymers, position in the sequence, size of the sequence and spatial localization in PT plates for insertion and deletion errors. These factors can be described by considering seven variables. No single variable can account for the error rate distribution, but most of the variation is explained by the combination of all seven variables.Conclusions: The pattern identified here calls for the use of internal controls and error-correcting base callers, to correct for errors, when available (e.g. when sequencing amplicons). For shotgun libraries, the use of both sequencing primers and deep coverage, combined with the use of random sequencing primer sites should partly compensate for even high error rates, although it may prove more difficult than previous thought to distinguish between low-frequency alleles and errors. © 2011 Gilles et al; licensee BioMed Central Ltd.


Martin J.-F.,Montpellier SupAgro | Pech N.,Aix - Marseille University | Meglecz E.,Aix - Marseille University | Ferreira S.,Campus Of Linstitut Pasteur | And 4 more authors.
BMC Genomics | Year: 2010

Background: Microsatellites are markers of choice in population genetics and genomics, as they provide useful insight into patterns and processes as diverse as genome evolutionary dynamics and demographic processes. The acquisition of microsatellites through multiplex-enriched libraries and 454 GS-FLX Titanium pyrosequencing is a promising new tool for the isolation of new markers in unknown genomes. This approach can also be used to evaluate the extent to which microsatellite-enriched libraries are representative of the genome from which they were isolated. In this study, we deciphered potential discrepancies in microsatellite content recovery for two reference genomes (Apis mellifera and Danio rerio), selected on the basis of their extreme heterogeneity in terms of the proportions and distributions of microsatellites on chromosomes.Results: The A. mellifera genome, in particular, was found to be highly heterogeneous, due to extremely high rates of recombination, with hotspots, but the only bias consistently introduced into pyrosequenced multiplex-enriched libraries concerned sequence length, with the overrepresentation of sequences 160 to 320 bp in length. Other deviations from expected proportions or distributions of motifs on chromosomes were observed, but the significance and intensity of these deviations was mostly limited. Furthermore, no consistent adverse competition between multiplexed probes was observed during the motif enrichment phase.Conclusions: This approach therefore appears to be a promising strategy for improving the development of microsatellites, as it introduces no major bias in terms of the proportions and distribution of microsatellites. © 2010 Martin et al; licensee BioMed Central Ltd.


Papadimitriou K.,Agricultural University of Athens | Anastasiou R.,Agricultural University of Athens | Mavrogonatou E.,Institute of Biosciences and Applications | Blom J.,Bielefeld University | And 15 more authors.
BMC Genomics | Year: 2014

Background: Within the genus Streptococcus, only Streptococcus thermophilus is used as a starter culture in food fermentations. Streptococcus macedonicus though, which belongs to the Streptococcus bovis/Streptococcus equinus complex (SBSEC), is also frequently isolated from fermented foods mainly of dairy origin. Members of the SBSEC have been implicated in human endocarditis and colon cancer. Here we compare the genome sequence of the dairy isolate S. macedonicus ACA-DC 198 to the other SBSEC genomes in order to assess in silico its potential adaptation to milk and its pathogenicity status.Results: Despite the fact that the SBSEC species were found tightly related based on whole genome phylogeny of streptococci, two distinct patterns of evolution were identified among them. Streptococcus macedonicus, Streptococcus infantarius CJ18 and Streptococcus pasteurianus ATCC 43144 seem to have undergone reductive evolution resulting in significantly diminished genome sizes and increased percentages of potential pseudogenes when compared to Streptococcus gallolyticus subsp. gallolyticus. In addition, the three species seem to have lost genes for catabolizing complex plant carbohydrates and for detoxifying toxic substances previously linked to the ability of S. gallolyticus to survive in the rumen. Analysis of the S. macedonicus genome revealed features that could support adaptation to milk, including an extra gene cluster for lactose and galactose metabolism, a proteolytic system for casein hydrolysis, auxotrophy for several vitamins, an increased ability to resist bacteriophages and horizontal gene transfer events with the dairy Lactococcus lactis and S. thermophilus as potential donors. In addition, S. macedonicus lacks several pathogenicity-related genes found in S. gallolyticus. For example, S. macedonicus has retained only one (i.e. the pil3) of the three pilus gene clusters which may mediate the binding of S. gallolyticus to the extracellular matrix. Unexpectedly, similar findings were obtained not only for the dairy S. infantarius CJ18, but also for the blood isolate S. pasteurianus ATCC 43144.Conclusions: Our whole genome analyses suggest traits of adaptation of S. macedonicus to the nutrient-rich dairy environment. During this process the bacterium gained genes presumably important for this new ecological niche. Finally, S. macedonicus carries a reduced number of putative SBSEC virulence factors, which suggests a diminished pathogenic potential. © 2014 Papadimitriou et al.; licensee BioMed Central Ltd.


