Despite active development during past years, the task of sequencing a genome still remains a challenge. Our current technologies are not able to read the whole genome in one piece. Instead, we shatter the target genome into a large amounts of small pieces that are then sequenced separately. The process of assembling these small pieces together, in order to obtain sequence of the whole genome, is painful and rsource-consuming. Multiple algorithms to solve the assembly problem were developed. This thesis presents yet another assembly algorithm, based on the usage of de Bruijn graphs, and focusing on sequencing short genome regions. The algorithm is compared to well-known solutions in the field. 1

Soluble dietary fibers (SDF) are fermented more than insoluble dietary fibers (IDF), but their effect on colonic bacterial community structure and function remains unclear. Thus, bacterial community composition and function in the colon of BALB/c mice (n = 7) fed with a high level (approximately 20%) of typical SDF, oat-derived beta-glucan (G), microcrystalline cellulose (M) as IDF, or their mixture (GM), were compared. Mice in group G showed a lowest average feed intake (p < 0.05) but no change on the average body weight gain (p > 0.05) compared to other groups, which may be associated with the highest concentration of colonic propionate (p < 0.05) in these mice. The bacterial alpha-diversity of group G was significantly lower than other groups (p < 0.01). In group G, the relative abundance of bacteria belonging to the phylum Bacteroidetes was significantly increased, whereas bacteria from the phylum Firmicutes were significantly decreased (p < 0.01). The core bacteria for different treatments showed distinct differences. Bacteroides, Dehalobacterium, and Prevotella, including known acetogens and carbohydrate fermenting organisms, were significantly increased in relative abundance in group G. In contrast, Adlercreutzia, Odoribacter, and Coprococcus were significantly more abundant in group M, whereas Oscillospira, Desulfovibrio, and Ruminoccaceae, typical hydrogenotrophs equipped with multiple carbohydrate active enzymes, were remarkably enriched in group GM (p < 0.05). The relative abundance of bacteria from the three classes of Proteobacteria, Betaproteobacteria, Gammaproteobacteria (including Enterobacteriaceae) and Deltaproteobacteria, were significantly more abundant in group G, indicating a higher ratio of conditional pathogenic bacteria in mice fed dietary beta-glucan in current study. The predicted colonic microbial function showed an enrichment of "Energy metabolism" and "Carbohydrate metabolism" pathways in mice from group G and M, suggesting that the altered bacterial community in the colon of mice with the two dietary fibers probably resulted in a more efficient degradation of dietary polysaccharides. Our result suggests that the influence of dietary beta-glucan (SDF) on colonic bacterial community of mice was more extensively than MCC (IDF). Co-supplementation of the two fibers may help to increase the bacterial diversity and reduce the conditional pathogens in the colon of mice.

The intestinal tract of virtually any metazoan, including mammals, is colonized with a complex microbial community to which we refer as intestinal or gut microbiota. One of the roles of the healthy intestinal microbiota is to protect the host against gut colonization with pathogenic bacteria through a phenomenon known as colonization resistance (CR). Dysbiosis of the intestinal microbiota, usually as a result of an antibiotic treatment, may lead to the disruption of the CR, and subsequent colonization with bacterial pathogens. However, and despite the importance, the role of the microbiota dysbiosis on the gut colonization by many bacterial pathogens, such as multidrug resistant Enterobacteriaceae, has not been elucidated: the members of the microbiota that confer CR and factors that promote colonization remain mostly unknown. The general aim of this thesis has been to improve the understanding of the role of the microbiota dysbiosis in gut colonization by bacterial pathogens. For this purpose, 3 projects have been established. In the first project we tried to elucidate the role of the microbiota dysbiosis on colonization by multidrug resistant Enterobacteriaceae (MRE) in mice. In the second project we investigated the risk factors and members of the microbiota associated with the MRE colonization in hospitalized patients. MRE infections represent a great threat for hospitalized patients. Specifically, acute leukemia patients are often colonized