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Riley R.,University of Ottawa | Charron P.,University of Ottawa | Idnurm A.,University of Missouri - Kansas City | Farinelli L.,FASTERIS S.A. | And 3 more authors.
New Phytologist | Year: 2014

Summary: Arbuscular mycorrhizal fungi (AMF) are important plant symbionts that have long been considered evolutionary anomalies because of their apparent long-term lack of sexuality, but recent explorations of available DNA sequence have challenged this notion by revealing the presence of homologues of fungal mating type-high-mobility group (MATA-HMG) and core meiotic genes in these organisms. To obtain more insights into the sexual potential of AMF, homologues of MATA-HMGs were sought in the transcriptome of three AMF isolates, and their functional and evolutionary trajectories were studied in genetically divergent strains of Rhizophagus irregularis using conventional and quantitative PCR procedures. Our analyses revealed the presence of at least 76 homologues of MATA-HMGs in R. irregularis isolates. None of these was found to be surrounded by genes generally found near other known fungal mating type loci, but here we report the presence of a 9-kb-long region in the AMF R. irregularis harbouring a total of four tandem-repeated MATA-HMGs; a feature that highlights a potentially elevated intragenomic diversity in this AMF species. The present study provides intriguing insights into the genome evolution of R. irregularis, and represents a stepping stone for understanding the potential of these fungi to undergo cryptic sex. See also the Commentary by Lee et al. © 2013 New Phytologist Trust.

Blevins T.,University of Basel | Blevins T.,Friedrich Miescher Institute for Biomedical Research | Rajeswaran R.,University of Basel | Aregger M.,University of Basel | And 7 more authors.
Nucleic Acids Research | Year: 2011

To successfully infect plants, viruses must counteract small RNA-based host defense responses. During infection of Arabidopsis, Cauliflower mosaic pararetrovirus (CaMV) is transcribed into pregenomic 35S and subgenomic 19S RNAs. The 35S RNA is both reverse transcribed and also used as an mRNA with highly structured 600nt leader. We found that this leader region is transcribed into long sense-and antisense-RNAs and spawns a massive quantity of 21, 22 and 24nt viral small RNAs (vsRNAs), comparable to the entire complement of host-encoded small-interfering RNAs and microRNAs. Leader-derived vsRNAs were detected bound to the Argonaute 1 (AGO1) effector protein, unlike vsRNAs from other viral regions. Only negligible amounts of leader-derived vsRNAs were bound to AGO4. Genetic evidence showed that all four Dicer-like (DCL) proteins mediate vsRNA biogenesis, whereas the RNA polymerases Pol IV, Pol V, RDR1, RDR2 and RDR6 are not required for this process. Surprisingly, CaMV titers were not increased in dcl1/2/3/4 quadruple mutants that accumulate only residual amounts of vsRNAs. Ectopic expression of CaMV leader vsRNAs from an attenuated geminivirus led to increased accumulation of this chimeric virus. Thus, massive production of leader-derived vsRNAs does not restrict viral replication but may serve as a decoy diverting the silencing machinery from viral promoter and coding regions. © 2011 The Author(s).

Agency: European Commission | Branch: FP7 | Program: CP-FP | Phase: HEALTH.2011.1.1-1 | Award Amount: 8.71M | Year: 2012

We propose a technology that will sit at the front-end of sequencing pipelines, present and future, and will significantly enhance the quality and throughput of DNA sequencing. Although much attention has been given to throughput/cost of the sequencing process itself, the same cannot be said for the preparation of samples. Identified bottlenecks are (1) sequencing technologies require days of upfront sample preparation which is further increased when sequencing selected parts of the genome; (2) genome assembly relies on computationally intensive comparisons to the reference genome because existing technologies produce short sequence reads; (3) it is difficult to begin with small amounts of sample material comprising micro-biopsies and single cells. The CELL-O-MATIC project will synergize efforts from SMEs, academics and large companies to address these bottlenecks by developing chip-based systems that process DNA from individual cells, ready for next generation high-throughput sequencing. Single cell analysis has numerous applications in systems biology but we will emphasize DNA isolation and sequencing from circulating tumor cells (CTC), which have a strong prognostic value in cancer management. A second innovation will be to develop methods that enable up to whole chromosome lengths of DNA to be contiguously mapped using nanofluidics. The inclusion of nanofluidics makes the project particularly distinctive and introduces European SMEs to an area that so far has been the domain of US companies. A modular prototype comprising, a chip, fluid and thermal control, sonication and optical detection will be developed. Samples prepared using CELL-O-MATIC technology will be benchmarked in a high throughput environment with samples prepared by existing methods. Finally, the information obtained from the CELL-O-MATIC processed sample material will be validated for its utility as an aid to clinical decision making.

