Background: Recently high-throughput sequencing (HTS) using next generation sequencing techniques became useful in digital gene expression profiling. Our study introduces analysis options for HTS data based on mapping to miRBase or counting and grouping of identical sequence reads. Those approaches allow a hypothesis free detection of miRNA differential expression. Methods: We compare our results to microarray and qPCR data from one set of RNA samples. We use Illumina platforms for microarray analysis and miRNA sequencing of 20 samples from benign follicular thyroid adenoma and malignant follicular thyroid carcinoma. Furthermore, we use three strategies for HTS data analysis to evaluate miRNA biomarkers for malignant versus benign follicular thyroid tumors. Results: High correlation of qPCR and HTS data was observed for the proposed analysis methods. However, qPCR is limited in the differential detection of miRNA isoforms. Moreover, we illustrate a much broader dynamic range of HTS compared to microarrays for small RNA studies. Finally, our data confirm hsa-miR-197-3p, hsa-miR-221-3p, hsa-miR-222-3p and both hsa-miR-144-3p and hsa-miR-144-5p as potential follicular thyroid cancer biomarkers. Conclusions: Compared to microarrays HTS provides a global profile of miRNA expression with higher specificity and in more detail. Summarizing of HTS reads as isoform groups (analysis pipeline B) or according to functional criteria (seed analysis pipeline C), which better correlates to results of qPCR are promising new options for HTS analysis. Finally, data opens future miRNA research perspectives for HTS and indicates that qPCR might be limited in validating HTS data in detail.

Advances in DNA sequencing revolutionized the field of genomics over the last 5 years. New sequencing instruments make it possible to rapidly generate large amounts of sequence data at substantially lower cost. These high-throughput sequencing technologies (e.g. Roche 454 FLX, Life Technology SOLiD, Dover Polonator, Helicos HeliScope and Illumina Genome Analyzer) make whole genome sequencing and resequencing, transcript sequencing as well as quantification of gene expression, DNA-protein interactions and DNA methylation feasible at an unanticipated scale. In the field of evolutionary genomics, high-throughput sequencing permitted studies of whole genomes from ancient specimens of different hominin groups. Further, it allowed large-scale population genetics studies of present-day humans as well as different types of sequence-based comparative genomics studies in primates. Such comparisons of humans with closely related apes and hominins are important not only to better understand human origins and the biological background of what sets humans apart from other organisms, but also for understanding the molecular basis for diseases and disorders, particularly those that affect uniquely human traits, such as speech disorders, autism or schizophrenia. However, while the cost and time required to create comparative data sets have been greatly reduced, the error profiles and limitations of the new platforms differ significantly from those of previous approaches. This requires a specific experimental design in order to circumvent these issues, or to handle them during data analysis. During the course of my PhD, I analyzed and improved current protocols and algorithms for next generation sequencing data, taking into account the specific characteristics of these new sequencing technologies. The presented approaches and algorithms were applied in different projects and are widely used within the department of Evolutionary Genetics at the Max Planck Institute of Evolutionary Anthropology. In this thesis, I will present selected analyses from the whole genome shotgun sequencing of two ancient hominins and the quantification of gene expression from short-sequence tags in five tissues from three primates.