with MRE, probably due to the impaired CR as a result of intensive antibiotic treatments these patients receive. In the third project we studied the role of the microbiota dysbiosis on the development of Epizootic Rabbit Enteropathy (ERE). ERE is a severe gastrointestinal disease with a high percentage of mortality that occurs in young rabbits during first weeks post-weaning. ERE rabbits have been shown to suffer microbiota dysbiosis during the development of the disease. Moreover, the disease could be reproduced by contact between healthy and sick animals and by administration of cecal contents from ERE rabbits to healthy rabbits, suggesting that a pathogenic agent may be involved in the development of this intestinal pathology, although no causative agent has been identified until now. / El tracto intestinal de prácticamente cualquier metazoo, incluidos los mamíferos, está colonizado por una compleja comunidad microbiana a la que nos referimos como microbiota intestinal. Uno de los papeles de la microbiota intestinal es proteger al huésped contra la colonización intestinal con bacterias patógenas a través de un fenómeno conocido como resistencia a la colonización (RC). La disbiosis de la microbiota intestinal, a menudo como resultado de un tratamiento antibiótico, puede conducir a la alteración de la RC y posterior colonización por patógenos bacterianos. Sin embargo, y pese a su importancia, el papel de la disbiosis de la microbiota en la colonización intestinal por muchos patógenos bacterianos, como son las Enterobacterias multirresistentes, no se ha esclarecido: los miembros de la microbiota que confieren RC y los factores que promueven la colonización siguen siendo desconocidos. El objetivo general de esta tesis ha sido mejorar la comprensión del papel de disbiosis de la microbiota en la colonización intestinal por patógenos bacterianos. Para ello se han establecido tres proyectos. En el primer proyecto investigamos el papel de disbiosis de la microbiota intestinal en la colonización por Enterobacterias multiresistentes (MRE) en ratones. En el segundo proyecto investigamos los factores de riesgo y los miembros de la microbiota asociados con la colonización por MRE en pacientes hospitalizados. Las infecciones por MRE representan una gran amenaza para los pacientes hospitalizados. Específicamente, MRE a menudo colonizan los pacientes con leucemia aguda, probablemente debido a que la RC está alterada como resultado de tratamientos antibióticos intensivos recibidos por estos pacientes. En el tercer proyecto investigamos el papel de la disbiosis microbiana en desarollo de Enteropatía Epizoótica de Conejo (ERE). ERE es una enfermedad gastrointestinal severa con un alto porcentaje de mortalidad que ocurre en conejos jóvenes durante las primeras semanas después del destete. Se ha demostrado que los conejos con ERE sufren disbiosis microbiana después del inicio de la enfermedad, aunque no está claro el papel de la disbiosis en el desarollo de la enfermedad. Además, la enfermedad puede ser reproducida por contacto entre animales sanos y enfermos y por la administración del contenido cecal de conejos con ERE a conejos sanos, lo que sugiere que un agente patógeno podría estar implicado en el desarrollo de esta patología intestinal, aunque hasta ahora no se ha logrado identificar ningún agente causal. / El tracte intestinal de pràcticament qualsevol metazoo, inclosos els mamífers, està colonitzat per una complexa comunitat microbiana a la qual ens referim com microbiota intestinal. Un dels papers de la microbiota intestinal és protegir a l'hoste contra la colonització intestinal amb bacteris patògens a través d'un fenomen conegut com a resistència a la colonització (RC). La disbiosis de la microbiota intestinal, frecuentment com a resultat d'un tractament antibiòtic, pot conduir a l'alteració de la RC i posterior colonització per patògens bacterians. No obstant això, i malgrat la seva importància, el paper de la disbiosis de la microbiota en la colonització intestinal per molts patògens bacterians, com són les Enterobacteries multirresistentes, no s'ha esclarit: els membres de la microbiota que confereixen RC i els factors que promouen la colonització segueixen sent desconeguts. L'objectiu general d'aquesta tesi ha estat millorar la comprensió del paper de la disbiosis de la microbiota en la colonització intestinal per patògens bacterians. Per a això s'han establert tres projectes. En el primer projecte vam investigar el paper de la disbiosis de la microbiota intestinal en la colonització per Enterobacteries multiresistentes (MRE) en