There has been tremendous advancement in sequencing technologies; the rate at which sequencing data can be generated has increased multifold while the cost of sequencing continues on a downward descent. Sequencing data provide novel insights into the ecological environment of microbes as well as human health and disease status but challenge investigators with a variety of computational issues. This thesis focuses on three common problems in the analysis of high-throughput data. The goals of the first project are to (1) develop a statistical framework and a complete software pipeline for metagenomics that identifies microbes to the strain level and thus facilitating a personalized drug treatment targeting the strain; and (2) estimate the relative content of microbes in a sample as accurately and as quickly as possible. The second project focuses on the analysis of the microbiome variation across multiple samples. Studying the variation of microbiomes under different conditions within an organism or environment is the key to diagnosing diseases and providing personalized treatments. The goals are to (1) identify various statistical diversity measures; (2) develop confidence regions for the relative abundance estimates; (3) perform multi-dimensional and differential expression analysis; and (4) develop a complete pipeline for multi-sample microbiome analysis. The third project is focused on batch effect analysis. When analyzing high dimensional data, non-biological experimental variation or “batch effects” confound the true associations between the conditions of interest and the outcome variable. Batch effects exist even after normalization. Hence, unless the batch effects are identified and corrected, any attempts for downstream analyses, will likely be error prone and may lead to false positive results. The goals are to (1) analyze the effect of correlation of the batch adjusted data and develop new techniques to account for correlation in two step hypothesis testing approach; (2) develop a software pipeline to identify whether batch effects are present in the data and adjust for batch effects in a suitable way. In summary, we developed software pipelines called PathoScope, PathoStat and BatchQC as part of these projects and validated our techniques using simulation and real data sets.

O material lignocelulósico, presente na biomassa vegetal, representa uma importante fonte de energia, entretanto necessita da ação das enzimas lignocelulolíticas para sua degradação. A busca por novas enzimas que atuam na quebra da parede celular da planta em comunidades microbianas evoluídas naturalmente em um ambiente de degradação de biomassa como o rúmen oferece uma estratégia promissora para a prospecção de genes. Com isso, o projeto teve como objetivo a identificação de genes degradarores de biomassa vegetal em microrganismos do rúmen de ovinos usando a abordagem metagenômica. Para tanto, foram coletadas amostras da fase sólida do rúmen de 6 animais fistulados (Ovis aries) divididos em dois grupos e submetidos a duas dietas por 60 dias: tratamento controle e tratamento com dieta contendo bagaço de cana-de-açúcar. O DNA metagenômico total das amostras foi extraído e sequenciado na plataforma MiSeq Personal Sequencer (Illumina®). A análise dos dados para a anotação taxônomica e funcional foi realizada no software MG-RAST. A caracterização dos genes degradadores de biomassa vegetal foi feita na plataforma CLC Genomic Workbench v.5.5.1(CLC Bio, Denmark) e a anotação de 4,68 gigabases de dados foi feita no banco de dados CAZy. A análise taxonômica mostrou uma predominância do domínio Bacteria compondo mais de 96% de todas as amostras, sendo os filos mais abundantes Bacteroidetes, Firmicutes, seguido de Proteobacteria. Entre todos os filos anotados, cinco tiveram a abundância aumentada no tratamento com adição de bagaço de cana-de-açúcar na dieta, Firmicutes, Proteobacteria, Actinobacteria, Spirochaetes e Verrucomicrobia, e dois filos foram mais abundantes no tratamento controle, Bacteroidetes e Synergistetes. De modo geral, a análise de ordenação não mostrou correlação entre a composição do microbioma e o tipo de dieta, porém, na análise funcional, essa correlação foi observada uma vez que houve separação entre os tratamentos. A abundância relativa das famílias de enzimas relacionadas à degradação de carboidratos segue um padrão similar em todas as