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Investigation of in-hospital norovirus transmission using whole genome sequencingWong, Tse Hua Nicholas January 2014 (has links)
Norovirus is the commonest cause of viral gastroenteritis, affecting all age groups worldwide. Outbreaks frequently occur in semi-closed communities such as schools, cruise ships, prisons and hospitals. Within the healthcare environment, the economic and logistical burdens and the inconvenience caused by norovirus is significant, since ward closure remains central to infection control. The aim of this study was to investigate norovirus transmission dynamics during hospital outbreaks. The ultimate goal was to provide information that could, in future, lead to the development of novel, less disruptive approaches to curtailing the spread of infection. The study explored the application of 'next generation' high throughput DNA sequencing technologies to the determination of large numbers of norovirus genomes. Whole genome sequences provide the highest possible level of discrimination among viruses, information which is essential to the identification of linked and independent cases of infection. The approach exploits the high norovirus mutation rate, which is typical of RNA viruses. Consequently, viruses within a single ward which differ by more than a few SNVs can be considered to represent independent introductions, rather than a single outbreak. Whole genome sequence data (determined for noroviruses collected between 2009 and 2013) were combined with epidemiological data, providing further insights into transmission dynamics. These data identified multiple independent virus introductions during single ward outbreaks. The possible origin of such outbreaks in Oxfordshire hospitals were investigated using viruses originating in the local community, and in other healthcare environments distributed throughout the UK. Whole genome sequences of noroviruses from consecutive years were genetically divergent, confirming the rapid evolution of the virus over time and excluding the possibility of prolonged environmental contamination as a reservoir of infection. Such detailed information on norovirus transmission within the healthcare environment could inform alternative future approaches to optimising infection control within the healthcare setting.
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Duplication de génome et évolution de la famille Sox chez les poissons téléostéens / Whole genome duplication and the evolution of the Sox family in teleostean fishVoldoire, Emilien 17 December 2013 (has links)
Les duplications de gènes et de génome sont considérées comme des moteurs de l’évolution des génomes eucaryotes. Trois duplications de génome complet (ou polyploïdisations) sont survenues au cours de l’évolution des vertébrés, dont deux à la base des vertébrés, et une troisième chez l’ancêtre commun des poissons téléostéens. La diversité morphologique, anatomique et écologique des espèces qui partagent un ancêtre commun polyploïde chez les chordés suggère un rôle des duplications de génome dans la diversification des espèces. En particulier, les duplications de génome semblent avoir facilité l’émergence du plan d’organisation des vertébrés, et être à l’origine de la radiation évolutive survenue chez les poissons téléostéens. Cependant, la portée évolutive des duplications de génome, et notamment les deux hypothèses majeures formulées ci-Avant, restent des questions ouvertes et en grande partie non résolues. Le groupe des téléostéens, qui compte plus de la moitié des espèces vertébrés existantes et partage un ancêtre commun polyploïde, constitue un modèle pertinent pour évaluer la contribution des duplications de génome dans l’expansion des familles multigéniques chez les vertébrés, pour comprendre les mécanismes évolutifs qui façonnent l’évolution des familles de gènes, et finalement tester les hypothèses moléculaires qui peuvent relier duplication de génome et biodiversité. Ainsi, nous avons étudié l’impact de la duplication de génome survenue à la base des téléostéens sur l’évolution de la famille multigénique sox, essentielle pour le développement et l’homéostasie des vertébrés. Notre analyse du contenu et de l’organisation des gènes sox dans 15 génomes de vertébrés, dont 10 téléostéens, révèle une importante expansion de l’ensemble de la famille des gènes sox dans ce vaste groupe de vertébrés, et démontre que cette expansion est essentiellement due à la duplication de génome survenue à la base des téléostéens. Les gènes sox dupliqués par duplication de génome semblent avoir été perdus par non-Fonctionnalisation dans certaines lignées, et préservés en deux copies par sous-Fonctionnalisation et/ou néo-Fonctionnalisation dans certaines autres lignées. Notre étude indique en effet une divergence lignée-Spécifique des patrons d’expression entre les gènes sox dupliqués chez différentes espèces de