Genome-wide association studies have very successfully found highly significant disease associations with single nucleotide polymorphisms (SNP) in the Major Histocompatibility Complex for adverse drug reactions, autoimmune diseases and infectious diseases. However, the extensive linkage disequilibrium in the region has made it difficult to unravel the HLA alleles underlying these diseases. Here I present two methods to comprehensively predict 4-digit HLA types from the two types of experimental genome data widely available. The Virtual SNP Imputation approach was developed for genome scan data and demonstrated a high precision and recall (96% and 97% respectively) for the prediction of HLA genotypes. A reanalysis of 6 genome-wide association studies using the HLA imputation method identified 18 significant HLA allele associations for 6 autoimmune diseases: 2 in ankylosing spondylitis, 2 in autoimmune thyroid disease, 2 in Crohn's disease, 3 in multiple sclerosis, 2 in psoriasis and 7 in rheumatoid arthritis. The EPIGEN consortium also used the Virtual SNP Imputation approach to detect a novel association of HLA-A*31:01 with adverse reactions to carbamazepine. For the prediction of HLA genotypes from next generation sequencing data, I developed a novel approach using a naïve Bayes algorithm called HLA-Genotyper. The validation results covered whole genome, whole exome and RNA-Seq experimental designs in the European and Yoruba population samples available from the 1000 Genomes Project. The RNA-Seq data gave the best results with an overall precision and recall near 0.99 for Europeans and 0.98 for the Yoruba population. I then successfully used the method on targeted sequencing data to detect significant associations of idiopathic membranous nephropathy with HLA-DRB1*03:01 and HLA-DQA1*05:01 using the 1000 Genomes European subjects as controls. Using the results reported here, researchers may now readily unravel the association of HLA alleles with many diseases from genome scans and next generation sequencing experiments without the expensive and laborious HLA typing of thousands of subjects. Both algorithms enable the analysis of diverse populations to help researchers pinpoint HLA loci with biological roles in infection, inflammation, autoimmunity, aging, mental illness and adverse drug reactions.

Next-generation sequencing technologies are revolutionising our ability to characterise and investigate infectious diseases. Utilising the power of high throughput sequencing, this study reports, the development of a sensitive, non-PCR based, unbiased amplification method, which allows the rapid and accurate sequencing of multiple microbial pathogens directly from clinical samples. The method employs Φ29 DNA polymerase, a highly efficient enzyme able to produce strand displacement during the polymerisation process with high fidelity. Problems with DNA secondary structure were overcome and the method optimised to produce sufficient DNA to sequence from a single bacterial cell in two hours. Evidence was also found that the enzyme requires at least six bases of single stranded DNA to initiate replication, and is not capable of amplification from nicks. Φ29 multiple displacement amplification was shown to be suitable for a range of GC contents and bacterial cell wall types as well as for viral pathogens. The method was shown to be able to provide relative quantification of mixed cells, and a method for quantification of viruses using a known standard was developed. To complement the novel molecular biology workflow, a data analysis pipeline was developed to allow pathogen identification and characterisation without prior knowledge of input. The use of de novo assemblies for annotation was shown to be equivalent to the use of polished reference genomes. Single cell Φ29 MDA samples had better assembly and annotation than non-amplification controls, a novel finding which, when combined with the very long DNA fragments produced, has interesting implications for a variety of analytical procedures. A sampling process was developed to allow isolation and amplification of pathogens directly from clinical samples, with good concordance shown between this method and traditional testing. The process was tested on a variety of modelled and real clinical samples showing good application to sterile site infections, particularly bacteraemia models. Within these samples multiple bacterial, viral and parasitic pathogens were identified, showing good application across multiple infection types. Emerging pathogens were identified including Onchocerca volvulus within a CSF sample, and Sneathia sanguinegens within an STI sample. Use of Φ29 MDA allows rapid and accurate amplification of whole pathogen genomes. When this is coupled with the sample processing developed here it is possible to detect the presence of pathogens in sterile sites with a sensitivity of a single genome copy.

