Ferramentas para comparação genomica

Orientador : João Carlos SetubalTese (doutorado) - Universidade Estadual de Campinas, Instituto de ComputaçãoResumo: Com o crescente número de genomas seqüenciados e publicados, surge a necessidade de se analisar as seqüências geradas, com o objetivo de se entender melhor caracterizações funcionais dos organismos estudados, assim como aspectos evolutivos. Um projeto genoma de um organismo, em especial de um procarioto, consiste essencialmente de três grandes fases: o seqüenciamento, a anotação e a análise. A última etapa, por sua vez, consiste na tentativa de se obter uma visão global do genoma a partir da anotação e a partir de outras análises, como por exemplo a comparação com outros genomas. E é nesse contexto, comparação de genomas, que esta tese se insere. Nosso trabalho propõe metodologias para comparação detalhada de dois genomas, tanto no nível de DNA, quanto no de seus genes, assim como a implementação dessas metodologias. O principal objetivo é fornecer ao usuário um conjunto de ferramentas para caracterização funcional do organismo estudado, servindo também como ferramental auxiliar na anotaçãoAbstract: With increasing availability of published genome sequences, we need to analyse them in order to understand functional and evolutionary issues of the organisms. A genome project, in particular for prokaryotes, consists of three main phases: sequencing, annotation and analysis. The last phase consists of getting an overview of the genome from the annotation and other analysis, like comparison to other genomes, for example. This thesis is about genome comparison. We propose methodologies for detailed comparison of two genomes, at the DNA and their genes levels. The main goal is to provide a set of tools for functional characterization of organisms, serving also as an auxiliar tool for annotationDoutoradoDoutor em Ciência da Computaçã

A method to find groups of orthogous genes across multiple genomes

In this work we propose a simple method to obtain groups of homologous genes across multiple (k) organisms, called kGC. Our method takes as input all-against-all Blastp comparisons and produces groups of homologous sequences. First, homologies among groups of paralogs of all the k compared genomes are found, followed by homologies of groups among k - 1 genomes and so on, until groups belonging exclusively to only one genome, that is, groups of one genome not presenting strong similarities with any group of any other genome, are identified. We have used our method to determine homologous groups across six Actinobacterial complete genomes. To validate kGC, we first investigate the Pfam classification of the homologous groups, and after compare our results with those produced by OrthoMCL. Although kGC is much simpler than OrthoMCL it presented similar results with respect to Pfam classification

Live neighbor-joining

Background: In phylogenetic reconstruction the result is a tree where all taxa are leaves and internal nodes are hypothetical ancestors. In a live phylogeny, both ancestral and living taxa may coexist, leading to a tree where internal nodes may be living taxa. The well-known Neighbor-Joining heuristic is largely used for phylogenetic reconstruction. Results: We present Live Neighbor-Joining, a heuristic for building a live phylogeny. We have investigated Live Neighbor-Joining on datasets of viral genomes, a plausible scenario for its application, which allowed the construction of alternative hypothesis for the relationships among virus that embrace both ancestral and descending taxa. We also applied Live Neighbor-Joining on a set of bacterial genomes and to sets of images and texts. Non-biological data may be better explored visually when their relationship in terms of content similarity is represented by means of a phylogeny. Conclusion: Our experiments have shown interesting alternative phylogenetic hypothesis for RNA virus genomes, bacterial genomes and alternative relationships among images and texts, illustrating a wide range of scenarios where Live Neighbor-Joining may be used

Draft genome sequence of Mycobacterium bovis 04-303, a highly virulent strain from Argentina