téléostéens. Ainsi, l’expansion du répertoire des gènes sox à la base des téléostéens semble avoir été suivi d’une évolution lignée-Spécifique du contenu et des fonctions de la famille des gènes sox chez les poissons téléostéens. Cette étude supporte l’hypothèse d’un rôle des duplications de génome dans l’enrichissement et la diversification subséquente des répertoires de gènes du développement tels que les gènes sox, et son rôle potentiel dans la diversification des espèces vertébrés. / Gene and genome duplications are major engines of eukaryotic genome evolution. Three rounds of whole genome duplication (WGD) have occurred during vertebrate evolution, two rounds at the base of the vertebrate lineage, and a third round in the common ancestor of the teleostean fish (the so-Called teleost-Specific WGD). In chordates, species that share a polyploid ancestor are characterized by a huge morphological, anatomical and ecological diversity suggesting a role of WGDs in species diversification. For instance, it is considered that these drastic genomic events provided the raw material for the emergence of the vertebrate body plan, and facilitated speciation processes during the teleost radiation. However, how WGD is related to phenotypic diversification or to major evolutionary transitions are fundamental questions that remain largely unsolved. Teleostean fish constitute more than half of all extant vertebrates and share a polyploid ancestor. Thus, they provide a relevant model to study the importance of WGDs in gene families expansion, to understand evolutionary mechanisms that drive the evolution of these families and, finally, to test molecular hypotheses that might relate WGD and biodiversity. In this project, we studied the impact of the teleost-Specific WGD on the evolution of the sox gene family which are involved in development and homeostasis in vertebrates. Our analysis of the content and the genomic organization of the sox genes in 15 vertebrate genomes, including 10 teleosts, reveals an important expansion of this family in the teleost lineage, and demonstrates that this expansion is mainly due to the teleost-Specific WGD. The duplicated sox genes seem to have been lost by non-Functionalization in certain lineages, and preserved in two copies in others by neo-Functionalization and/or sub-Functionalization. Indeed, this study indicates lineage-Specific divergence in expression patterns between duplicated sox genes in different teleostean species. Hence, the sox family expansion that occurred in the last common ancestor of teleostean fish seems to have been followed by a lineage-Specific evolution of the content and functions of the sox family in this group. Our study supports the hypothesis for a role of WGDs in the enrichment and diversification of developmental genes repertories and its potential role in species diversification in vertebrates.
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Whole Genome Amplification von Plasma-DNA und Entwicklung eines Ausschlusskriteriums zur Verbesserung der Genotypisierungsqualität / Sample selection algorithm to improve quality of genotyping from plasma-derived DNA: to separate the wheat from the chaff.Schoenborn, Veit January 2008 (has links) (PDF)
Plasma- und Serumproben waren in früheren epidemiologischen Studien häufig das einzige biologische Material, das gesammelt und untersucht wurde. Diese Studien besitzen gerade durch ihren sehr langen Untersuchungszeitraum einen riesigen Informationsgehalt und wären ein unbezahlbarer Schatz für genetische Analysen. Oft ist aufgrund damals mangelnder Akquirierung jedoch keine genomische DNA verfügbar. Um die in Plasmaproben in geringer Menge vorkommende DNA verwenden zu können, extrahierten wir die DNA mit Hilfe von magnetischen Partikeln und setzten sie in eine Whole Genome Amplification (WGA) mittels Φ29-DNA-Polymerase ein. Wir stellten 88 Probenpärchen, bestehend aus einer WGA-Plasma-DNA und der korrespondierenden Vollblut-DNA derselben Person, zusammen und genotypisierten bei diesen neun hochpolymorphe Short Tandem Repeats (STR) und 25 SNPs. Die durchschnittliche innerhalb der Probenpaare auftretende Diskordanzrate betrug 3,8% für SNPs sowie 15,9% für STRs. Basierend auf den Ergebnissen der Hälfte der Probenpaare entwickelten wir einen Ausschlussalgorithmus und validierten diesen in der anderen Hälfte der Probenpaare. Mit diesem ist es möglich, zum Einen diejenigen Proben mit einer guten DNA-Qualität herauszufiltern, um Genotypisierungsfehler zu vermeiden, und zum Anderen jene Proben mit insuffizienter DNA-Qualität auszuschließen. Nachdem Proben, die fünf oder mehr homozygote Loci in dem 9-STR-Markerset aufwiesen, ausgeschlossen wurden, resultierte dies in einer Ausschlussrate von 22,7% und senkte die durchschnittliche Diskordanzrate auf 3,92% für STRs bzw. 0,63% für SNPs. Bei SNPs entspricht dieser Wert ungefähr der Fehlerquote, wie er auch bei Genotypisierungen mit Vollblut-DNA in vielen