Agency: European Commission | Branch: FP7 | Program: CP-FP | Phase: HEALTH.2012.1.2-1 | Award Amount: 7.62M | Year: 2012

Hematological diseases are highly heterogeneous malignancies in the matter of the molecular mechanisms related to their development and progression. A considerable heterogeneity can be further observed within the same hematological disease at the inter-individual level, being reflected by different clinical outcomes and responses to treatment in different patients. Nowadays, the advent of high-throughput next generation sequencing (NGS) technologies that are revolutionizing genomics and transcriptomics by providing a single base resolution tool for a unified deep analysis of diseases complexity allows a fast and cost-efficient fine-scale assessment of the genetic variability hidden within cohorts of patients affected by the same leukemia. That being so, by potentially highlighting inter-individual differences that may play a role in the differential success of diverse therapeutic interventions, they promise to be crucial for selecting the most appropriate medical treatments. This project aims at developing a European Hematological/NGS platform of scientists for improving outcomes for therapeutic interventions on acute and chronic leukemias and at developing strategies to personalize treatments and tailor therapies to different stratified groups of leukemia patients, with the goal of optimizing their efficacy and safety through a deeper and deeper understanding of the influence of genetic alterations on leukemias pathogenesis and treatment response (i.e. personalized therapy). Moreover, the final aim will be the identification of novel prognostic biomarkers for acute and chronic leukemias, as well as of molecular biomarkers and/or genome-wide profiles for the assessment of minimal residual disease. The originality of this project is to perform systematic deep whole exome/transcriptome studies on well-clinically-characterized leukemia patients, by exploiting NGS technologies able to quickly produce data with a good cost-effectiveness and an unprecedented resolution.

Kaufmann K.,Wageningen University | Muino J.M.,Wageningen University | Osteras M.,Fasteris SA | Farinelli L.,Fasteris SA | And 3 more authors.
Nature Protocols | Year: 2010

Chromatin immunoprecipitation (ChIP) is a powerful technique to study interactions between transcription factors (TFs) and DNA in vivo. For genome-wide de novo discovery of TF-binding sites, the DNA that is obtained in ChIP experiments needs to be processed for sequence identification. The sequences can be identified by direct sequencing (ChIP-SEQ) or hybridization to microarrays (ChIP-CHIP). Given the small amounts of DNA that are usually obtained in ChIP experiments, successful and reproducible sample processing is challenging. Here we provide a detailed procedure for ChIP of plant TFs, as well as protocols for sample preparation for ChIP-SEQ and for ChIP-CHIP. Our ChIP procedure is optimized for high signal-to-noise ratio starting with tissue fixation, followed by nuclei isolation, immunoprecipitation, DNA amplification and purification. We also provide a guide for primary data analysis of ChIP-SEQ data. The complete protocol for ChIP-SEQ/ChIP-CHIP sample preparation starting from plant harvest takes 7 d. © 2010 Nature Publishing Group.

Ravn U.,NovImmune | Gueneau F.,NovImmune | Baerlocher L.,Fasteris SA | Osteras M.,Fasteris SA | And 6 more authors.
Nucleic Acids Research | Year: 2010

In recent years, unprecedented DNA sequencing capacity provided by next generation sequencing (NGS) has revolutionized genomic research. Combining the Illumina sequencing platform and a scFv library designed to confine diversity to both CDR3, >1.9×10 7 sequences have been generated. This approach allowed for in depth analysis of the library's diversity, provided sequence information on virtually all scFv during selection for binding to two targets and a global view of these enrichment processes. Using the most frequent heavy chain CDR3 sequences, primers were designed to rescue scFv from the third selection round. Identification, based on sequence frequency, retrieved the most potent scFv and valuable candidates that were missed using classical in vitro screening. Thus, by combining NGS with display technologies, laborious and time consuming upfront screening can be by-passed or complemented and valuable insights into the selection process can be obtained to improve library design and understanding of antibody repertoires. © 2010 The Author(s).