amostras metagenômicas. O módulo catalítico da família de Glycoside Hydrolases (GH), o qual foi anotado em 129 subfamílias diferentes, foi o mais abundante em todas as amostras (45,5%), seguido da família GT (Glicosyl Tranferase), anotada em 97 subfamílias diferentes e CBM (Carbohydrete-Bining Module), em 78 subfamílias. A montagem do metagenoma resultou em aproximadamente 110.000 contigs e possibilitou a identificação de 15 diferentes genes completos codificados nas subfamílias GH1, GH2, GH3, GH16, GH20, GH25, GH32, GH97 e GH127. A análise comparativa dos diferentes tratamentos mostrou uma maior abundância dessas enzimas no rúmen dos animais alimentados com a dieta enriquecida com bagaço de cana-de-açúcar. Em conclusão, a manipulação da dieta de ovinos por meio da substituição de parte da fração fibrosa da dieta por bagaço de cana-de-açúcar promove o enriquecimento de enzimas que degradam a biomassa vegetal no rúmem, favorecendo a prospecção e identificação de genes ativos em carboidratos. / The lignocellulose present in the plant biomass is a promising source of energy generation. However, the breakdown of plant biomass into simple sugars for bioethanol production is still inefficient and costly due to the recalcitrant nature of the plant fiber. The sheep rumen microbiome is specialized in degradation of plant material, but most members of this complex community are uncultured in the laboratory. Therefore, the search for new lignocellulolytic enzymes in microbial communities naturally evolved in biomass degradation environments, such as the rumen, using the exploration of the metagenome, is a promising strategy for identifying new genes. In this context, this study aimed to prospect plant biomass-degrading genes, selected from the sheep rumen microorganisms. The rumen samples were collected from 6 fistulated animals (Ovis aries), divided into two groups and subjected to two diets: control treatment and a treatment with a diet amended with sugarcane bagasse. The animals were fed for 60 days before sampling. To characterize the composition and functions of the rumen microbiome followed by the search of biomass-degrading genes, the metagenomic DNA was extracted from the solid contents of rumen and sequenced in MiSeq Personal Sequencer platform (Illumina®). The taxonomic and functional data were performed using MG-RAST software. For the characterization of the plant biomass degrading genes, they were analyzed on the CLC platform Genomic Workbench v.5.5.1 (CLC Bio, Denmark) and 4.68 gigabases of data was annotated against the CAZy database. The taxonomic analysis showed a predominance of the Bacteria domain composing more than 96% of all the samples, being the most abundant phyla Bacterioidetes, Firmiutes, followed by Proteobacteria. Five bacterial phyla were significantly more abundant in the treatment were sugarcane bagasse was added, Firmicutes, Proteobacteria, Actinobacteria, Spirochaetes and Verrucomicrobia, and two phyla were more abundant in the control treatment, Bacteroidetes and Synergistetes. In general, the ordination analysis did not show correlation between diet type and rumen microbiota, but in the functional analysis, this correlation was observed since there was separation between the treatments. The relative abundance of enzyme families related to carbohydrate degradation follows a similar pattern of abundance across all metagenomic samples. The catalytic module of the GH (Glycoside Hydrolases) family, which was annotated in 129 different subfamilies, was the most abundant in all samples (45.5%), followed by the GT family (Glycosyltransferase), annotated in 97 different subfamilies and CBM (Carbohydre-Bining Module) in 78 sub-families. Metagenome assembly resulted in ~110,000 contigs enabled the retrieval of 15 complete different genes encoded in the subfamilies GH1, GH2, GH3, GH16, GH20, GH25, GH32, GH97 and GH127. A comparative analysis between the groups of animals in the different treatments showed a greater abundance of enzymes, with no metagenome of the fiber proven from the group of animals fed a diet enriched with sugarcane bagasse. These results show the sheep rumen microbiome as an untapped source of potential new fibrolytic enzymes. Using a diet amended with sugarcane bagasse increases the abundance of CAE and provide a substantially expanded catalog of genes participating in the deconstruction of plant biomass.