Laboratorien auftritt. Unsere Methode und das Ausschlusskriterium bieten damit neue Möglichkeiten, um zuverlässige DNA aus archivierten Plasmaproben wiederzugewinnen. Dieser Algorithmus ist auch besser geeignet, als nur die eingesetzte DNA-Menge in die WGA-Reaktion als Kriterium zu benützen. / Plasma and serum samples were often the only biological material collected for earlier epidemiological studies. These studies have a huge informative content, especially due to their long follow-up and would be an invaluable treasure for genetic investigations. However, often no banked DNA is available. To use the small amounts of DNA present in plasma, in a first step, we applied magnetic bead technology to extract this DNA, followed by a whole-genome amplification (WGA) using phi29-polymerase. We assembled 88 sample pairs, each consisting of WGA plasma DNA and the corresponding whole-blood DNA. We genotyped nine highly polymorphic short tandem repeats (STRs) and 23 SNPs in both DNA sources. The average within-pair discordance was 3.8% for SNPs and 15.9% for STR genotypes, respectively. We developed an algorithm based on one-half of the sample pairs and validated on the other one-half to identify the samples with high WGA plasma DNA quality to assure low genotyping error and to exclude plasma DNA samples with insufficient quality: excluding samples showing homozygosity at five or more of the nine STR loci yielded exclusion of 22.7% of all samples and decreased average discordance for STR and SNP markers to 3.92% and 0.63%, respectively. For SNPs, this is very close to the error observed for genomic DNA in many laboratories. Our workflow and sample selection algorithm offers new opportunities to recover reliable DNA from stored plasma material. This algorithm is superior to testing the amount of input DNA.
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Expanding the horizons of next generation sequencing with RUFUSFarrell, Andrew R. January 2014 (has links)
Thesis advisor: Gabor T. Marth / To help improve the analysis of forward genetic screens, we have developed an efficient and automated pipeline for mutational profiling using our reference guided tools including MOSAIK and FREEBAYES. Studies using next generation sequencing technologies currently employ either reference guided alignment or de novo assembly to analyze the massive amount of short read data produced by second generation sequencing technologies; the far more common approach being reference guided alignment due to the massive computational and sequencing costs associated with de novo assembly. The success of reference guided alignment is dependent on three factors; the accuracy of the reference, the ability of the mapper to correctly place a read, and the degree to which a variant allele differs from the reference. Reference assemblies are not perfect and none are entirely complete. Moreover, read mappers can only map reads in genomic locations that are unique enough to confidently place reads; paralogous sections, such as related gene families, cannot be characterized and are often ignored. Further, variant alleles that drastically alter the subject's DNA, such as insertions or deletions (INDELs), will not map to the reference and are either entirely missed or require further downstream analysis to characterize. Most importantly, reference guided methods are restricted to organisms for which such reference genomes have been assembled. The current alternative, de novo assembly of a genome, is prohibitively expensive for most labs requiring deep read coverage from numerous different library preparations as well as massive computing power. To address the shortcomings of current methods, while eliminating the costs intrinsic to de novo sequence assembly, we developed RUFUS, a novel, completely reference-independent variant discovery tool. RUFUS directly compares raw sequence data from two or more samples and identifies groups of reads unique to one or the other sample. RUFUS has at least the same variant detection sensitivity as mapping methods, with greatly increased specificity for SNPs and INDEL variation events. RUFUS is also capable of extremely sensitive copy number detection, without any restriction on event length. By modeling the underlying k-mer distribution, RUFUS produces a specific copy number spectrum for each individual sample. Applying a Bayesian detection method to detect changes in k-mer content between two samples, RUFUS produces copy number calls that are equally as sensitive as traditional copy number detection methods with far fewer false positives. Our data suggest that RUFUS' reference-free approach to variant discovery is able to substantially improve upon existing variant detection methods: reducing reference biases, reducing false positive variants, and detecting copy number variants with excellent sensitivity and specificity. / Thesis (PhD) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.