O objetivo deste trabalho foi caracterizar os mecanismos adquiridos de resistência de isolados de enterobactérias produtoras de carbapenemases utilizando a tecnologia de sequenciamento de nova geração. Foram incluídos no estudo quatro isolados – três Escherichia coli e uma Serratia marcescens – produtores de diferentes carbapenemases – OXA-370, KPC-2, NDM-1 e GES-5, respectivamente, obtidos a partir de um estudo de vigilância para detecção de carbapenemases. O DNA total dos isolados foi extraído utilizando kits comerciais e submetido à fragmentação enzimática para a obtenção de bibliotecas genômicas de aproximadamente 300 pares de bases. Após a preparação das bibliotecas, elas foram carregadas em chips 316 v2 para a plataforma Ion Torrent PGM e submetidas ao sequenciamento, utilizando um programa de 850 flows. Para cada genoma, foram obtidos aproximadamente um milhão de reads, os quais foram submetidos ao processo de montagem para a obtenção de genomas de aproximadamente 5Mb com uma cobertura de, em média, 175 vezes. As sequências obtidas foram submetidas à anotação utilizando o sistema RAST e a ferramenta online ResFinder. Sequências de inserção foram pesquisadas utilizando a ferramenta ISFinder. Além das carbapenemases, genes que codificam para outras β-lactamases (blaTEM-1, blaCTX-M-2 blaCTX-M-8, blaOXA-1, blaOXA-2) foram encontrados em todos os genomas. AMEs (aadA1, aph(3’)-la, aac(3)-IIa, strA, strB, aac(6’)Ib-cr, aac(6’)-Ib and aac(6`)-Ic), bem como genes que codificam resistência às sulfonamidas e ao trimetoprim também foram comuns aos isolados avaliados. Os seguintes ambientes genéticos foram observados: blaOXA-370 é flanqueada pela IS5075-like, blaKPC-2 está inserida no transposon Tn4401, e blaNDM-1, no transposon Tn3000. Cada isolado de E. coli pertenceu a um sequence type (ST) distinto: 1099F pertence à ST617, 1326F, à ST648, e 2610F, à ST707. Nossos resultados indicaram a diversidade de genes de resistência que podem ser encontrados em um isolado clínico, ressaltaram a variedade de contextos genéticos em que as carbapenemases podem estar inseridas e demonstraram que o sequenciamento de nova geração pode ser utilizado como uma ferramenta para a caracterização de isolados bacterianos multirresistentes, auxiliando, entre outros aspectos, na tipagem e na identificação de determinantes de resistência. / The aim of this study was to characterize the resistome of carbapenemase-producing Enterobacteriaceae using a next-generation sequencing platform. Four isolates were included in this study – three Escherichia coli and one Serratia marcescens – producing different carbapenemases – OXA-370, KPC-2, NDM-1 and GES-5, respectively, obtained from a surveillance study for carbapenemase detection. Total DNA was extracted using commercially available kits and submitted to enzymatic fragmentation to obtain libraries of around 300 base pairs of each isolate. After library preparation, they were loaded in 316 v2 chips for Ion Torrent PGM platform, and sequencing was performed using an 850 flows program. For each genome, approximately a million reads were obtained, and they were further assembled. The genome length for each isolate was of around 5Mb, with a mean coverage of 175x. The RAST system and the online tool ResFinder were used for annotation, as well as ISFinder. Besides the carbapenemases, genes encoding for other β-lactamases (blaTEM-1, blaCTX-M-2 blaCTX-M-8, blaOXA-1, blaOXA-2) were found in all genomes. AMEs (aadA1, aph(3’)-la, aac(3)-IIa, strA, strB, aac(6’)Ib-cr, aac(6’)-Ib and aac(6`)-Ic) were also detected in every isolate included in the study, as well as genes encoding for resistance determinants to sulfonamides and trimetoprim. The genetic environment of the carbapenemases was very similar to other isolates described in the literature – blaOXA-370 is flanked by IS5075-like, blaKPC-2 is inserted in transposon Tn4401, and blaNDM-1 was found in transposon Tn3000. Each isolate belonged to a distinct ST – 1099F is part of ST617, 1326F belonged to ST648 and 2610F, to ST707. Our results demonstrated the diversity of resistance determinants that can be found in a clinical isolate, highlighted the variability of genetic environments in which carbapenemases can be present and demonstrated that next-generation sequencing is a valuable tool for the characterization of multidrug-resistant isolates, and can provide information regarding the molecular typing and the identification of resistance determinants.

The 2019 Research Symposium will bring together leaders in research and training from colleges and schools of pharmacy to discuss opportunities and challenges in research training at pharmacy schools. Symposium topics will include engaging pharmacy students in research projects and strengthening the pipeline for pharmacy research, integrating basic and clinical sciences in pharmacy schools, the value of research experience for building professional competencies and more. Through a mix of presentations, panel discussion and open forums, symposium participants will share experience and ideas and seek to identify points of potential collaboration to advance pharmacy research training. Deans, department chairs and faculty involved or interested in expanding research training are encouraged to attend.