Papadimitriou K.,Agricultural University of Athens | Anastasiou R.,Agricultural University of Athens | Maistrou E.,Agricultural University of Athens | Plakas T.,Agricultural University of Athens | And 16 more authors.
PLoS ONE | Year: 2015

Background: Streptococcus macedonicus is an intriguing streptococcal species whose most frequent source of isolation is fermented foods similarly to Streptococcus thermophilus. However, S. macedonicus is closely related to commensal opportunistic pathogens of the Streptococcus bovis/Streptococcus equinus complex. Methodology/Principal Findings: We analyzed the pSMA198 plasmid isolated from the dairy strain Streptococcus macedonicus ACA-DC 198 in order to provide novel clues about the main ecological niche of this bacterium. pSMA198 belongs to the narrow host range pCI305/pWV02 family found primarily in lactococci and to the best of our knowledge it is the first such plasmid to be reported in streptococci. Comparative analysis of the pSMA198 sequence revealed a high degree of similarity with plasmids isolated from Lactococcus lactis strains deriving from milk or its products. Phylogenetic analysis of the pSMA198 Rep showed that the vast majority of closely related proteins derive from lactococcal dairy isolates. Additionally, cloning of the pSMA198 ori in L. lactis revealed a 100% stability of replication over 100 generations. Both pSMA198 and the chromosome of S. macedonicus exhibit a high percentage of potential pseudogenes, indicating that they have co-evolved under the same gene decay processes. We identified chromosomal regions in S. macedonicus that may have originated from pSMA198, also supporting a long co-existence of the two replicons. pSMA198 was also found in divergent biotypes of S. macedonicus and in strains isolated from dispersed geographic locations (e.g. Greece and Switzerland) showing that pSMA198's acquisition is not a recent event. Conclusions/Significance: Here we propose that S. macedonicus acquired plasmid pSMA198 from L. lactis via an ancestral genetic exchange event that took place most probably in milk or dairy products. We provide important evidence that point towards the dairy origin of this species. © 2015 Papadimitriou et al.


Gauzere C.,French Scientific and Technical Center for Building | Godon J.-J.,French National Institute for Agricultural Research | Blanquart H.,Campus Of Linstitut Pasteur | Ferreira S.,Campus Of Linstitut Pasteur | And 3 more authors.
Science of the Total Environment | Year: 2014

Although we spend the majority of our lives indoors, the airborne microbial content of enclosed spaces still remains inadequately described. The objective of this study was to characterize the bacterial diversity of indoor air in three different enclosed spaces with three levels of occupancy, and, in particular, to highlight the 'core' species, the opportunistic pathogens and their origins. Our findings provide an overall description of bacterial diversity in these indoor environments. Data gathered from the three enclosed spaces revealed the presence of a common indoor signature (60% of total sequences in common). This work will provide a clearer understanding of the dominant groups of bacteria encountered in enclosed spaces: Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes. Thus, certain evidence revealed a connection between 'core' species and the human micro-environment (20% of phylotypes and 12% of sequences of human origin). Overall PCA analysis showed that the indoor environment is influenced mainly by the microbial diversity from nose and skin. Among the 'core species' found during this study, a large number (72% of all pathogen-related sequences were concentrated in 'core species') of genera and species are known to be responsible for opportunistic or nosocomial diseases or to include human commensal bacteria such as Mycobacterium sp., Acinetobacter baumanii, Aerococcus viridians, Thermoactinomyces vulgaris or Clostridium perfringens. © 2014 Elsevier B.V.


PubMed | Campus Of Linstitut Pasteur, French Scientific and Technical Center for Building and French National Institute for Agricultural Research
Type: | Journal: The Science of the total environment | Year: 2014

Although we spend the majority of our lives indoors, the airborne microbial content of enclosed spaces still remains inadequately described. The objective of this study was to characterize the bacterial diversity of indoor air in three different enclosed spaces with three levels of occupancy, and, in particular, to highlight the core species, the opportunistic pathogens and their origins. Our findings provide an overall description of bacterial diversity in these indoor environments. Data gathered from the three enclosed spaces revealed the presence of a common indoor signature (60% of total sequences in common). This work will provide a clearer understanding of the dominant groups of bacteria encountered in enclosed spaces: Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes. Thus, certain evidence revealed a connection between core species and the human micro-environment (20% of phylotypes and 12% of sequences of human origin). Overall PCA analysis showed that the indoor environment is influenced mainly by the microbial diversity from nose and skin. Among the core species found during this study, a large number (72% of all pathogen-related sequences were concentrated in core species) of genera and species are known to be responsible for opportunistic or nosocomial diseases or to include human commensal bacteria such as Mycobacterium sp., Acinetobacter baumanii, Aerococcus viridians, Thermoactinomyces vulgaris or Clostridium perfringens.


PubMed | Campus Of Linstitut Pasteur, University dAuvergne, French National Institute for Agricultural Research and Clermont University
Type: | Journal: Methods in molecular biology (Clifton, N.J.) | Year: 2015

Oligonucleotide microarrays have been widely used for gene detection and/or quantification of gene expression in various samples ranging from a single organism to a complex microbial assemblage. The success of a microarray experiment, however, strongly relies on the quality of designed probes. Consequently, probe design is of critical importance and therefore multiple parameters should be considered for each probe in order to ensure high specificity, sensitivity, and uniformity as well as potentially quantitative power. Moreover, to assess the complete gene repertoire of complex biological samples such as those studied in the field of microbial ecology, exploratory probe design strategies must be also implemented to target not-yet-described sequences. To design such probes, two algorithms, KASpOD and HiSpOD, have been developed and they are available via two user-friendly web services. Here, we describe the use of this software necessary for the design of highly effective probes especially in the context of microbial oligonucleotide microarrays by taking into account all the crucial parameters.

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