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Understanding inflammatory bowel disease using high-throughput sequencingde Lange, Katrina Melanie January 2017 (has links)
For over two decades, the study of genetics has been making significant progress towards understanding the causes of common disease. Across a wide range of complex disorders there have been hundreds of associated loci identified, largely driven by common genetic variation. Now, with the advent of next-generation sequencing technology, we are able to interrogate rare and low frequency variation in a high throughput manner for the first time. This provides an exciting opportunity to investigate the role of rarer variation in complex disease risk on a genome-wide scale, potentially o↵ering novel insights into the biological mechanisms underlying disease pathogenesis. In this thesis I will assess the potential of this technology to further our understanding of the genetics of complex disease, using inflammatory bowel disease (IBD) as an example. After first reviewing the history of genetic studies into IBD, I will describe the analytical challenges that can occur when using sequencing to perform case-control association testing at scale, and the methods that can be used to overcome these. I then test for novel IBD associations in a low coverage whole genome sequencing dataset, and uncover a significant burden of rare, damaging missense variation in the gene NOD2, as well as a more general burden of such variation amongst known inflammatory bowel disease risk genes. Through imputation into both new and existing genotyped cohorts, I also describe the discovery of 26 novel IBD-associated loci, including a low frequency missense variant in ADCY7 that approximately doubles the risk of ulcerative colitis. I resolve biological associations underlying several of these novel associations, including a number of signals associated with monocyte-specific changes in integrin gene expression following immune stimulation. These results reveal important insights into the genetic architecture of inflammatory bowel disease, and suggest that a combination of continued array-based genome- wide association studies, imputed using substantial new reference panels, and large scale deep sequencing projects will be required in order to fully understand the genetic basis of complex diseases like IBD.
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Clostridium botulinum, du génotypage de la toxine en passant par les flagellines jusqu'au séquençage de génomes : un aperçu de la diversité génétique des Clostridies associés au botulisme animal et humain / Clostridium botulinum, from toxin and flagellin genotyping to Whole Genome Sequencing : an insight into genetic diversity of human and animal botulism associated clostridia’sWoudstra, Cedric 21 March 2016 (has links)
Le botulisme est une maladie nerveuse, commune à l’homme et aux animaux, due à l’action de la toxine botulique produite par Clostridium botulinum. Il existe 8 types de toxines dénommées A à H. Les bactéries capables de produire cette toxine se différencient en six groupe sur la base de leurs caractéristiques phénotypiques et biologiques. Les souches de C. botulinum responsables du botulisme humain appartiennent aux groupes I et II selon qu’elles soient protéolytiques ou non. Elles produisent les toxines A, B, E et F, ainsi que le nouveau type H récemment découvert. C. butyricum et C. baratii sont également capables de produire les toxines botuliques de type F et E et appartiennent au groupe V et VI. C. argentinense appartient au groupe IV et est capable de synthétiser la toxine de type G. Elle a été soupçonnée d’être impliquée dans des cas de botulisme infantile en Argentine. Les souches de C. botulinum responsables du botulisme animal appartiennent au groupe III (C. novyi sensu lato) et produisent les toxines C, D et leurs formes mosaïques C/D et D/C. La toxine botulique est le poison le plus puissant connu à ce jour. La dose létale nécessaire pour tuer une personne en bonne santé par intoxication alimentaire est de 70 µg seulement. C’est pourquoi cette toxine a fait l’objet d’études particulièrement approfondies, notamment celles impliquées dans des cas de botulisme humain. Elle peut également être utilisée pour le traitement de certaine pathologie ou la chirurgie esthétique (Botox). Malheureusement, elle peut également être utilisée à mauvais escient, en tant qu’arme de guerre ou à des fins de bioterrorisme. C’est pourquoi l’emploi de la toxine botulique ou de sa bactérie productrice fait l’objet d’une législation particulièrement stricte. Mon projet de doctorat s’est organisé autour de plusieurs projets de