L’altération des roches silicatées constitue le dénominateur commun d’une multitude de problématiques environnementales et sociétales. Du fait de la difficulté d’extrapoler au milieu naturel les cinétiques de dissolution des minéraux mesurées in vitro, cette thèse propose de réviser en profondeur l’approche actuelle de la réactivité minérale du laboratoire au terrain. Ce travail démontre que l’évolution intrinsèque des propriétés texturales et structurales de l’interface réactive au cours de la dissolution induit des variations de vitesse qui ne peuvent être expliquées dans le cadre des théories thermocinétiques classiques. Nous proposons une nouvelle méthode permettant de sonder la réactivité biogéochimique des minéraux sur le terrain et de révéler les interactions réciproques entre le minéral et le monde microbien au sein de la minéralosphère. Nous démontrons la pertinence des phénomènes de passivation pour l’altération de surface et l’incapacité des microorganismes à les surmonter. / Chemical weathering of silicate minerals is central to numerous environmental and societal challenges. This study addresses the long-standing question of the inconsistency between field and laboratory estimates of dissolution kinetics, by revisiting current approaches of mineral reactivity. It is demonstrated that evolution of feldspar reaction rates are inaccurately describedby current kinetics rate laws, due to textural and structural changes occurring at the fluid-mineral interface over the course of the dissolution process. A novel method is developed to enable probing biogeochemical weathering rates in the field. Bacterial and fungal metagenomic data reveal that subtle reciprocal relationships are established between microorganisms and mineral substrates within the mineralosphere. This thesis emphasizes the impact of passivation phenomena on dissolution rates, under field-relevant reacting conditions and the incapacity of microorganisms to overcome the passivation barrier.

HMMs de perfil são um método poderoso para modelar a diversidade de sequências biológicas e constituem uma abordagem muito sensível para a detecção de ortólogos remotos. Uma potencial aplicação de tais modelos é a detecção de vírus emergentes e novos elementos genéticos móveis. Nosso grupo desenvolveu recentemente o GenSeed-HMM, um programa que emprega HMMs de perfil como sementes para montagem progressiva de genes-alvo, utilizando tanto dados genômicos como metagenômicos. No presente trabalho foi desenvolvido o TABAJARA, um programa para o desenho racional de HMMs de perfil. Partindo de um alinhamento de múltiplas sequências, o TABAJARA é capaz de encontrar blocos que são (1) conservados ou (2) discriminativos para dois ou mais grupos de sequências. O programa utiliza diferentes métricas para atribuir pontuações posição-específicas ao longo de todo o alinhamento e utiliza então uma janela deslizante para encontrar as regiões com maiores pontuações. Blocos de alinhamento selecionados são então extraídos e utilizados para construir HMMs de perfil. Para validar o método, o programa TABAJARA foi empregado para a construção de modelos para vírus do gênero Flavivirus e para fagos da família Microviridae. Em ambos os grupos virais foi possível se obter modelos de ampla abrangência, capazes de detectar todos os membros de um respectivo grupo taxonômico, e modelos de abrangência mais restrita, específicos para espécies distintas de Flavivirus (ex. DENV, ZIKV ou YFV) ou subfamílias de Microviridae (ex. Alpavirinae, Gokushovirinae e Pichovirinae). Em outra validação, foram utilizadas sequências da endonuclease Cas1 para se obter modelos capazes de diferenciar CRISPRs de casposons, esses últimos representando uma superfamília de transposons de DNA autossintetizantes, os quais originaram o sistema de imunidade CRISPR-Cas de procariotos. O TABAJARA conseguiu gerar modelos específicos de Cas1 derivada de casposons, permitindo sua diferenciação em relação aos seus ortólogos de CRISPRs. No presente trabalho foi desenvolvido ainda o HMM-Prospector, uma ferramenta que utiliza um conjunto de HMMs de perfil para a triagem de dados de sequenciamento genômico ou metagenômico. O programa informa quais são os modelos mais reconhecidos pelas leituras, sob valores de corte de pontuação definidos pelo usuário, assim como quantas leituras são detectadas por cada modelo. Com esta informação, os modelos mais relevantes podem ser utilizados como sementes em montagens progressivas com o programa GenSeed-HMM, dentro de uma abordagem integrada para a construção de modelos e sua aplicação. Finamente, foi desenvolvido o e-Finder, um aplicativo genérico para a detecção e extração de elementos multigênicos a partir de genomas ou metagenomas montados utilizando HMMs de perfil. O e-Finder executa buscas de similaridade entre os HMMs de perfil e as sequências traduzidas dos dados montados e checa, em seguida, se os critérios de sintenia pré-definidos foram atendidos, incluindo o número mínimo de genes, a ordem dos genes e as distâncias intergênicas. As sequências