Three investigations were conducted in order to investigate the effect of dietary probiotics on tilapia (Oreochromis niloticus) growth performance, intestinal morphology, intestinal microbiology and immunity. The first experiment demonstrated that Bacillus subtilis, Lactobacillus reuteri and Pediococcus acidilactici supplemented individually and as a mixed probiotic (in addition to Enterococcus faecium; AquaStar® Growout) were capable of modulating intestinal microbial populations as determined by culture dependent methods and DGGE. Furthermore, high-throughput sequencing reported that >99% of 16S rRNA reads in the mixed probiotic group belonged to the probiotic genera, predominantly assigned to Enterococcus (52.50%) and Bacillus (45.94%). Tilapia in the mixed probiotic group displayed significantly higher intraepithelial leucocyte (IEL) populations in the mid intestine when compared to the control and L. reuteri treatment. The mixed probiotic also improved microvilli density and had a higher absorptive surface area when compared to the control. In the second trial, after six weeks of supplementing tilapia diets with AquaStar® Growout at 3g kg-1, fish demonstrated significantly higher final weight, weight gain and SGR when compared to that of the control (void of probiotic) treatment or an initial probiotic feed (lasting two weeks) followed by control feeding. Probiotic supplementation at 3g kg-1 also caused an increase in the abundance of intestinal IELs and goblet cells and an up-regulation in the gene expression of intestinal caspase-3, PCNA and HSP70 and immunity genes TLR2, TNFα, IL-1β, TGFβ and IL-10 when compared with the expression of control replicates. These changes were not observed when supplementing tilapia diets with a lower dose (1.5g kg-1), nor when supplementing the probiotic in either a pulsed manner or as an initial feed (two weeks) followed by control feeding. Trial three revealed that the probiotic had a more discrete effect on the intestinal allochthonous microbiota as 16S rRNA reads assigned to probiotic genera only accounted for 5-10% of total reads. Nevertheless, the supplementation of dietary AquaStar® Growout at 3g kg-1 improved the localised immune response in tilapia, through the regulation of immunity genes TLR2, MYD88, NFκB, TNFα, IL-1β, TGFβ and IL-10, larger populations of goblet cells and a higher recruitment of IELs. Furthermore, the probiotic also improved the systemic immune response through the regulation of immunity genes (mentioned above) in the head kidney and significantly higher circulating leucocyte levels in whole blood. The extent of these changes were dependent on the probiotic treatment (i.e. continuously supplemented in feed or alternating weekly between probiotic at 3 g kg-1 and control feeding), the duration of feeding and the parameter investigated. This research demonstrates that B. subtilis, L. reuteri, P. acidilactici and AquaStar® Growout can modulate the intestinal microbiota. In addition, AquaStar® Growout can improve intestinal morphology, growth performance and modulate both the localised and systemic immune responses of tilapia when supplemented through the feed at the appropriate dosage and feeding regime.

Le facteur nutritionnel affecte de façon significative la sécrétion et la composition des constituants du lait qui conditionnent sa qualité nutritionnelle. Dans la glande mammaire, ces processus font intervenir de nombreux gènes dont l’expression est modulée par l’alimentation, cependant les mécanismes de régulation sous-jacents ne sont pas connus. Les microARNs (miARN) sont des petits ARN non codants qui se lient sur leurs ARNm cibles pour en réguler l’expression. Ils ouvrent donc des pistes d’investigation pour la compréhension de ces mécanismes. La première partie de mon travail de thèse a consisté à obtenir une meilleure connaissance des miARN exprimés dans la glande mammaire, notamment en dressant les miRNomes de référence par séquençage haut débit chez la souris, la vache et la chèvre. Ensuite, pour la première fois, l’impact de la nutrition sur l’expression des miARN mammaires a été étudié. Deux modèles ruminants, un modèle dit « extrême » et un modèle de supplémentation lipidique proche des conditions d’élevage, ont permis d’identifier 30 et 2 miARN, respectivement, dont l’expression est nutrirégulée. L’analyse in silico des cibles des miARN nutrirégulés a révélé un rôle potentiel de ceux-ci dans le métabolisme des lipides. Certaines des cibles sont effectivement différentiellement exprimées dans ces modèles, parmi celles-ci certains gènes sont essentiels pour la lactation tels que ESR1. Enfin, une étude pilote de la fonction de trois miARN nutrigulés a été initiée in vitro dans des cellules épithéliales mammaires bovines. Ces travaux permettent donc d’apporter des premiers éléments pour la compréhension de la régulation de l’expression des gènes en réponse à la nutrition et de l’impact des miARN sur la lactation. / Nutrition significantly affects the secretion and the composition of milk which determine its nutritional quality. In the mammary gland, regulation of these processes involves numerous genes which expression can be affected by nutrition. However, their regulations remain unclear. MicroRNAs (miRNA) are small non coding RNA which can bind mRNAs and regulate their expression of target genes. Consequently, they offer opportunities to understand the regulation of gene expression in response to nutrition. The first step of my PhD aimed to obtain a better knowledge of miRNA expressed in the mammary gland. Mammary miRNome were established from the lactating mouse, cow and goat using high-throughput sequencing. Later, the effect of nutrition on the expression of miRNA in the mammary gland was analyzed for the first time. Two models in ruminants, a food deprivation (“extreme” model) and a lipid supplementation (model similar to breeding conditions) highlighted 30 and 2 nutriregulated miRNA, respectively. The analysis of nutriregulated miRNA’s predicted targets, in silico, revealed their potential role in lipid metabolism. Some of those target genes have been previously identified as differently expressed in the same conditions and could thus be involved in the regulation of the expression of genes essential for the mammary gland function, such as ESR1. Finally, three nutriregulated miRNA were selected and used in a preliminary study of their functions in vitro in bovine mammary epithelial cells. These works bring first evidences in understanding the nutritional regulation of gene expression in the mammary gland as well as the role of miRNA in lactation.