Mycobacterium bovis strain 04-303 was isolated from a wild boar living in a free-ranging field in Argentina. This work reports the draft genome sequence of this highly virulent strain and the genomic comparison of its major virulence-related genes with those of M. bovis strain AF2122/97 and Mycobacterium tuberculosis strain H37Rv.Instituto de BiotecnologíaFil: Nishibe, Christiane. Universidade Federal de Mato Grosso do Sul. Faculdade de Computação; BrasilFil: Canevari Castelão, Ana Beatriz. Universidade Federal de Mato Grosso do Sul. Programa de Pós-Graduação em Ciência Animal; BrasilFil: Dalla Costa, Ricardo. Life Technologies do Brasil; BrasilFil: Pinto, Beatriz Jeronimo. Life Technologies do Brasil; BrasilFil: Varuzza, Leonardo. Life Technologies do Brasil; BrasilFil: Cataldi, Angel Adrian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Bernardelli, Amelia. Ceva Salud Animal; ArgentinaFil: Bigi, Fabiana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Blanco, Federico Carlos. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Zumarraga, Martin Jose. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Almeida, Nalvo Franco. Universidade Federal de Mato Grosso do Sul. Faculdade de Computação; BrasilFil: Araujo, Flabio Ribeiro de. Empresa Brasileira de Pesquisa Agropecuária (Embrapa). Gado de Corte; Brasi

Bioinformatics of the Paracoccidioides brasiliensis EST Project

Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, an endemic mycosis of Latin America. This fungus presents a dimorphic character; it grows as a mycelium at room temperature, but it is isolated as yeast from infected individuals. It is believed that the transition from mycelium to yeast is important for the infective process. The Functional and Differential Genome of Paracoccidioides brasiliensis Project - PbGenome Project was developed to study the infection process by analyzing expressed sequence tags - ESTs, isolated from both mycelial and yeast forms. The PbGenome Project was executed by a consortium that included 70 researchers (professors and students) from two sequencing laboratories of the midwest region of Brazil; this project produced 25,741 ESTs, 19,718 of which with sufficient quality to be analyzed. We describe the computational procedures used to receive process, analyze these ESTs, and help with their functional annotations; we also detail the services that were used for sequence data exploration. Various programs were compared for filtering and grouping the sequences, and they were adapted to a user-friendly interface. This system made the analysis of the differential transcriptome of P. brasiliensis possible

Transcriptional remodeling patterns in murine dendritic cells infected with Paracoccidioides brasiliensis : more is not necessarily better

Most people infected with the fungus Paracoccidioides spp. do not get sick, but approximately 5% develop paracoccidioidomycosis. Understanding how host immunity determinants influence disease development could lead to novel preventative or therapeutic strategies; hence, we used two mouse strains that are resistant (A/J) or susceptible (B10.A) to P. brasiliensis to study how dendritic cells (DCs) respond to the infection. RNA sequencing analysis showed that the susceptible strain DCs remodeled their transcriptomes much more intensely than those from the resistant strain, agreeing with a previous model of more intense innate immunity response in the susceptible strain. Contrastingly, these cells also repress genes/processes involved in antigen processing and presentation, such as lysosomal activity and autophagy. After the interaction with P. brasiliensis, both DCs and macrophages from the susceptible mouse reduced the autophagy marker LC3-II recruitment to the fungal phagosome compared to the resistant strain cells, confirming this pathway’s repression. These results suggest that impairment in antigen processing and presentation processes might be partially responsible for the inefficient activation of the adaptive immune response in this model

Origin and diversification of Xanthomonas citri subsp. citri pathotypes revealed by inclusive phylogenomic, dating, and biogeographic analyses.

Xanthomonas citri subsp. citri pathotypes cause bacterial citrus canker, being responsible for severe agricultural losses worldwide. The A pathotype has a broad host spectrum, while A* and Aw are more restricted both in hosts and in geography. Two previous phylogenomic studies led to contrasting well-supported clades for sequenced genomes of these pathotypes. No extensive biogeographical or divergence dating analytic approaches have been so far applied to available genomes. Results: Based on a larger sampling of genomes than in previous studies (including six new genomes sequenced by our group, adding to a total of 95 genomes), phylogenomic analyses resulted in different resolutions, though overall indicating that A?+?AW is the most likely true clade. Our results suggest the high degree of recombination at some branches and the fast diversification of lineages are probable causes for this phylogenetic blurring effect. One of the genomes analyzed, X. campestris pv. durantae, was shown to be an A* strain; this strain has been reported to infect a plant of the family Verbenaceae, though there are no reports of any X. citri subsp. citri pathotypes infecting any plant outside the Citrus genus. Host reconstruction indicated the pathotype ancestor likely had plant hosts in the family Fabaceae, implying an ancient jump to the current Rutaceae hosts. Extensive dating analyses indicated that the origin of X. citri subsp. citri occurred more recently than the main phylogenetic splits of Citrus plants, suggesting dispersion rather than host-directed vicariance as the main driver of geographic expansion. An analysis of 120 pathogenic-related genes revealed pathotype-associated patterns of presence/absence. Conclusions: Our results provide novel insights into the evolutionary history of X. citri subsp. citri as well as a sound phylogenetic foundation for future evolutionary and genomic studies of its pathotypes

Detection and identification of Xanthomonas pathotypes associated with citrus diseases using comparative genomics and multiplex PCR.