dos elementos são então extraídas, as regiões codificantes (ORFs) identificadas e traduzidas conceitualmente em sequências completas de proteínas. Para validar esta ferramenta, foram empegados dois estudos de caso, profagos da família Microviridae e casposons, utilizando-se HMMs de perfil específicos, construídos com o programa TABAJARA. Em ambos os casos, o e-Finder foi executado usando-se a base de dados PATRIC, um repositório com mais de 135.000 genomas de bactérias e arqueias. Foram identificados um total de 91 contigs positivos para casposons a partir de 79 genomas distintos. No caso dos Microviridae, foram encontrados 104 profagos candidatos, estendendo o conhecimento da gama de hospedeiros bacterianos. Em ambos os casos, análises filogenéticas confirmaram a correta atribuição taxonômica das sequências positivas. Os programas desenvolvidos neste trabalho podem ser utilizados isoladamente ou em combinação para detectar e discriminar sequências conhecidas ou remotamente relacionadas. Juntamente com o GenSeed-HMM, estes programas constituem um conjunto integrado de ferramentas com potencial aplicação na busca de novos vírus e elementos genéticos móveis, bem como em qualquer outra tarefa relacionada à detecção e/ou discriminação de subgrupos de famílias de sequências nucleotídicas ou proteicas / Profile HMMs are a powerful way of modeling sequence diversity and constitute a very sensitive approach to detect remote orthologs. A potential application of such models is the detection of emerging viruses and novel mobile genetic elements. Our group has recently developed GenSeed-HMM, a tool that employs profile HMMs as seeds for gene-targeted progressive assembly using either genomic or metagenomic data. In this work we developed TABAJARA, a program for the rational design of profile HMMs. Starting from a multiple sequence alignment, TABAJARA is able to find blocks that are either (1) conserved across all sequences or (2) discriminative for two or more specific groups of sequences. The program uses different metrics to ascribe position-specific scores along the whole alignment and then uses a sliding-window to find top-scoring regions. Selected alignment blocks are then extracted and used to build profile HMMs. To validate the method, we employed TABAJARA to construct models for viruses of the Flavivirus genus and phages of the Microviridae family. In both viral groups we were able to obtain wide-range models, able to detect all members of the respective taxonomic group, and models that are specific to particular Flavivirus species (e.g. DENV, ZIKV or YFV) or Microviridae subfamilies (e.g. Alpavirinae, Gokushovirinae and Pichovirinae). In another validation, we used sequences of the endonuclease Cas1 to obtain models capable of differentiating CRISPRs from casposons, the latter elements representing a superfamily of self-synthesizing DNA transposons that originated the prokaryotic CRISPR-Cas immunity. TABAJARA succeeded to generate models specific to casposon-derived Cas1, enabling their differentiation from CRISPR orthologs. We also developed HMM-Prospector, a tool that can use a batch of profile HMMs to screen genomic or metagenomic sequencing data, reporting which profile HMMs are mostly recognized under user-defined score cutoff values, and how many reads are detected by each model. With this information, the most relevant models can be used as seeds in progressive assemblies with GenSeed-HMM program, providing an integrated approach for model construction and application. Finally, we developed e-Finder, a generic application for detecting and extracting multigene elements from assembled genomes or metagenomes using profile HMMs. e-Finder runs similarity searches of profile HMMs against translated sequences of the assembled data and then checks if pre-defined syntenic criteria have been fulfilled, including minimum number of genes, gene order and intergenic distances. Element sequences are then extracted, their ORFs identified and conceptually translated into full-length protein sequences. To validate the tool, we employed two distinct case studies, prophages of the Microviridae family and casposons, using specific profile HMMs constructed by TABAJARA. In both cases, we executed e-Finder using the PATRIC database, a repository with over 135,000 bacterial and archaeal genomes. We identified in total 91 casposon-positive contigs from 79 distinct genomes. In the case of Microviridae, we found a total of 104 provirus candidates, extending the known range of bacterial hosts. In both cases, phylogenetic analyses confirmed the correct taxonomic assignment of the positive sequences. The programs developed in this work can be used alone or in combination to detect and discriminate known or distantly related sequences. Together with GenSeed-HMM, these programs provide an integrated toolbox with potential application in the search of novel viruses and mobile genetic elements, as well as in any other task related to the detection and/or discrimination of subgroups of DNA or protein sequences.

