Modélisation du processus d'annotation par une architecture blackboard

Devant la multiplication des projets de séquençage de génomes complets, il est aujourd hui essentiel de disposer d outils informatiques performants capables d'aider l utilisateur biologiste vers un meilleur usage des méthodes d analyses. Ceci s avère particulièrement important en raison des très nombreuses méthodes d analyse disponibles ainsi que des grandes quantités de données concernées. Ce travail vise à apporter une contribution à cette problématique, par l élaboration d un modèle bio-informatique adapté à l annotation de génomes complets. Avec l étude de l état de l art, nous avons dégagé l existence de deux approches différentes pour modéliser le processus d'annotation. Cette constatation nous a permis, dans un premier temps, de re-formaliser la démarche d annotation de séquences génomiques. Puis, dans un second temps, de proposer une nouvelle architecture logicielle adéquate afin de traiter la question ainsi reformulée : l architecture blackboard.Because of the huge increase in sequencing projects of complete genomes, it is essential today to have powerful computer softwares able to help biologists towards a better use of analysis methods. This is particularly important because there exists many methods of analysis as well as a great amount of data. In order to solve this issue, we propose in this thesis to develop of a new bioinformatic model suitable for the annotation of complete genomes. From studying the "state of the art", we concluded that we could approach the modelisation of the annotation process of two different ways. This enabled us to propose a new model based on this analysis. We then decided to re-formalize the annotation process; it gave us the opportunity to propose an adequate software architecture in order to handle the process thus reformulated. The thesis is then a double contribution : it is both a modelisation of the annotation process, and a technical proposal based on a blackboard architecture.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Complete Genome Sequences of Two Middelburg Viruses Isolated from Arthropods in the Central African Republic

International audienceABSTRACT Arboviral diseases are a major threat to human and animal health today. Analysis of whole-genome sequences of decades-old arboviral strains may bring new insights into the viral evolution that might have facilitated outbreaks. Here, we report the whole-genome sequences of two Middelburg viruses isolated several decades ago in the Central African Republic

Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal rearrangements

Microsatellites are short tandem repeats, ubiquitous in all eukaryotes and represent ∼2% of the human genome. Among them, trinucleotide repeats are responsible for more than two dozen neurological and developmental disorders. Targeting microsatellites with dedicated DNA endonucleases could become a viable option for patients affected with dramatic neurodegenerative disorders. Here, we used the Streptococcus pyogenes Cas9 to induce a double-strand break within the expanded CTG repeat involved in myotonic dystrophy type 1, integrated in a yeast chromosome. Repair of this double-strand break generated unexpected large chromosomal rearrangements around the repeat tract. These rearrangements depended on RAD52, DNL4 and SAE2, and both non-homologous end-joining and single-strand annealing pathways were involved. Resection and repair of the double-strand break (DSB) were totally abolished in a rad50Δ strain, whereas they were impaired in a sae2Δ mutant, only on the DSB end containing most of the repeat tract. This proved that Sae2 plays significant different roles in resecting a DSB end containing a repeated and structured sequence as compared to a non-repeated DSB end.In addition, we also discovered that gene conversion was less efficient when the DSB could be repaired using a homologous template, suggesting that the trinucleotide repeat may interfer with gene conversion too. Altogether, these data show that SpCas9 is probably not a good choice when inducing a double-strand break at or near a microsatellite, especially in mammalian genomes that contain many more dispersed repeated elements than the yeast genome

Continuous Circulation of Yellow Fever among Rural Populations in the Central African Republic

International audienceYellow fever remains a public-health threat in remote regions of Africa. Here, we report the identification and genetic characterisation of one yellow-fever case observed during the investigation of a cluster of nine suspected haemorrhagic fever cases in a village in the Central African Republic. Samples were tested using real-time RT-PCR targeting the main African haemorrhagic fever viruses. Following negative results, we attempted virus isolation on VERO E6 cells and newborn mice and rescreened the samples using rRT-PCR. The whole viral genome was sequenced using an Illumina NovaSeq 6000 sequencer. Yellow-fever virus (YFV) was isolated from one woman who reported farming activities in a forest setting several days before disease onset. Phylogenetic analysis shows that this strain belongs to the East-Central African YFV genotype, with an estimated emergence some 63 years ago. Finally, five unique amino-acid changes are present in the capsid, envelop, NS1A, NS3, and NS4B proteins. More efforts are required to control yellow-fever re-emergence in resource-limited settings

Characterizing meiotic chromosomes' structure and pairing using a designer sequence optimized for Hi‐C

International audienceIn chromosome conformation capture experiments (Hi-C), the accuracy with which contacts are detected varies due to the uneven distribution of restriction sites along genomes. In addition, repeated sequences or homologous regions remain indistinguishable because of the ambiguities they introduce during the alignment of the sequencing reads. We addressed both limitations by designing and engineering 144 kb of a yeast chromosome with regularly spaced restriction sites (Syn-HiC design). In the Syn-HiC region, Hi-C signal-to-noise ratio is enhanced and can be used to measure the shape of an unbiased distribution of contact frequencies , allowing to propose a robust definition of a Hi-C experiment resolution. The redesigned region is also distinguishable from its native homologous counterpart in an otherwise isogenic diploid strain. As a proof of principle, we tracked homologous chromosomes during meiotic prophase in synchronized and pachytene-arrested cells and captured important features of their spatial reorganization, such as chromatin restructuration into arrays of Rec8-delimited loops, centromere declustering, individualization, and pairing. Overall, we illustrate the promises held by redesigning genomic regions to explore complex biological questions

Molecular epidemiology of merkel cell polyomavirus: evidence for geographically related variant genotypes.