The work in this thesis is concerned with the study of bacterial adaptation on short and long timescales. In the first section, consisting of three chapters, I describe a recently developed high-throughput technology for probing gene function, transposon-insertion sequencing, and its application to the study of functional differences between two important human pathogens, Salmonella enterica subspecies enterica serovars Typhi and Typhimurium. In a first study, I use transposon-insertion sequencing to probe differences in gene requirements during growth on rich laboratory media, revealing differences in serovar requirements for genes involved in iron-utilization and cell-surface structure biogenesis, as well as in requirements for non-coding RNA. In a second study I more directly probe the genomic features responsible for differences in serovar pathogenicity by analyzing transposon-insertion sequencing data produced following a two hour infection of human macrophage, revealing large differences in the selective pressures felt by these two closely related serovars in the same environment. The second section, consisting of two chapters, uses statistical models of sequence variation, i.e. covariance models, to examine the evolution of intrinsic termination across the bacterial kingdom. A first collaborative study provides background and motivation in the form of a method for identifying Rho-independent terminators using covariance models built from deep alignments of experimentally-verified terminators from Escherichia coli and Bacillus subtilis. In the course of the development of this method I discovered a novel putative intrinsic terminator in Mycobacterium tuberculosis. In the final chapter, I extend this approach to de novo discovery of intrinsic termination motifs across the bacterial phylogeny. I present evidence for lineage-specific variations in canonical Rho-independent terminator composition, as well as discover seven non-canonical putative termination motifs. Using a collection of publicly available RNA-seq datasets, I provide evidence for the function of some of these elements as bona fide transcriptional attenuators.