It is estimated that 4000 genetic diseases/syndromes affect humans with one third of these diseases involving the eye. Many eye disorders, such as age-related macular degeneration that affects an estimated 170 million elderly adults worldwide, are associated with genetic variants. Since the conception of the human genome project we have learned a great deal about the genetic make-up of the human race and have identified over ~20,000 genes. Over 270 of these genes have been implicated in retinal diseases alone with many more genes involved in other forms of ocular disease. Though we have made a great deal of progress in understanding the genetics of eye disease, there remain many eye diseases with significant evidence of genetic components for which a disease-causing gene has not been identified. In my thesis research, I utilized computational tools and strategies to analyze microarrays and whole-exome sequencing to investigate the genetic causes of three different eye diseases. First, I utilized a combination of familial analyses and whole-exome sequencing to study the genetic cause of Keratoconus, a progressive cornea abnormality that can lead to distorted vision and light sensitivity. Second, I analyzed three different cohorts of patients with Bardet-Biedl syndrome (BBS), a syndromic retinopathy leading to blindness, using whole-exome sequencing to identify both known and novel genetic causes of BBS. Finally, I performed the largest whole-exome sequencing study at the time for Pigment Dispersion Syndrome (PDS), a disorder associated with glaucoma, and identified variants within previously established candidate genes and a novel candidate gene that is now the subject of further scientific investigation. By using computational tools and strategies in tandem with high-quality bench research performed by fellow lab members, we have identified both candidate and known eye disease-causing genes/mutations and furthered the goal to cure blindness.

Next generation monoclonal antibodies (mAbs) are unique in that they are specifically designed to enhance their mechanisms of action, primarily complement fixation and antibody-dependent cellular cytotoxicity (ADCC). Recent studies suggest that complement-fixing properties of a mAb can counter its ability to activate NK cells and mediate ADCC. GA101, a third generation (type II anti-CD20) mAb, and rituximab-MAGE (glyco-engineered type I mAb) show enhanced ADCC and direct cell killing; while ofatumumab, a second generation anti-CD20 mAb, shows enhanced complement-mediated cytotoxicity (CMC). These studies set out to determine the primary mechanisms of actions of these various mAbs, and compare the effect of complement on their ability to activate NK cells and mediate ADCC or CMC. We also studied the efficiency of rituximab vs. rituximab-MAGE to deplete B-cells in vivo in mice expressing human transgenic CD20. In vitro, rituximab and ofatumumab fixed more complement and mediated a greater degree of CMC, than GA101 and rituximab-MAGE. Additionally, complement inhibited the ability of both rituximab and ofatumumab to bind to and activate NK cells, whereas, addition of complement to GA101 or rituximab-MAGE did not affect their NK cell activating ability. Complement also blocked rituximab-induced NK-cell mediated ADCC, but not GA101-induced NK-cell mediated ADCC. Finally, GA101 and rituximab-MAGE depleted a higher percentage of B cells in whole blood compared to rituximab and ofatumumab, whereas rituximab-MAGE depleted fewer B cells, in vivo, in a complement-dependent fashion. We conclude from these studies that there are significant differences among these antibodies and that the ability of a given antibody to mediate CMC and complement fixation correlates with the ability of complement to block the interaction between the antibody and NK cells.