Organohalide-respiring Bacteria (OHRB) play key roles in the reductive dehalogenation of natural organohalides and anthropogenic chlorinated contaminants. Reductive dehalogenases (RDases) catalyze the cleavage of carbon-halogen bonds, enabling respiratory energy conservation and growth. Large numbers of RDase genes, a majority lacking experimental characterization of function, are found on the genomes of OHRB. In silico genomics tools were employed to identify shared sequence features among RDase genes and proteins, predict RDase functionality, and elucidate RDase evolutionary history. These analyses showed that the RDase superfamily could be divided into proteins exported to the membrane and cytoplasmic proteins, indicating that not all RDases function in respiration. Further, Hidden Markov models (HMMs) and multiple sequence alignments (MSAs) based upon biochemically characterized RDases identified previously uncharacterized members of an RDase superfamily, delineated protein domains and amino acid motifs serving to distinguish RDases from unrelated iron-sulfur proteins. Such conserved and discriminatory features among RDases may facilitate monitoring of organohalide-degrading microbial communities or improve accuracy of genome annotation. Phylogenetic analyses of RDase superfamily sequences provided evidence of convergent evolution and horizontal gene transfer (HGT) across distinct OHRB genera. Yet, the low frequency of RDase transfer outside the genus level and the absence of RDase transfer between phyla indicate that RDases evolve primarily by vertical evolution or HGT is restricted among related OHRB strains. Polyphyletic evolutionary lineages within the RDase superfamily comprise distantly-related RDases, some exhibiting activities towards the same substrates, suggesting a longstanding history of OHRB adaptation to natural organohalides. Similar functional and phylogenetic analyses provided evidence that nitrous oxide (N₂O, a potent greenhouse gas) reductase (nosZ) genes from versatile OHRB members of the Anaeromyxobacter and Desulfomonile genera comprised a nosZ sub-family evolutionarily distinct from nosZ found in non-OHRB denitrifiers. Hence, elucidation of RDase and NosZ sequence diversity may enhance the mitigation of anthropogenic organohalides and greenhouse gases (i.e., N₂O), respectively. The tetrachloroethene-respiring bacterium Geobacter lovleyi strain SZ exhibited genomic features distinguishing it from non-organohalide-respiring members of the Geobacter genus, including a conjugative pilus transfer gene cluster, a chromosomal genomic island harboring two RDase genes, and a diminished set of c-type cytochrome genes. The G. lovleyi strain SZ genome also harbored a 77 kbp plasmid carrying 15 out of the 24 genes involved in biosynthesis of corrinoid, likely related to this strains ability to degrade PCE to cis-DCE in the absence of supplied corrinoid (i.e., vitamin B₁₂). Although corrinoids are essential cofactors to RDases, the strictly organohalide-respiring Dehalococcoides mccartyi strains are corrinoid auxotrophs and depend upon uptake of extracellular corrinoids via Archaeal and Bacterial salvage pathways. A key corrinoid salvage gene in D. mccartyi, cbiZ, occurs at duplicated loci adjacent to RDase genes and appears to have been horizontally-acquired from Archaea. These comparative genome analyses highlight RDase dependencies upon corrinoids and also suggest mobile genomic elements (e.g., plasmids) are associated with organohalide respiration and corrinoid acquisition among OHRB. In summary, analyses of OHRB genomes promise to enable more complete modeling of metabolic and evolutionary processes associated with the turnover of organohalides in anoxic environments. These efforts also expand knowledge of biomarkers for monitoring OHRB activity in anoxic environments, and will improve our understanding of the fate of chlorinated contaminants.