ratolins. En el segon projecte, investiguem els factors de risc i els membres de la microbiota associats amb la colonització per MRE en pacients hospitalitzats. Les infeccions per MRE representen una gran amenaça per als pacients hospitalitzats. Específicament, MRE sovint colonitza els pacients amb leucèmia aguda, probablement a causa de que la RC està alterada com a resultat de tractaments antibiòtics intensius rebuts per aquests pacients. En el tercer projecte, vam investigar el paper de la disbiosis microbiana en desenvolupament de l'Enteropatía Epizoótica de Conill (ERE). ERE és una malaltia gastrointestinal severa amb un alt percentatge de mortalitat que ocorre en conills joves durant les primeres setmanes després del deslleti. S'ha demostrat que els conills amb ERE sofreixen disbiosis microbiana després de l'inici de la malaltia, encara que no és clar el paper de la disbiosis en el desenvolupament de la malaltia. A més, la malaltia pot ser reproduïda per contacte entre animals sans i malalts i per l'administració del contingut cecal de conills amb ERE a conills sans, la qual cosa suggereix que un agent patogen podria estar implicat en el desenvolupament d'aquesta patologia intestinal, encara que fins ara no s'ha aconseguit identificar cap agent causal. / Djukovic, A. (2017). Role of intestinal dysbiosis on gut colonization by bacterial pathogens [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/90415 / TESIS

Les papillomavirus humains (HPV) sont classés en 5 genres α, β, γ, µ et η. Leur génome comprend six gènes précoces dont deux oncogènes E6 et E7 et deux gènes tardifs codant les protéines de capside. Les β- et γ-HPV constituent une part importante du virome cutané. Généralement asymptomatiques ils peuvent se manifester par des papillomatoses et sont associés à certains cancers de la peau, en particulier chez l’immunodéprimé. Les α-HPV ont un tropisme muqueux ; les α-HPV à haut risque (HR) HPV16 et 18 sont impliqués dans 99% des cancers du col de l’utérus.La détection des α-HR-HPV dans les frottis cervico-utérins (FCU) lors d’atypie cellulaire de signification indéterminée (ASCUS) constitue une information décisive dans le dépistage du cancer du col de l’utérus, bien que les tests de génotypage ne ciblent que les types les plus fréquents. Le génotypage des β- et γ-HPV devient nécessaire pour l’étude du virome notamment dans des contextes de susceptibilité aux pathogénies HPV (syndrome WHIM : Warts, Hypogammaglobulimemia, Infections and Myelokathexis). Cet immunodéficit congénital rare causé par une mutation gain de fonction du récepteur CXCR4 se manifeste dans 70% des cas par des papillomatoses cutanées étendues et ano-génitales évoluant souvent en cancer. Des études du laboratoire ont identifié le rôle intrinsèque de l’axe CXCL12/CXCR4 dérégulé dans la pathogénie virale en démontrant notamment l’action bénéfique du blocage de cet axe par un antagoniste de CXCR4 (AMD3100) in vitro et in vivo sur l’oncogenèse due à HPV.Nos objectifs étaient : (i) d’identifier dans des FCU ASCUS, les HPV dont le génotype n’avait pu être déterminé (HPV-X) par un test classique (INNO-LiPA HPV Genotyping Extra II®), (ii) de caractériser le virome HPV d’un patient atteint de WHIM au cours d’un essai thérapeutique par AMD3100 administré à titre compassionnel pendant 7 mois avec pour objectif d’évaluer son impact sur les anomalies associées à HPV.Dans les deux cas, nous avons mis au point une méthode de génotypage par séquençage haut débit sur Illumina Miseq®. La distribution des génotypes et leur polymorphisme nucléotidique ont été étudiés par analyses comparatives et phylogénétiques. (i) Notre stratégie a permis d’identifier dans 54 ASCUS/HPV-X étudiés une majorité d'HPV bas risque réalisant dans 41% des cas une infection à multiples génotypes (2 à 7), et aussi l’existence de quasi-espèces (41% des FCU) comprenant jusqu’à 17 variants pour un même génotype. Ainsi, de probables compétitions ou défauts d’hybridation des variants minoritaires peuvent expliquer le manque de performance du test INNO-LiPA. (ii) Chez le patient WHIM, le séquençage a été complété par des qPCR spécifiques de types, permettant une étude qualitative et quantitative. L’AMD3100 n’a pas modifié qualitativement le virome HPV cutané composé de 16 types, principalement des β- et γ-HPV, déjà présents 3 ans auparavant dans des verrues cutanées analysées rétrospectivement. En revanche, l’analyse quantitative montre des modifications en proportion relative des génomes viraux suggérant un effet du traitement sur l‘expression de certains types pouvant être associés sélectivement