O comportamento social pode ser descrito como qualquer atividade de interação intraespecífica incluindo a escolha entre parceiros reprodutivos, reconhecimento da espécie, comportamento altruísta e organização da sociedade animal. Entre as espécies de animais mais sintonizadas com seu ambiente social estão os insetos que, como por exemplo nas espécies de abelhas das tribos Apini e Meliponini, apresentam um padrão complexo de socialidade conhecido como comportamento altamente eussocial. As abelhas constituem um grupo ideal para o estudo das bases da evolução deste comportamento, pois apresentam uma grande diversidade de organização social, desde espécies solitárias até altamente eussociais. Embora a evolução da eussocialidade tenha sido motivo de muitos estudos, as mudanças genéticas envolvidas nesse processo não são completamente conhecidas. Dados da literatura fornecem um ponto de partida para o entendimento da relação entre alterações gênicas específicas e a eussocialidade, mas questões fundamentais na evolução do comportamento social ainda precisam ser respondidas. Recentemente, novas tecnologias de sequenciamento têm permitido o estudo de organismos modelo e não modelo de forma mais detalhada e não direcional. Análises deste tipo são promissoras para o estudo evolutivo de características complexas como o comportamento. Neste contexto, realizamos um amplo estudo sobre as bases moleculares envolvidas em diferentes características comportamentais relacionadas à evolução da socialidade em abelhas. Para tanto, o padrão global de expressão de genes, em espécies e fases do desenvolvimento distintas, foram analisados comparativamente através de múltiplas abordagens. No Capítulo 1, utilizamos contaminantes do transcriptoma da abelha solitária Tetrapedia diversipes para analisar os recursos florais utilizados por esta espécie em suas duas gerações reprodutivas. Neste estudo concluímos que a riqueza de espécies visitadas durante a primeira geração é muito maior do que durante a segunda geração, o que está provavelmente relacionado à floração de primavera durante o primeiro período reprodutivo. No Capítulo 2, verificamos que o padrão de expressão dos genes das fêmeas fundadoras possivelmente afeta o desenvolvimento larval em T. diversipes. O padrão bivoltino de reprodução desta espécie, com diapausa em uma das gerações, pode ser importante para a evolução do comportamento social. Além disso, entre os genes possivelmente envolvidos nessa característica, podemos encontrar genes mitocondriais e lncRNAs. Os resultados obtidos no Capítulo 3 sugerem que a especialização em subcastas de operárias ocorreu posteriormente nas diferentes linhagens de abelhas, envolvendo genes específicos. No entanto, esses genes afetam processos biológicos comuns nas diferentes espécies. Por sua vez, o Capítulo 4 apresenta um método promissor para a identificação de genes comportamentais em diferentes espécies de abelhas, através de uma análise de expressão comparativa. Com base nessas análises, 787 genes comportamentais, que possivelmente fazem parte de um toolkit eussocial em abelhas, foram encontrados. O padrão de metilação desses genes, em espécies com diferentes níveis sociais, indicou ainda que o contexto genômico da metilação pode ser relevante para eussocialidade. Os resultados obtidos nesses estudos apresentam novas perspectivas metodológicas e evolutivas para o estudo da evolução do comportamento social em abelhas / The social behaviour can be widely described as any intraspecific interaction in the animal life, including but not restricted to, female choice, species recognition, altruistic behaviour and the organization of animal society. Among the animal species most attuned to their social environment are the insects that, for example, in the Apini and Meliponini tribes, present a complex behaviour known as highly eusocial. Bees are an ideal group to study the evolution of the social behaviour because they have a great diversity of social life styles that evolved independently. The tribes Apini and Meliponini comprise only highly eusocial species whereas various levels of sociality can be detected in other tribes, being most bees indeed solitary. Although the evolution of eusociality has been the subject of many studies, the genetic changes involved in the process have not been completely understood. Results from studies conducted so far provide a starting point for the connection between specific genetic alterations and the evolution of eusocial behaviour. However fundamental questions about this process are still open. Recently, new sequencing technologies have allowed genetic studies of model and non-model organisms in a deep and non-directional way, which is promising for the study of complex characteristics. Herein, we present a broad analysis of the molecular bases of different behavioural characteristics related to the evolution of sociality in bees. To that end, the global expression pattern of genes involved in different behavioural features, in a number of bee species and distinct developmental stages, was comparatively studied using multiple approaches. Through these approaches different results were obtained. In Chapter 1, we used contaminant transcripts from the solitary bee Tetrapedia diversipes to identify the plants visited by this bee, during its two reproductive generations. These contaminant transcripts revealed that the richness of plant species visited during the first reproductive generation was considerably greater than during the second generation. Which is probably related to the floral boom occurring in spring during the first reproductive period. In Chapter 2, data suggests that the expression pattern in foundresses affect larval development in T. diversipes. The bivoltinism presented by this species, with diapause in one generation, might be an important feature for the evolution of sociality. Our results suggest that mitochondrial genes and lncRNAs are involved in this reproductive pattern. Results described in Chapter 3 indicate that specialization in worker subcastes occurred posteriorly in distinct bee lineages, driven by specific genes. However, these genes affected common biological processes in the different species. In Chapter 4 is described a promising analyses method to identify, comparatively, genes involved in bee social behaviour. Using this approach, we identified 787 behavioural genes that might be involved in social behaviour of different species. The methylation pattern of these genes suggests that the DNA context in which methylation marks occur, might be especially relevant to bee sociality. Results obtained here presents new methodological and evolutionary approaches to the study of social behaviour in bees