No description available.

No description available.

Thesis (MSc (Genetics. Institute of Plant Biotechnology))--Stellenbosch University, 2008. / Traditional methods used for the discovery of novel genes have previously relied upon the ability to culture the relevant microbes and then demonstrate the activity of a specific enzyme. Although these methods have proved successful in the past, they severely limit our access to the genomes of organisms which are not able to be cultured under laboratory conditions. It was therefore the aim of this project to use metagenomic strategies for the identification of novel polymer-producing genes with the prospect of commercial exploitation. In this study, soil-derived metagenomic libraries were functionally screened for potential -glucan producing clones using aniline blue staining. Positive reacting clones were selected and sequenced. Initial sequencing revealed a gene with high homology to previously described glucan synthases, the products of these genes all having significant industrial value. The clone was transformed into a suitable bacterial host, cultured and allowed to produce the polymer of interest. The polysaccharide was purified and subjected to various chemical analyses so as to confirm its monosaccharide composition. Data suggests that this polymer is composed mainly of glucose units and that it may be secreted out of the cell. Purification of the active enzyme was attempted using classical protein purification methods with faint activity being detected using Native polyacrylamide gel electrophoresis (PAGE). Further attempts to demonstrate activity were made through the construction of a GST (glutathione S-transferase) tagged fusion protein. The second part of this study focuses on the construction and screening of a metagenomic DNA library from whey, a by-product of the cheese manufacturing process. It was envisaged that this could provide a resource for the identification of high value polymers when lactose is provided as a sole carbon source. The library was screened for function using Congo Red for the detection of extra-cellular polysaccharides.

O estudo da diversidade viral em amostras ambientais tem se tornado cada vez mais importante devido a funções-chave desempenhadas por esses organismos. Estudos recentes têm fornecido evidências de que vírus de bactérias (bacteriófagos) podem ser os principais determinantes em ciclos biogeoquímicos de grandes ecossistemas, além de atuarem no fluxo de genes entre comunidades ambientais e na plasticidade funcional das mesmas frente a estresses ambientais. Neste trabalho, propomos a investigação e caracterização da diversidade viral presente em amostras de compostagem através de abordagens não dependentes e dependentes de cultivo. Na primeira abordagem, coletamos amostras seriadas de uma unidade de compostagem do zoológico de São Paulo para realização de sequenciamento metagenômico. O conjunto de sequências gerado foi extensivamente minerado (data-mining) para a produção de resultados de diversidade e abundância de táxons virais ao longo do processo de compostagem. Adicionalmente, procedemos com a montagem e recuperação de sequências virais candidatas a genomas completos e/ou parciais de novos vírus ambientais. Os dois protocolos computacionais utilizados para a mineração de dados encontram-se definidos e automatizados, podendo ser aplicados em quaisquer conjuntos de dados de sequenciamento metagenômico ou metatranscritômico obtidos através da plataforma Illumina. A segunda abordagem correspondeu ao isolamento e caracterização de novos fagos de Pseudomonas obtidos de amostras de compostagem. Três novos fagos foram identificados e tiveram os seus genomas sequenciados. A caracterização genômica desses fagos revelou genomas com alto grau de novidade, insights sobre a evolução de Caudovirales e a presença de genes de tRNA, cuja função pode estar relacionada com um mecanismo dos fagos para contornar o viés traducional apresentado pela bactéria hospedeira. A caracterização experimental dos novos fagos isolados demonstrou grande potencial para lise e dissolução de biofilme da cepa Pseudomonas aeruginosa PA14, conhecida como agente causador de infecções hospitalares em pacientes imunodeprimidos. Em suma, os dados reunidos nesta dissertação