L'acquisition des données moléculaires a été bouleversée par le développement des techniques de séquençage haut-débit. Celles-ci ont augmenté la quantité des données et ont fait diminuer le coût de manière considérable. Ces nouvelles approches ont également redonné accès à des sources de matériel biologique qui étaient auparavant inutilisables en raison de la faible quantité et la forte dégradation de l'ADN qu'elles fournissent, notamment des tissus anciens, des échantillons de musée voire du matériel fossile. Un avantage supplémentaire c'est la possibilité de multiplexer les échantillons, c'est-à-dire, les mélanger et les séquencer simultanément grâce à l'utilisation de « tags » ou étiquettes permettant de les séparer après avec des outils bioinformatiques. Un marqueur qui a grandement bénéficié de ces technologies est le génome mitochondrial. En effet, nous montrons que grâce au ratio élevé entre l'ADN mitochondrial et l'ADN nucléaire par cellule, il est possible l'obtention de mitogénomes entiers, avec de couvertures adéquates, sans qu'un enrichissement préalable de l'échantillon soit nécessaire. Ceci permet d'envisager la réalisation de projets de mitogénomique comparative pour de groupes riches en espèces, requérant un échantillonnage taxonomique exhaustif et dont les divergences génétiques rendrait difficile l'usage du séquençage classique. C'est dans ce contexte que cette thèse aborde la systématique, la phylogénie et l'évolution moléculaires d'une famille de chauves-souris néotropicales : les Phyllostomidae. Cette famille est riche en espèces, avec plus de 160 taxons mais aussi en traits d'histoire de vie contrastés notamment le régime alimentaire. Cette diversité résulte en des morphologies convergentes dont les caractères sont en conséquence peu appropriés pour reconstruire l'histoire évolutive de ce groupe. La mitogénomique a prouvé être un outil efficace dans ce dessein, mais à présent aucune étude de ce type a été conduite pour cette famille. Nous avons d'abord réussi à séquencer les mitogénomes de représentants de toutes les lignées majeures couvrant également la diversité de traits d'histoire de vie. Nous montrons ensuite que l'utilisation de ces mitogénomes permet de résoudre les relations de parenté au niveau intrafamilial avec une résolution similaire à celle d'une concaténation de marqueurs mitochondriaux et nucléaires avec un soutien statistique robuste pour la plupart des nœuds de la phylogénie. Ceci nous a permis de clarifier plusieurs relations qui restaient controversées dans des études précédentes et confirmer plusieurs des clades proposés par celles-ci. Ensuite, nous abordons l'évolution du mitogénome en relation avec les traits d'histoire de vie en utilisant comme exemple le clade des vampires, les seules Mammifères hématophages, dont le génome mitochondrial semble avoir été touché par une accélération du taux d'évolution comme conséquence de l'action combinée de forces neutres et sélectives pour répondre aux contraintes imposées par ce régime alimentaire. Finalement, le cadre phylogénétique robuste proportionné par les 100 mitogénomes que nous avons séquencés pourra être utilisé comme référence pour étudier la diversification des Phyllostomidae. / New sequencing technologies have revolutionized the acquisition of molecular data by increasing the amount of sequences at a considerably lower cost. These new technologies have also given access to samples previously neglected because they resulted in low-quantity and degraded DNA yields, as for example, old tissues, museum specimens and even fossil rests. An additional advantage comes from the possibility of multiplexing; this is, mixing several taxa in a single sample thanks to the use of tags or labels allowing late separating the sequences using bioinformatic tools. A molecular marker that has greatly benefited from these technologies is the mitochondrial genome. Indeed, we show that, thanks to the high per-cell ratio of mitochondrial to nuclear DNA, it's possible to obtain whole well-covered mitochondrial genomes without previous sample enrichment. This allows the accomplishment of projects of comparative mitogenomics for species-rich groups needing exhaustive taxon sampling and for which strong genetic divergences would difficult the use of classical sequencing.It is in this context that this thesis tackles the molecular systematics, phylogenetics and evolution of a Neotropical family of bats: the Phyllostomidae. This species-rich family, accounting for more than 160 species, is also the family of Mammals with the highest diversity of life history traits, for example, feeding on almost every possible source of food. This diversity results in convergent morphologies that make this kind of characters inadequate for reconstructing the evolutionary history of this group. Mitogenomics has proven useful in similar cases but no study of this kind has been conducted for this family. We got to sequence whole mitogenomes for representatives of all major lineages and covering the diversity of life history traits. We then show that using these mitogenomes allows solving intrafamilial relationships with a resolution similar to that resulting from a concatenation of mitochondrial and nuclear markers and with solid statistical support for most of the nodes of the phylogeny. This allowed clarifying several controversial relationships and confirming several clades proposed in previous studies. Next, we illustrate the evolution of mitogenomes and the influence of life history traits using the clade of vampire bats, the only hematophagous Mammals, whose mitogenome seem to have undergone an acceleration of evolutionary rate as a consequence of the combined action of neutral and selective forces in order to counter the constraints imposed by this feeding habit. Finally, the robust phylogenetic frame provided by the 100 mitogenomes that we sequenced, will be used for future studies about, for exemple, the diversification process of Phyllostomids.

No description available.