recherche visant à développer des méthodes de détection et de typage de du germe et de sa toxine (projets Européens BIOTRACER et AniBioThreat ; projets NRBC-bio ; LNR botulisme aviaire en France). Lors de mes recherches j’ai concentré mon travail sur le développement de méthodes capable de suivre et remonter à la source d’une contamination, qu’elle soit délibérée, accidentelle ou naturelle. Afin d’y parvenir j’ai investigué les gènes des flagellines de C. botulinum groupe I à III, responsables du botulisme humain et animal. L’analyse des gènes flaA et flaB a mis en évidence 5 groupes majeurs et 15 sous-groupes, certain étant spécifiques de régions géographiques. FlaB s’est montré spécifique de C. botulinum type E. Les gènes flagellines fliC, spécifiques à C. botulinum du groupe III, se divisent 5 groupes, avec fliC-I et fliC-IV associés aux types mosaïques C/D et D/C. J’ai étudié la prévalence des souches productrices de toxine de type mosaïques chez les volailles et les bovins. Les résultats montrent que les types C/D et D/C sont majoritaires en Europe. Enfin, j’ai séquencé 17 génomes provenant de souches responsables de botulisme animal en France (14 types C/D et 3 types D/C). Leur analyse montre que ces souches sont très proche génétiquement, entre elles et avec les souches Européennes. Grâce à ces données j’ai mis en évidence un large contenu extra chromosomique dans les souches C/D, qui peut être utilisé pour créer une carte d’identité génétique. D’autre part, l’étude des séquences Crisps à des fins de typage ne s’est pas avérée suffisamment résolutive, du fait de système Crispr-Cas déficient chez les souches C/D. Enfin, un très haut degré de discrimination a été atteint par typage SNP, qui a permis de distinguer jusqu’à l’origine de chaque souche. L’ensemble de ces résultats est développé dans le présent manuscrit / Clostridium botulinum is the etiologic agent of botulism, a deadly paralytic disease that can affects both human and animals. Different bacteria, producing neurotoxins type A to H, are responsible for the disease. They are separated into different groups (I to VI) on the basis of their phenotypical and biological characteristics. Human botulism is mainly due to Groups I and II producing neurotoxins A, B, E and F, with type H recently discovered. Also C. butyricum and C. baratii species (Groups V and VI), producing toxins type F and E respectively, are scarcely reported. C. argentinense Group IV, producing toxin type G, which has been suspected to be associated with infant botulism in Argentina. Animal botulism is mainly due to Group III, which is constituted by C. novyi sensu lato species. They produce toxin types C, D and their mosaic variants. Botulinum neurotoxins are the most powerful toxin known to date with as little as 70 µg enough to kill a person by food poisoning. Therefore, it received a great deal of attention. Botulinum neurotoxins have been deeply studied, especially human related toxins compared to animal. The toxins found to be useful for medical or cosmetic (Botox) treatments, but it was also used as a biological warfare agent, and for bioterrorism. Its extreme potency is equal to its dangerousness. Therefore, governments show concerns of its potential misuse as a bioterrorism weapon; research programs are funded to study and raise awareness about both the toxins and the producing organisms. My PhD work was structured by the different projects I was involved in, which were related to C. botulinum detection and typing, like BIOTRACER and AniBioThreat European projects, the French national CBRN program, or the NRL for avian botulism. The main transversal objective I followed lead me to develop new methods to trace back the origin of C. botulinum contamination, in case of a deliberate, accidental or naturally occurring botulism outbreak. I investigated flagellin genes as potential genetic targets for typing C. botulinum Group I-II and III, responsible for human and animal botulism respectively. Flagellin genes flaA and flaB showed the investigated C. botulinum Group I and II strains to cluster into 5 major groups and up to 15 subgroups, some being specific for certain geographical areas, and flaB being specific to C. botulinum type E. Flagellin fliC gene investigated in C. botulinum Group III showed to cluster into five groups, with fliC-I and fliC-IV associated to type C/D and D/C respectively, being not discriminative enough to differentiate highly genetically related strains. I also studied the prevalence of mosaic toxin genes in C. botulinum Group III in animal botulism, mainly in poultry and bovine. The results brought out the mosaic toxin types C/D and D/C to be predominant in the