à la papillomatose. A cet égard, un des HPV composant le virome cutané du patient qui se trouve être un des deux seuls types présents dans une biopsie profonde de verrue, étaye l’hypothèse d’une sélection dans le processus lésionnel. De plus, les protéines oncogènes E6 et E7 de ce virus présentent des mutations, en comparaison à la séquence du génome HPV de référence, qui pourraient favoriser le potentiel pathogène de ce varian; hypothèse en cours d’investigation.En conclusion, les techniques de séquençage haut débit que nous avons développées ont permis de mieux caractériser la composition du virome HPV démontrant à la fois sa complexité en génotypes viraux ou en dérivés de ceux-ci (concept de quasi-espèces) et sa dynamique d’évolution qui pourraient sous-tendre le potentiel pathogène de ce virome HPV. / Human papillomaviruses (HPV) are classified into 5 genera α, β, γ, μ and η. Their genome comprises six early genes including two oncogenes E6 and E7, and two late genes encoding the L1 and L2 capsid proteins. β- and γ-HPV constitute an important part of the cutaneous virome; usually asymptomatic, they can manifest as papillomatosis like warts and are associated with certain skin cancers, especially in immunocompromised patients. α-HPV has a mucosal tropism; high-risk (HR) α-HPV16 and 18 are involved in 99% of cervical cancers.Detection of α-HR-HPV in cervical samples guide the management of women whose Pap smear result shows atypical squamous cells of undetermined significance (ASCUS), although genotyping targets only the most common HPV types. Genotyping of β- and γ-HPV becomes necessary for the study of the virome especially in contexts of susceptibility to HPV pathogenesis (i.e. WHIM syndrome (for Warts, Hypogammaglobulimemia, Infections and Myelokathexis)). WHIM syndrome is a rare congenital immunodeficiency caused by a gain-of-function mutation of the CXCR4 receptor of the chemokine CXCL12 and manifests in 70% of cases by extensive cutaneous papillomatosis and ano-genital lesions that often evolve into cancer. Studies in our laboratory have identified the intrinsic role of the dysregulated CXCL12/CXCR4 axis in viral pathogenesis by demonstrating in particular the beneficial action of the blocking of this axis by an antagonist of CXCR4 (AMD3100) in vitro and in vivo on HPV-associated oncogenesis.Our objectives were: (i) to identify HPV whose genotype could not be determined (HPV-X) by a conventional test (INNO-LiPA HPV Genotyping Extra II®) in cervical samples with Pap smear report of ASCUS (ii) to characterize the HPV virome of a patient suffering from WHIM syndrom during a 7-month compassionate AMD3100 clinical trial to assess its impact on HPV-associated abnormalities.In both cases, we have developed a high-throughput sequencing genotyping method on Illumina Miseq®. The distribution of genotypes and their nucleotide polymorphism were studied by comparative and phylogenetic analyzes. (i) Our strategy identified in the 54 investigated ASCUS/HPV-X a majority of low-risk HPV, achieving a multiple infection (2 to 7 genotypes) in 41% of cases, and also the existence of quasi-species (41% of FCU) comprising up to 17 variants for the same genotype. Thus, probable competitions or hybridization defects of the minority variants may explain the lack of performance of the INNO-LiPA test. (ii) In the WHIM patient, sequencing was supplemented with type-specific qPCRs, allowing a qualitative and quantitative study. AMD3100 did not qualitatively modify the cutaneous HPV virome composed of 16 types, mainly β- and γ-HPV. In contrast, the quantitative analysis shows changes in the relative proportions of viral genomes suggesting a treatment effect on the expression of certain types that can be selectively associated with. papillomatosis. In this respect, one of the HPVs belonging to the cutaneous virome of the patient was found to be one of only two types present in a deep wart biopsy. This result supports the hypothesis of HPV selection in the lesion process. In addition, the oncogenic proteins E6 and E7 of this virus have mutations which could promote the pathogenic potential of this viral variant in comparison with the sequence of the reference HPV genome; a hypothesis that is under investigation.In conclusion, the high throughput sequencing techniques that we have developed have made it possible to better characterize the composition of the HPV virome demonstrating both its complexity in viral genotypes or in derivatives (i.e. quasi-species concept). The dynamics of which may underlie the pathogenic potential of this HPV virome.