Le SELEX est une technique d’évolution moléculaire dirigée qui permet, après plusieurs tours de sélection, d’enrichir une banque d’acides nucléiques en séquences capable de se lier de manière spécifique à une cible. Le séquençage est utilisé pour identifier ces séquences que l’on nomme « aptamères ». Depuis l’arrivée récente du séquençage à haut débit (HD), il est possible d’analyser des millions de séquences. L’objectif de la thèse était de développer des méthodes pour traiter et analyser les données de séquençage HD afin de faciliter l’identification des meilleurs aptamères d’un SELEX. Au cours de cette thèse, un test robotisé de liaison sur cellules adhérentes vivantes a été mis au point pour mesurer l’affinité d’aptamères issus de SELEX ciblant des cellules (cell-SELEX). Puis, l’évolution de l’abondance des séquences d’un cell-SELEX a été analysée par séquençage HD. Ceci nous a permis de concevoir une nouvelle approche phylogénétique baptisée FREDROGRAM. Cette approche évolutive a permis d’identifier des mutants avec une meilleure affinité au sein d’une famille d’aptamères issu de ce cell-SELEX. Enfin, le séquençage HD de deux SELEX dirigés contre des protéines a contribué à mieux comprendre l’impact des paramètres de sélection sur la population de séquences et à identifier de nouveaux aptamères, notamment en réduisant le nombre de tours de SELEX. En conclusion, ces travaux montrent l’utilité du séquençage HD pour l’identification des meilleurs aptamères et suggèrent de nouvelles pratiques pour la conduite des SELEX futurs. / SELEX is a directed molecular evolution technic which allows, after several rounds of selection, enriching a library from random nucleic acids to sequences able to bind specifically a target. Sequencing technics are then used to identify these sequences called « aptamers ». Since the arrival of High-Throughput Sequencing (HTS), it is now possible to analyse millions of sequences. The aim of the thesis was to develop methods for the treatment and the analysis of HTS data, in order to facilitate the identification of the best aptamers inside a SELEX. During this thesis, a semi-automatic binding test on adherent living cells has been developed to measure the affinity of aptamers identified in SELEX directed against specific cells (cell-SELEX). Then, the evolution of the sequence enrichment during a cell-SELEX has been analysed by HTS. This analysis gave us the possibility to design a new phylogenetic approch named FREDROGRAM. This evolutive approch allowed to identify variants of an aptamer’s family with a better affinity. Finally, HTS of two SELEX directed against proteins has contributed to a better understanding of the impact of selection parameters on the library and to identified new aptamers, notably by reducing the number of SELEX rounds. To conclude, this work shows the importance of HTS in the identification of the best aptamers and suggests new protocols to monitor the next SELEX in a different manner.

No description available.

Je me suis intéressé au système de défense anti-viral majeur de Drosophila melanogaster qui est la voie du RNA silencing (siRNA). A ce jour, le seul senseur d’acide nucléique viral et activateur de la voie siRNA est Dicer-2. Ainsi, le travail que j’ai effectué́ a permis d’apporter de nouvelles informations concernant la détection des ARNs viraux par Dicer-2. L’utilisation de méthodes de séquençage à haut débit (HTS) des petits ARNs dans des cellules S2 infectées par le Drosophila C Virus (DCV) à des temps précoces m’a permis de proposer un point d’entrée précis et interne de Dicer-2 sur l’ARN double brin de ce dicistrovirus. La validation de ce point faible dans la défense du virus a été effectuée en réalisant un HTS des petits ARNs dans des mouches de différents génotypes infectées avec DCV. J’ai ensuite caractérisé plus en profondeur cette région du génome virale en déterminant tout d’abord sa structure 2D puis sa sensibilité à des clivages médiés par des extraits embryonnaires de mouches. Finalement, l’utilisation de différents variants de Dicer-2 présentant des mutations du domaine DRA m’a permis de proposer un nouveau mécanisme de fonctionnement de cette protéine. / My Ph.D revolved around the study of the major antiviral defense system of Drosophila melanogaster: the siRNA pathway. To date, the only viral nucleic acid sensor and siRNA pathway activator in drosophila is Dicer-2. Thus, the work I have done has provided new information regarding the detection of viral RNAs by Dicer-2. The use of high throughput sequencing (HTS) methods of small RNAs in S2 cells infected with Drosophila C Virus (DCV) at early time points has allowed me to propose a precise and internal entry point for Dicer-2 on the double-stranded RNA of this dicistrovirus. The validation of this weak point in the defence of the virus was carried out by performing an HTS of small RNAs in flies of different genotypes infected with DCV. I then characterized this region of the viral genome in more depth by first determining its 2D structure and then its sensitivity to cleavages mediated by embryonic fly extracts. Finally, the use of different variants of Dicer-2 with mutations in the DRA domain allowed me to propose a new mechanism of action for this protein.