Wenner N.,University of Lausanne | Maes A.,University of Lausanne | Cotado-Sampayo M.,Fasteris SA | Lapouge K.,University of Lausanne
Environmental Microbiology | Year: 2014

Summary: The opportunistic pathogen Pseudomonas aeruginosaPAO1 has a remarkable capacity to adapt to various environments and to survive with limited nutrients. Here, we report the discovery and characterization of a novel small non-coding RNA: NrsZ (nitrogen-regulated sRNA). We show that under nitrogen limitation, NrsZ is induced by the NtrB/C two-component system, an important regulator of nitrogen assimilation and P.aeruginosa's swarming motility, in concert with the alternative sigma factor RpoN. Furthermore, we demonstrate that NrsZ modulates P.aeruginosa motility by controlling the production of rhamnolipid surfactants, virulence factors notably needed for swarming motility. This regulation takes place through the post-transcriptional control of rhlA, a gene essential for rhamnolipids synthesis. Interestingly, we also observed that NrsZ is processed in three similar short modules, and that the first short module encompassing the first 60 nucleotides is sufficient for NrsZ regulatory functions. © 2013 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

Genolet R.,University of Lausanne | Stevenson B.J.,University of Lausanne | Stevenson B.J.,Swiss Institute of Bioinformatics | Farinelli L.,Fasteris SA | And 2 more authors.
EMBO Journal | Year: 2012

Although the T-cell receptor αδ (TCRαδ) locus harbours large libraries of variable (TRAV) and junctional (TRAJ) gene segments, according to previous studies the TCRα chain repertoire is of limited diversity due to restrictions imposed by sequential coordinate TRAV-TRAJ recombinations. By sequencing tens of millions of TCRα chain transcripts from naive mouse CD8 + T cells, we observed a hugely diverse repertoire, comprising nearly all possible TRAV-TRAJ combinations. Our findings are not compatible with sequential coordinate gene recombination, but rather with a model in which contraction and DNA looping in the TCRαδ locus provide equal access to TRAV and TRAJ gene segments, similarly to that demonstrated for IgH gene recombination. Generation of the observed highly diverse TCRα chain repertoire necessitates deletion of failed attempts by thymic-positive selection and is essential for the formation of highly diverse TCRαβ repertoires, capable of providing good protective immunity. © 2012 European Molecular Biology Organization | All Rights Reserved.

Pelin A.,University of Ottawa | Selman M.,University of Ottawa | Aris-Brosou S.,University of Ottawa | Farinelli L.,FASTERIS S.A. | Corradi N.,University of Ottawa
Environmental Microbiology | Year: 2015

Nosema ceranae is a microsporidian pathogen whose infections have been associated with recent global declines in the populations of western honeybees (Apis mellifera). Despite the outstanding economic and ecological threat that N. ceranae may represent for honeybees worldwide, many aspects of its biology, including its mode of reproduction, propagation and ploidy, are either very unclear or unknown. In the present study, we set to gain knowledge in these biological aspects by re-sequencing the genome of eight isolates (i.e. a population of spores isolated from one single beehive) of this species harvested from eight geographically distant beehives, and by investigating their level of polymorphism. Consistent with previous analyses performed using single gene sequences, our analyses uncovered the presence of very high genetic diversity within each isolate, but also very little hive-specific polymorphism. Surprisingly, the nature, location and distribution of this genetic variation suggest that beehives around the globe are infected by a population of N. ceranae cells that may be polyploid (4n or more), and possibly clonal. Lastly, phylogenetic analyses based on genome-wide single-nucleotide polymorphism data extracted from these parasites and mitochondrial sequences from their hosts all failed to support the current geographical structure of our isolates. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

Selman M.,University of Ottawa | Sak B.,Academy of Sciences of the Czech Republic | Kvac M.,Academy of Sciences of the Czech Republic | Farinelli L.,Fasteris S.A | And 2 more authors.
Eukaryotic Cell | Year: 2013

The genomes of microsporidia in the genus Encephalitozoon have been extensively studied for their minimalistic features, but they have seldom been used to investigate basic characteristics of the biology of these organisms, such as their ploidy or their mode of reproduction. In the present study, we aimed to tackle this issue by mapping Illumina sequence reads against the genomes of four strains of E. cuniculi. This approach, combined with more conventional molecular biology techniques, resulted in the identification of heterozygosity in all strains investigated, a typical signature of a diploid nuclear state. In sharp contrast with similar studies recently performed on a distant microsporidian lineage (Nematocida spp.), the level of heterozygosity that we identified across the E. cuniculi genomes was found to be extremely low. This reductive intraindividual genetic variation could result from the long-term propagation of these strains under laboratory conditions, but we propose that it could also reflect an intrinsic capacity of these vertebrate pathogens to self-reproduce. © 2013, American Society for Microbiology. All Rights Reserved.

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