Analysis of soil-derived DNA has been shown to minimize problems seen during traditional vegetation surveys, e.g. by matching the eDNA to a reference database for taxonomic identification rather than relying solely on taxonomic expertise. However, it has been debated to what extent and how accurately eDNA acts as a proxy for biodiversity. The reliability on eDNA and the awareness on influencing factors is also important for palaeoenvironmental reconstructions where above-ground vegetation cannot be used. This study aimed to investigate how well modern soil-derived eDNA reflects the contemporary vascular vegetation communities in a subarctic environment, and if the efficiency of the taxonomic identification differed between spatial scales. Near-surface soil samples at altitudinal gradients along numerous transects were collected in combination with vegetation surveys. The eDNA was amplified through metabarcoding using the P6 loop region of the chloroplast trnL intron, followed by a high-throughput sequencing. No difference in the number of identified taxa between eDNA and vegetation survey was seen at landscape scale. In contrast, the number of identified taxa was consistently higher in the vegetation survey at smaller spatial scales. The efficiency of identified taxa per scale remained stable for the vegetation survey, whereas for eDNA, a decreasing trend was seen. This study highlights the variations on taxa identification between both methods and which factors might cause it. Combining the methods allows for a more precise modern biodiversity estimation, as well as to minimize wrongful conclusions. This allows for a more accurate palaeoenvironmental reconstructions, which in turn can improve future species conservation decisions.

In this thesis the processing mechanisms of short non-coding RNAs (ncRNAs) is investigated by using data generated by the current method of high-throughput sequencing (HTS). The recently adapted short RNA-seq protocol allows the sequencing of RNA fragments of microRNA-like length (∼18-28nt). Thus, after mapping the data back to a reference genome, it is possible to not only measure, but also visualize the expression of all ncRNAs that are processed to fragments of this specific length. Short RNA-seq data was used to show that a highly abundant class of small RNAs, called microRNA-offset-RNAs (moRNAs), which was formerly detected in a basal chordate, is also produced from human microRNA precursors. To simplify the search, the blockbuster tool that automatically recognizes blocks of reads to detect specific expression patterns was developed. By using blockbuster, blocks from moRNAs were detected directly next to the miR or miR* blocks and could thus easily be registered in an automated way. When further investigating the short RNA-seq data it was realized that not only microRNAs give rise to short ∼22nt long RNA pieces, but also almost all other classes of ncRNAs, like tRNAs, snoRNAs, snRNAs, rRNAs, Y-RNAs, or vault RNAs. The formed read patterns that arise after mapping these RNAs back to a reference genome seem to reflect the processing of each class and are thus specific for the RNA transcripts of which they are derived from. The potential of this patterns in classification and identification of non-coding RNAs was explored. Using a random forest classifier which was trained on a set of characteristic features of the individual ncRNA classes, it was possible to distinguish three types of ncRNAs, namely microRNAs, tRNAs, and snoRNAs. To make the classification available to the research community, the free web service ‘DARIO’ that allows to study short read data from small RNA-seq experiments was developed. The classification has shown that read patterns are specific for different classes of ncRNAs. To make use of this feature, the tool deepBlockAlign was developed. deepBlockAlign introduces a two-step approach to align read patterns with the aim of quickly identifying RNAs that share similar processing footprints. In order to find possible exceptions to the well-known microRNA maturation by Dicer and to identify additional substrates for Dicer processing the small RNA sequencing data of a Dicer knockdown experiment in MCF-7 cells was re-evaluated. There were several Dicer-independent microRNAs, among them the important tumor supressor mir-663a. It is known that many aspects of the RNA maturation leave traces in RNA sequencing data in the form of mismatches from the reference genome. It is possible to recover many well- known modified sites in tRNAs, providing evidence that modified nucleotides are a pervasive phenomenon in these data sets.

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No description available.

Surgical preparation reduces commensal bacterial load. Currently, no standardized preoperative ocular preparation method in the dog has been reported. Previous studies use culture-based methods to determine commensal bacterial populations. Recent reports suggest that high-throughput sequencing may be superior to culture techniques to determine bacterial communities in the eye and other tissues. The goal of this study was to describe the conjunctival commensal ocular microbiome and bacterial community using DNA sequencing and aerobic cultures of six normal, healthy dogs and investigate the short and long-term effects of an antiseptic protocol on the ocular microbiome. Samples were obtained prior to, immediately following, 24 hours following, and 4 weeks following ocular preparation. The Mississippi State University microbiology laboratory evaluated aerobic cultures, and the Gastrointestinal Laboratory of Texas A&M University performed DNA sequencing. This is the first study to show short and long-term effects of standard ocular surgical preparation on the ocular surface microbiome.

No description available.