samples investigated throughout Europe. Finally, I explored the full genome sequences of 14 types C/D and 3 types D/C C. botulinum Group III strains, mainly originating from French avian and bovine botulism outbreaks. Analyses of their genome sequences showed them to be closely related to other European strains from Group III. While studying their genetic content, I was able to point out that the extrachromosomal elements of strains type C/D could be used to generate a genetic ID card. Investigation of Crispr typing method showed to be irrelevant for type C/D, due to a deficient Crispr-Cas mechanism, but deserve more investigation for type D/C. The highest level of discrimination was achieved while using SNP core phylogeny, which allowed distinguishing up to the strain level. Here are the results I’m going to develop in this manuscript
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Etude des mécanismes évolutifs perturbant l’organisation des gènes dans les génomes de vertébrés / Analysis of evolutionary mecanisms altering gene organisation in vertebrate genomesBerthelot, Camille 28 September 2012 (has links)
Les phénomènes évolutifs qui perturbent l’organisation des gènes dans les génomes eucaryotes sont de deux types : les changements dans l’ordre des gènes, ou réarrangements, et les modifications du contenu en gènes du génome, par duplications, délétions ou gains de gènes. Ces processus sont mal connus, tant au niveau de leurs mécanismes d’apparition que de leur impact fonctionnel et sélectif. Ce travail de thèse s’articule autour de deux projets. Le premier s’intéresse à la distribution des points de cassure de réarrangements évolutifs entre un génome ancestral et ses descendants modernes. Cette distribution a été modélisée en fonction des caractéristiques locales du génome pour mettre en évidence quels facteurs influencent la probabilité de cassure. Nos résultats montrent que la distribution des cassures peut s’expliquer simplement comme une fonction de la longueur des espaces intergéniques, fonction qui est cependant non-linéaire contrairement aux attentes sous un régime aléatoire classique. La répartition des points de cassure dans les génomes semble principalement liée à des propriétés de structure, et n’est que peu soumise à des contraintes de sélection. Elle pourrait être liée à la structure chromatinienne du génome. Le second projet s’inscrit dans le cadre du séquençage du génome du poisson zèbre, et fournit un aperçu global de l’organisation de ce génome. Les génomes de poissons téléostéens sont anciennement dupliqués : l’analyse est axée sur les conséquences de cette duplication. Les résultats montrent que le génome du poisson zèbre présente une organisation assez typique d’un génome téléostéen. Les gènes retenus en deux copies après la duplication du génome appartiennent à des catégories fonctionnelles particulières, et sont biaisés vers des gènes déjà conservés après les duplications 1R et 2R ayant eu lieu au début de l’histoire des vertébrés. / Evolutionary processes disrupting the gene organisation in eukaryotic genomes belong to two categories: changes in the order of the genes, known as rearrangements, and changes in the content of the genome by gene duplications, deletions and gains. The mechanisms through which these events arise, and their functional and selective impact on genomes, are poorly understood. This thesis covers two different projects. Firstly, we investigated the distribution of rearrangement breakpoints between an ancestral genome and its modern descendants. This distribution was modelled according to local genomic characteristics to highlight factors influencing the breakage process. Our results show that the distribution of breakpoints can be simply explained as a function of intergenic spacers length, although in a non-linear fashion differing from classical random expectations. The repartition of breakpoints in genomes seems to be linked to structural properties, and is only marginally affected by selective constraints. It might in fact reflect local chromatin structure in the genome. The second project is part of the joint sequencing effort for the zebrafish genome, and provides an overview of the organisation of this genome. Teleost fish genomes are anciently duplicated: the analysis focuses on the consequences of this duplication. Results show that the zebrafish genome displays a typical teleost fish genome organisation. Genes retained in two copies after the whole genome duplication belong to specific functional categories, and are biased towards genes already conserved as duplicates after the 1R and 2R duplication events that have taken place early in vertebrate history.