Massive sequencing has transformed the field of genome biology due to the continuous introduction and evolution of new methods. In recent years, the technologies available to read through genomes have undergone an unprecedented rate of development in terms of cost-reduction. Generating sequence data has essentially ceased to be a bottleneck for analyzing genomes instead to be replaced by limitations in sample preparation and data analysis. In this work, new strategies are presented to increase both the throughput of library generation prior to sequencing, and the informational content of libraries to aid post-sequencing data processing. The protocols developed aim to enable new possibilities for genome research concerning project scale and sequence complexity. The first two papers that underpin this thesis deal with scaling library production by means of automation. Automated library preparation is first described for the 454 sequencing system based on a generic solid-phase polyethylene-glycol precipitation protocol for automated DNA handling. This was one of the first descriptions of automated sample handling for producing next generation sequencing libraries, and substantially improved sample throughput. Building on these results, the use of a double precipitation strategy to replace the manual agarose gel excision step for Illumina sequencing is presented. This protocol considerably improved the scalability of library construction for Illumina sequencing. The third and fourth papers present advanced strategies for library tagging in order to multiplex the information available in each library. First, a dual tagging strategy for massive sequencing is described in which two sets of tags are added to a library to trace back the origins of up to 4992 amplicons using 122 tags. The tagging strategy takes advantage of the previously automated pipeline and was used for the simultaneous sequencing of 3700 amplicons. Following that, an enzymatic protocol was developed to degrade long range PCR-amplicons and forming triple-tagged libraries containing information of sample origin, clonal origin and local positioning for the short-read sequences. Through tagging, this protocol makes it possible to analyze a longer continuous sequence region than would be possible based on the read length of the sequencing system alone. The fifth study investigates commonly used enzymes for constructing libraries for massive sequencing. We analyze restriction enzymes capable of digesting unknown sequences located some distance from their recognition sequence. Some of these enzymes have previously been extensively used for massive nucleic acid analysis. In this first high throughput study of such enzymes, we investigated their restriction specificity in terms of the distance from the recognition site and their sequence dependence. The phenomenon of slippage is characterized and shown to vary significantly between enzymes. The results obtained should favor future protocol development and enzymatic understanding. Through these papers, this work aspire to aid the development of methods for massive sequencing in terms of scale, quality and knowledge; thereby contributing to the general applicability of the new paradigm of sequencing instruments. / <p>QC 20121126</p>

DNA sequence assembly is one of the fundamental areas of bioinformatics. It involves the correct formation of a genome sequence from its DNA fragments ("reads") by aligning and merging the fragments. There are different sequencing technologies -- some support long DNA reads and the others, shorter DNA reads. There are sequence assembly programs specifically designed for these different types of raw sequencing data.<p> This work explores and experiments with these different types of assembly software in order to compare their performance on the type of data for which they were designed, as well as their performance on data for which they were not designed, and on mixed data. Such results are useful for establishing good procedures and tools for sequence assembly in the current genomic environment where read data of different lengths are available. This work also investigates the effect of the presence or absence of quality information on the results produced by sequence assemblers.<p> Five strategies were used in this research for assembling mixed data sets and the testing was done using a collection of real and artificial data sets for six bacterial organisms. The results show that there is a broad range in the ability of some DNA sequence assemblers to handle data from various sequencing technologies, especially data other than the kind they were designed for. For example, the long-read assemblers PHRAP and MIRA produced good results from assembling 454 data. The results also show the importance of having an effective methodology for assembling mixed data sets. It was found that combining contiguous sequences obtained from short-read assemblers with long DNA reads, and then assembling this combination using long-read assemblers was the most appropriate approach for assembling mixed short and long reads. It was found that the results from assembling the mixed data sets were better than the results obtained from separately assembling individual data from the different sequencing technologies. DNA sequence assemblers which do not depend on the availability of quality information were used to test the effect of the presence of quality values when assembling data. The results show that regardless of the availability of quality information, good results were produced in most of the assemblies.<p> In more general terms, this work shows that the approach or methodology used to assemble DNA sequences from mixed data sources makes a lot of difference in the type of results obtained, and that a good choice of methodology can help reduce the amount of effort spent on a DNA sequence assembly project.

This thesis describes the design of a Virtual Application Networking Infrastructure (VANI) node that can be used to facilitate network architecture experimentation. Cur- rently the VANI nodes provide four classes of physical resources – processing, reconfig- urable hardware, storage and interconnection fabric – but the set of sharable resources can be expanded. Virtualization software allows slices of these resources to be appor- tioned to VANI nodes that can in turn be interconnected to form virtual networks, which can operate according to experimental network and application protocols. This thesis discusses the design decisions that have been made in the development of this system and provides a detailed description of the prototype, including how users interact with the resources and the interfaces provided by the virtualization layers.