Background. In Citrus cultures, three species of Xanthomonas are known to cause distinct diseases. X. citri subsp. citri patothype A, X. fuscans subsp. aurantifolii pathotypes B and C, and X. alfalfae subsp. citrumelonis, are the causative agents of cancrosis A, B, C, and citrus bacterial spots, respectively. Although these species exhibit different levels of virulence and aggressiveness, only limited alternatives are currently available for proper and early detection of these diseases in the fields. The present study aimed to develop a new molecular diagnostic method based on genomic sequences derived from the four species of Xanthomonas. Results. Using comparative genomics approaches, primers were synthesized for the identification of the four causative agents of citrus diseases. These primers were validated for their specificity to their target DNA by both conventional and multiplex PCR. Upon evaluation, their sensitivity was found to be 0.02 ng/?l in vitro and 1.5 ? 104 CFU ml?1 in infected leaves. Additionally, none of the primers were able to generate amplicons in 19 other genomes of Xanthomonas not associated with Citrus and one species of Xylella, the causal agent of citrus variegated chlorosis (CVC). This denotes strong specificity of the primers for the different species of Xanthomonas investigated in this study. Conclusions. We demonstrated that these markers can be used as potential candidates for performing in vivo molecular diagnosis exclusively for citrus-associated Xanthomonas. The bioinformatics pipeline developed in this study to design specific genomic regions is capable of generating specific primers. It is freely available and can be utilized for any other model organism

Transcriptional profiles of the human pathogenic fungus paracoccidioides brasiliensis in mycelium and yeast cells

This work was supported by MCT, CNPq, CAPES, FUB, UFG, and FUNDECT-MS. PbGenome Network: Alda Maria T. Ferreira, Alessandra Dantas, Alessandra J. Baptista, Alexandre M. Bailão, Ana Lídia Bonato, André C. Amaral, Bruno S. Daher, Camila M. Silva, Christiane S. Costa, Clayton L. Borges, Cléber O. Soares, Cristina M. Junta, Daniel A. S. Anjos, Edans F. O. Sandes, Eduardo A. Donadi, Elza T. Sakamoto-Hojo, Flábio R. Araújo, Flávia C. Albuquerque, Gina C. Oliveira, João Ricardo M. Almeida, Juliana C. Oliveira, Kláudia G. Jorge, Larissa Fernandes, Lorena S. Derengowski, Luís Artur M. Bataus, Marcus A. M. Araújo, Marcus K. Inoue, Marlene T. De-Souza, Mauro F. Almeida, Nádia S. Parachin, Nadya S. Castro, Odair P. Martins, Patrícia L. N. Costa, Paula Sandrin-Garcia, Renata B. A. Soares, Stephano S. Mello, and Viviane C. B. ReisParacoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, a disease that affects 10 million individuals in Latin America. This report depicts the results of the analysis of 6,022 assembled groups from mycelium and yeast phase expressed sequence tags, covering about 80% of the estimated genome of this dimorphic, thermo-regulated fungus. The data provide a comprehensive view of the fungal metabolism, including overexpressed transcripts, stage-specific genes, and also those that are up- or down-regulated as assessed by in silico electronic subtraction and cDNA microarrays. Also, a significant differential expression pattern in mycelium and yeast cells was detected, which was confirmed by Northern blot analysis, providing insights into differential metabolic adaptations. The overall transcriptome analysis provided information about sequences related to the cell cycle, stress response, drug resistance, and signal transduction pathways of the pathogen. Novel P. brasiliensis genes have been identified, probably corresponding to proteins that should be addressed as virulence factor candidates and potential new drug targets