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Chicken genome variations and selection : from sequences to consequencesKhoo, Choon-Kiat January 2017 (has links)
Chicken is a major protein source and intensively selected for economically important traits by humans. As such, this generated a huge range of phenotypes that representing a diverse spectrum of genetic variation. Understanding the functional basis of the genetic variants that underlie these traits, however, remains a formidable endeavour particularly for complex traits. Nonetheless, molecular phenotyping of an organism from sequenced data is doable with the advances in bioinformatics analysis and unparalleled surveys of genome wide genetic variants. This provides the opportunity to gain insights into the genome architecture and assists in identifying chromosomal regions underlying selection through a “sequences to consequences” approach. Combining a whole genome re-sequencing (WGS) approach with the knowledge of selection history, this thesis aimed to study the chromosomal regions and genetic variants underlying traits of interest in various selected chicken populations. To achieve this, genetic (quantitative and population genetics), genomic and bioinformatics approaches were employed and integrated to investigate the genome wide selection signatures in a number of different lines of chicken selected for different complex traits. This includes analysing: (i) divergently selected broilers for fatness traits (Chapter 2), (ii) a closed population of layer chickens (Chapter 3), (iii) selection signatures unique to broiler or layer chickens (Chapter 4) and (iv) selection signatures in colony stimulating factor 1 (CSF1) associated with gene expression differences in broiler and layer populations (Chapter 5). Candidate genes and nucleotides underlying potential selection regions were identified, and attempts were made to further elucidate the potential interplay between genes and the biological pathways involved in regulating traits in these selected chicken lines. Incorporating integrative approaches, variants within selection signatures were annotated to provide further evidence of their functional consequences. Overall, non-coding regions were enriched in selection signatures implied that causative variants may have regulatory roles. Capitalising on the millions of genetic variants discovered from WGS, chromosomal regions subject to selection were detected using a number of population genetics statistics. In broiler chicken lines divergently selected for very low-density plasma lipoprotein (VLDL) (Chapter 2), incorporating signatures of selection helped to improve the resolution of previously mapped quantitative traits loci (QTL) intervals. This research demonstrated that the integration of the analysis of selection signatures with functional annotation of genetic variants enabled refinement and characterisation of the QTL for fatness traits. In a closed population of brown leghorn layers (Chapter 3), evidence of selection signatures was found through Tajima’s D analysis. The analysis unravelled selection signatures encoding genes involved in numerous pathways and genes having key roles such as in behaviour, including feather pecking. Combining population differentiation statistic (FST) and Tajima’s D, a number of regions subject to divergent selection between broilers and white egg layers were identified (Chapter 4). Selection signatures were found to harbour mutations involved in cellular and tissue development, including genes having important roles in growth, fatness, egg shell strength and muscle development. These regions and the overlapping genes thereby may be potentially contributing to the different phenotypic variations observed between broilers and layers. In Chapter 5, a revised gene model for colony stimulating factor 1 (CSF1) showing complex pattern of alternate transcripts was predicted from transcriptome analysis of RNA isolated from 21 different tissues. In parallel, selection signatures analysis with the FST statistic, identified selection signatures that differentiate broilers to white egg layers (3 regions) or brown egg layers to white egg layers (4 regions). All these selection signatures were located within non-coding regions, indicating potential divergent selection of CSF1 within regulatory regions. Overall, the results presented in this thesis using the “sequences to consequences” approach, link several genomic regions and genes to phenotypic variation in domesticated chicken lines. The work reported here serves as a foundation for further study to decipher the relationship between “genotype and phenotype” and its functional consequences due to selection.