caracterizam a diversidade presente no viroma da compostagem e contribuem para o entendimento dos perfis taxonômico, funcional e ecológico do processo. / The study of the viral diversity in environmental samples has become increasingly important due to key-roles that are performed by these organisms in our ecosystems. Recent publications provide evidence that viruses of bacteria (bacteriophages) may be key-players in biogeochemical cycles of large ecosystems, as oceans and forests. Besides, they may also be determinant in the genes flux among populations and in the plasticity of the communities face to environmental stresses. In this work, we propose the investigation and characterization of the viral diversity in composting samples through non-culturable and culturable-dependent approaches. In the first approach, we sampled a composting unit from the Sao Paulo Zoo Park in different time points and proceeded with metagenomic sequencing. The dataset generated was extensively mined to provide results of diversity and abundance of viral taxa through the composting process. Additionally, we proceeded with the assembly and retrieval of candidate sequences to partial or/and complete viral genomes. The two computational protocols were automatized as pipelines and can be applied to any metagenomic dataset of illumina reads. The second approach refers to the isolation and characterization of new Pseudomonas phages obtained from composting samples. Three new phages were identified and their genomes were sequenced. A detailed characterization of these genomes revealed high degree of novelty, insights about evolution of tailed-phages and the presence of tRNA genes, which may be related to a mechanism to bypass host translational bias. The experimental characterization of the new phages demonstrated great potential to lyse bacterial cells and to degrade Pseudomonas aeruginosa PA14 biofilms. In short, the data presented in this dissertation shed light to the composting virome diversity, as well as to the functional and ecological profiles of viruses in the composting environment.

O material lignocelulósico, presente na biomassa vegetal, representa uma importante fonte de energia, entretanto necessita da ação das enzimas lignocelulolíticas para sua degradação. A busca por novas enzimas que atuam na quebra da parede celular da planta em comunidades microbianas evoluídas naturalmente em um ambiente de degradação de biomassa como o rúmen oferece uma estratégia promissora para a prospecção de genes. Com isso, o projeto teve como objetivo a identificação de genes degradarores de biomassa vegetal em microrganismos do rúmen de ovinos usando a abordagem metagenômica. Para tanto, foram coletadas amostras da fase sólida do rúmen de 6 animais fistulados (Ovis aries) divididos em dois grupos e submetidos a duas dietas por 60 dias: tratamento controle e tratamento com dieta contendo bagaço de cana-de-açúcar. O DNA metagenômico total das amostras foi extraído e sequenciado na plataforma MiSeq Personal Sequencer (Illumina®). A análise dos dados para a anotação taxônomica e funcional foi realizada no software MG-RAST. A caracterização dos genes degradadores de biomassa vegetal foi feita na plataforma CLC Genomic Workbench v.5.5.1(CLC Bio, Denmark) e a anotação de 4,68 gigabases de dados foi feita no banco de dados CAZy. A análise taxonômica mostrou uma predominância do domínio Bacteria compondo mais de 96% de todas as amostras, sendo os filos mais abundantes Bacteroidetes, Firmicutes, seguido de Proteobacteria. Entre todos os filos anotados, cinco tiveram a abundância aumentada no tratamento com adição de bagaço de cana-de-açúcar na dieta, Firmicutes, Proteobacteria, Actinobacteria, Spirochaetes e Verrucomicrobia, e dois filos foram mais abundantes no tratamento controle, Bacteroidetes e Synergistetes. De modo geral, a análise de ordenação não mostrou correlação entre a composição do microbioma e o tipo de dieta, porém, na análise funcional, essa correlação foi observada uma vez que houve separação entre os tratamentos. A abundância relativa das famílias de enzimas relacionadas à degradação de carboidratos segue um padrão similar em todas as amostras metagenômicas. O módulo catalítico da família de Glycoside Hydrolases (GH), o qual foi anotado em 129 