International audienceMerkel cell polyomavirus (MCPyV) is linked to a cutaneous cancer mainly occurring in Caucasians. DNA from skin swabs of 255 adults, originating from the 5 continents, were subjected to MCPyV PCRs. Phylogenetic analyses demonstrate the existence of 5 major geographically related MCPyV genotypes (Europe/North America, Africa [sub-Saharan], Oceania, South America, and Asia/Japan)

The cnf1 gene is associated to an expanding Escherichia coli ST131 H30Rx/C2 sublineage and confers a competitive advantage for host colonization

Epidemiological projections point to acquisition of ever-expanding multidrug resistance (MDR) by Escherichia coli, a commensal of the digestive tract acting as a source of urinary tract pathogens. We performed a high-throughput genetic screening of predominantly clinical E. coli isolates from wide geographical origins. This revealed a preferential distribution of the Cytotoxic Necrotizing Factor 1 (CNF1)-toxin encoding gene, cnf1, in four sequence types encompassing the pandemic E. coli MDR lineage ST131. This lineage is responsible for a majority of extraintestinal infections that escape first-line antibiotic treatment and has known enhanced capacities to colonize the gastrointestinal tract (GIT). Statistical modeling uncovered a dominant global expansion of cnf1-positive strains within multidrug-resistant ST131 subclade H30Rx/C2. Despite the absence of phylogeographical signals, cnf1-positive isolates adopted a clonal distribution into clusters on the ST131-H30Rx/C2 phylogeny, sharing a similar profile of virulence factors and the same cnf1 allele. Functional analysis of the cnf1-positive clinical strain EC131GY ST131-H30Rx/C2, established that a cnf1-deleted EC131GY is outcompeted by the wildtype strain in a mouse model of competitive infection of the bladder while both strains behave similarly during monoinfections. This points for positive selection of cnf1 during UTI rather than urovirulence. Wildtype EC131GY also outcompeted the mutant when concurrently inoculated into the gastrointestinal tract, arguing for selection within the gut. Whatever the site of selection, these findings support that the benefit of cnf1 enhancing host colonization by ST131-H30Rx/C2 in turn drives a worldwide dissemination of the cnf1 gene together with extended spectrum of antibiotic resistance genes

The cnf1 gene is associated with an expanding Escherichia coli ST131 H 30Rx/C2 subclade and confers a competitive advantage for gut colonization

International audienceEpidemiological projections point to acquisition of ever-expanding multidrug resistance (MDR) by Escherichia coli, a commensal of the digestive tract and a source of urinary tract pathogens. Bioinformatics analyses of a large collection of E. coli genomes from EnteroBase, enriched in clinical isolates of worldwide origins, suggest the Cytotoxic Necrotizing Factor 1 (CNF1)-toxin encoding gene, cnf1, is preferentially distributed in four common sequence types (ST) encompassing the pandemic E. coli MDR lineage ST131. This lineage is responsible for a majority of extraintestinal infections that escape first-line antibiotic treatment, with known enhanced capacities to colonize the gastrointestinal tract. Statistical projections based on this dataset point to a global expansion of cnf1-positive multidrug-resistant ST131 strains from subclade H30Rx/C2, accounting for a rising prevalence of cnf1-positive strains in ST131. Despite the absence of phylogeographical signals, cnf1-positive isolates segregated into clusters in the ST131-H30Rx/C2 phylogeny, sharing a similar profile of virulence factors and the same cnf1 allele. The suggested dominant expansion of cnf1-positive strains in ST131-H30Rx/C2 led us to uncover the competitive advantage conferred by cnf1 for gut colonization to the clinical strain EC131GY ST131-H30Rx/C2 versus cnf1-deleted isogenic strain. Complementation experiments showed that colon tissue invasion was compromised in the absence of deamidase activity on Rho GTPases by CNF1. Hence, gut colonization factor function of cnf1 was confirmed for another clinical strain ST131-H30Rx/C2. In addition, functional analysis of the cnf1-positive clinical strain EC131GY ST131-H30Rx/C2 and a cnf1-deleted isogenic strain showed no detectable impact of the CNF1 gene on bacterial fitness and inflammation during the acute phase of bladder monoinfection. Together these data argue for an absence of role of CNF1 in virulence during UTI, while enhancing gut colonization capacities of ST131-H30Rx/C2 and suggested expansion of cnf1-positive MDR isolates in subclade ST131-H30Rx/C2