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CARBAPENEM-RESISTANT <em>ENTEROBACTERIACEAE</em>: EPIDEMIOLOGY, GENETICS, <em>IN VITRO</em> ACTIVITY, AND PHARMACODYNAMIC MODELINGKulengowski, Brandon 01 January 2019 (has links)
Background: Infections caused by carbapenem-resistant Enterobacteriaceae (CRE) such as Escherichia coli and Klebsiella pneumoniae are among the most urgent threats of the infectious disease realm. The incidence of these infections has been increasing over the years and due to very limited treatment options, mortality is estimated at about 50%. By 2050, mortality from antimicrobial resistant infections is expected to surpass cancer at 10 million deaths annually.
Methods: We evaluated 18 contemporary antimicrobials against 122 carbapenem-resistant Enterobacteriaceae using a variety of antimicrobial susceptibility testing methods according to Clinical Laboratory Standards Institute guidelines. Time-kill studies were performed on clinical isolates with variable resistance to meropenem, amikacin, and polymyxin B. Phenotypic expression assays were performed on all isolates and whole genome sequencing was performed on 8 isolates to characterize molecular resistance mechanisms. Pharmacodynamic modeling of meropenem and polymyxin B was also conducted.
Results: CRE were primarily K. pneumoniae, and Enterobacter spp. 60% expressed Klebsiella pneumoniae carbapenemase (KPC) only, 16% expressed Verona Integron-encoded Metallo-beta-lactamase (VIM) only, 5% expressed KPC and VIM, and 20% expressed other mechanisms of resistance. Antimicrobial susceptibility testing indicated the most active antimicrobials against CRE were ceftazidime/avibactam, imipenem/relebactam, amikacin, tigecycline, and the polymyxins. Etest® strips did not reliably measure polymyxin B resistance. The automated testing system, BD Phoenix™, consistently reported lower MICs than the gold standard broth microdilution. Time-kill studies showed regrowth at clinically achievable concentrations of meropenem alone (4, 16, and 64 mg/L), polymyxin B alone (0.25 and 1 mg/L), or amikacin alone (8 and 16 mg/L), but combinations of meropenem with either polymyxin B or amikacin were bactericidal and synergistic. Meropenem administered simultaneously or prior to polymyxin B exhibited superior activity to polymyxin B administered first.
Conclusions: Novel carbapenemase-inhibitor combinations (ceftazidime/avibactam and imipenem/relebactam) exhibit the best activity against KPC-producing CRE. The polymyxins, amikacin, and tigecycline exhibit the best activity against VIM-producing CRE. Meropenem in combination with polymyxin B is bactericidal and synergistic when the meropenem MIC is ≤32 mg/L, and meropenem should never be administered after polymyxin B. Meropenem and amikacin is bactericidal and synergistic when the amikacin MIC is ≤16 mg/L. Etest® strips should not be used for characterizing polymyxin B or colistin activity. Clinicians should be aware that automated testing systems may produce biased susceptibility results relative to the gold standard method, broth microdilution, which may influence interpretation of in vitro results.
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Genomic Detection Using Sparsity-inspired ToolsJanuary 2011 (has links)
Genome-based detection methods provide the most conclusive means for establishing the presence of microbial species. A prime example of their use is in the detection of bacterial species, many of which are naturally vital or dangerous to human health, or can be genetically engineered to be so. However, current genomic detection methods are cost-prohibitive and inevitably use unique sensors that are specific to each species to be detected. In this thesis we advocate the use of combinatorial and non-specific identifiers for detection, made possible by exploiting the sparsity inherent in the species detection problem in a clinical or environmental sample. By modifying the sensor design process, we have developed new molecular biology tools with advantages that were not possible in their previous incarnations. Chief among these advantages are a universal species detection platform, the ability to discover unknown species, and the elimination of PCR, an expensive and laborious amplification step prerequisite in every molecular biology detection technique. Finally, we introduce a sparsity-based model for analyzing the millions of raw sequencing reads generated during whole genome sequencing for species detection, and achieve significant reductions in computational speed and high accuracy.
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