subfamílias diferentes, foi o mais abundante em todas as amostras (45,5%), seguido da família GT (Glicosyl Tranferase), anotada em 97 subfamílias diferentes e CBM (Carbohydrete-Bining Module), em 78 subfamílias. A montagem do metagenoma resultou em aproximadamente 110.000 contigs e possibilitou a identificação de 15 diferentes genes completos codificados nas subfamílias GH1, GH2, GH3, GH16, GH20, GH25, GH32, GH97 e GH127. A análise comparativa dos diferentes tratamentos mostrou uma maior abundância dessas enzimas no rúmen dos animais alimentados com a dieta enriquecida com bagaço de cana-de-açúcar. Em conclusão, a manipulação da dieta de ovinos por meio da substituição de parte da fração fibrosa da dieta por bagaço de cana-de-açúcar promove o enriquecimento de enzimas que degradam a biomassa vegetal no rúmem, favorecendo a prospecção e identificação de genes ativos em carboidratos. / The lignocellulose present in the plant biomass is a promising source of energy generation. However, the breakdown of plant biomass into simple sugars for bioethanol production is still inefficient and costly due to the recalcitrant nature of the plant fiber. The sheep rumen microbiome is specialized in degradation of plant material, but most members of this complex community are uncultured in the laboratory. Therefore, the search for new lignocellulolytic enzymes in microbial communities naturally evolved in biomass degradation environments, such as the rumen, using the exploration of the metagenome, is a promising strategy for identifying new genes. In this context, this study aimed to prospect plant biomass-degrading genes, selected from the sheep rumen microorganisms. The rumen samples were collected from 6 fistulated animals (Ovis aries), divided into two groups and subjected to two diets: control treatment and a treatment with a diet amended with sugarcane bagasse. The animals were fed for 60 days before sampling. To characterize the composition and functions of the rumen microbiome followed by the search of biomass-degrading genes, the metagenomic DNA was extracted from the solid contents of rumen and sequenced in MiSeq Personal Sequencer platform (Illumina®). The taxonomic and functional data were performed using MG-RAST software. For the characterization of the plant biomass degrading genes, they were analyzed on the CLC platform Genomic Workbench v.5.5.1 (CLC Bio, Denmark) and 4.68 gigabases of data was annotated against the CAZy database. The taxonomic analysis showed a predominance of the Bacteria domain composing more than 96% of all the samples, being the most abundant phyla Bacterioidetes, Firmiutes, followed by Proteobacteria. Five bacterial phyla were significantly more abundant in the treatment were sugarcane bagasse was added, Firmicutes, Proteobacteria, Actinobacteria, Spirochaetes and Verrucomicrobia, and two phyla were more abundant in the control treatment, Bacteroidetes and Synergistetes. In general, the ordination analysis did not show correlation between diet type and rumen microbiota, but in the functional analysis, this correlation was observed since there was separation between the treatments. The relative abundance of enzyme families related to carbohydrate degradation follows a similar pattern of abundance across all metagenomic samples. The catalytic module of the GH (Glycoside Hydrolases) family, which was annotated in 129 different subfamilies, was the most abundant in all samples (45.5%), followed by the GT family (Glycosyltransferase), annotated in 97 different subfamilies and CBM (Carbohydre-Bining Module) in 78 sub-families. Metagenome assembly resulted in ~110,000 contigs enabled the retrieval of 15 complete different genes encoded in the subfamilies GH1, GH2, GH3, GH16, GH20, GH25, GH32, GH97 and GH127. A comparative analysis between the groups of animals in the different treatments showed a greater abundance of enzymes, with no metagenome of the fiber proven from the group of animals fed a diet enriched with sugarcane bagasse. These results show the sheep rumen microbiome as an untapped source of potential new fibrolytic enzymes. Using a diet amended with sugarcane bagasse increases the abundance of CAE and provide a substantially expanded catalog of genes participating in the deconstruction of plant biomass.