Utilization of Pyrosequencing to Monitor the Microbiome Dynamics of Probiotic Treated Poultry (Gallus gallus domesticus) during Downstream Poultry Processing

Antibiotic growth promoters that have been historically employed to control pathogens and increase the rate of animal development for human consumption are currently banned in many countries. Probiotics have been proposed as an alternative to control pathogenic bacteria. Traditional culture methods typically used to monitor probiotic effects on pathogens possess significant limitations such as a lack in sensitivity to detect fastidious and non-culturable bacteria, and are both time consuming and costly. Here, we tested next generation pyrosequencing technology as a streamline and economical method to monitor the effects of a probiotic on microbial communities in juvenile poultry (Gallus gallus domesticus) after exposure to several microbiological challenges and litter conditions. Seven days and repeated again at 39 days following hatching, chicks were challenged with either Salmonella enterica serovar Enteritidis, Campylobacter jejuni, or no bacteria in the presence of, or without a probiotic (i.e., Bacillus subtilis) added to the feed. Three days following each of two challenges (i.e., days 10 and 42, respectively) the microbiome distributions of the poultry caecum were characterized based on 16S rDNA analysis. Generated PCR products were analyzed by automated identification of the samples after pooling, multiplexing and sequencing. A bioinformatics pipeline was then employed to identify microbial distributions at the phylum and genus level for the treatments. In conclusion, our results demonstrated that pyrosequencing technology is a rapid, efficient and cost-effective method to monitor the effects of probiotics on the microbiome of poultry propagated in an agricultural setting

COMPARATIVE METAGENOMICS ANALYSIS OF PALM OIL MILL EFFLUENT (POME) USING THREE DIFFERENT BIOINFORMATICS PIPELINES

ABSTRACT: The substantial cost reduction and massive production of next-generation sequencing (NGS) data have contributed to the progress in the rapid growth of metagenomics. However, production of the massive amount of data by NGS has revealed the challenges in handling the existing bioinformatics tools related to metagenomics. Therefore, in this research we have investigated an equal set of DNA metagenomics data from palm oil mill effluent (POME) sample using three different freeware bioinformatics pipelines’ websites of metagenomics RAST server (MG-RAST), Integrated Microbial Genomes with Microbiome Samples (IMG/M) and European Bioinformatics Institute (EBI) Metagenomics, in term of the taxonomic assignment and functional analysis. We found that MG-RAST is the quickest among these three pipelines. However, in term of analysis of results, IMG/M provides more variety of phylum with wider percent identities for taxonomical assignment and IMG/M provides the highest carbohydrates, amino acids, lipids, and coenzymes transport and metabolism functional annotation beside the highest in total number of glycoside hydrolase enzymes. Next, in identifying the conserved domain and family involved, EBI Metagenomics would be much more appropriate. All the three bioinformatics pipelines have their own specialties and can be used alternately or at the same time based on the user’s functional preference. ABSTRAK: Pengurangan kos dalam skala besar dan pengeluaran data ‘next-generation sequencing’ (NGS) secara besar-besaran telah menyumbang kepada pertumbuhan pesat metagenomik. Walau bagaimanapun, pengeluaran data dalam skala yang besar oleh NGS telah menimbulkan cabaran dalam mengendalikan alat-alat bioinformatika yang sedia ada berkaitan dengan metagenomik. Justeru itu, dalam kajian ini, kami telah menyiasat satu set data metagenomik DNA yang sama dari sampel effluen kilang minyak sawit dengan menggunakan tiga laman web bioinformatik percuma iaitu dari laman web ‘metagenomics RAST server’ (MG-RAST), ‘Integrated Microbial Genomes with Microbiome Samples’ (IMG/M) dan ‘European Bioinformatics Institute’ (EBI) Metagenomics dari segi taksonomi dan analisis fungsi. Kami mendapati bahawa MG-RAST ialah yang paling cepat di antara ketiga-tiga ‘pipeline’, tetapi mengikut keputusan analisa, IMG/M mengeluarkan maklumat philum yang lebih pelbagai bersama peratus identiti yang lebih luas berbanding yang lain untuk pembahagian taksonomi dan IMG/M juga mempunyai bacaan tertinggi dalam hampir semua anotasi fungsional karbohidrat, amino asid, lipid, dan koenzima pengangkutan dan metabolisma malah juga paling tinggi dalam jumlah enzim hidrolase glikosida. Kemudian, untuk mengenal pasti ‘domain’ terpelihara dan keluarga yang terlibat, EBI metagenomics lebih bersesuaian. Ketiga-tiga saluran ‘bioinformatics pipeline’ mempunyai keistimewaan mereka yang tersendiri dan boleh digunakan bersilih ganti dalam masa yang sama berdasarkan pilihan fungsi penggun

Metagenômica e possíveis aplicações biotecnológicas - identificação do microbioma de ectoparasitos que impactam a bovinocultura.

A comunidade microbiana que coexiste em habitats tais como a superfície externa e ambientes internos de ectoparasitos de bovinos pode e deve ser conhecida quanto a sua composição e fisiologia. Este conhecimento pode contribuir para o estabelecimento de estratégias de controle, busca de novas moléculas funcionais, prevenção de transmissão de patógenos emergentes e reemergentes, entre outras possibilidades biotecnológicas. Análises completas e complexas de taxonomia, filogenia e funcionalidade têm sido possíveis pelo uso da metagenômica. Por esta metodologia, baseada em sequenciamento concomitante de diferentes genomas, seguido de análises in silico (ferramentas de bioinformática), é possível identificar inclusive microrganismos não cultiváveis, abordagem esta que antes não era possível a partir de métodos de isolamento tradicionais. Até recentemente, pouco se conhecia sobre a comunidade microbiana associada a diferentes ectoparasitos de bovinos. De interesse maior estão o carrapato-do-boi e a mosca-dos-estábulos, responsáveis por impactarem negativamente a produção na bovinocultura de corte e de leite. Esta publicação apresenta uma breve revisão na busca da identificação e entendimento da interação da comunidade microbiana presente e/ou carreada por ectoparasitos de bovinos e, também, uma breve descrição da metodologia que tem permitido avanço significativo do conhecimento nesta área.bitstream/item/207808/1/metagenomica-e-possiveis-aplicacoes-biotecnologicas.pd

Methods for fast sequence comparison and identification in metagenomic data

Předmětem této práce je vytvoření metody sloužící k identifikaci organismů z metagenomických dat. Doposud k tomuto účelu spolehlivě dostačovaly metody založené na zarovnání sekvencí s referenční databází. Množství dat ovšem s rozvojem sekvenačních technik rapidně roste a tyto metody se tak stávají díky své výpočetní náročnosti nevhodnými. V této diplomové práci je popsán postup nové techniky, která umožňuje klasifikaci metagenomických dat bez nutnosti zarovnání. Metoda spočívá v převedení sekvenovaných úseků na genomické signály ve formě fázových reprezentací, ze kterých jsou následně extrahovány vektory příznaků. Těmito příznaky jsou tři Hjorthovy deskriptory. Ty jsou dále vystaveny metodě maximalizace věrohodnosti směsi Gaussovských rozložení, která umožňuje spolehlivé roztřídění fragmentů podle jejich příslušnosti k organismu.The objective of this thesis is to create a method for identification of organisms in metagenomic data. Until this point methods based on sequence alignment with reference database have been sufficient for this purpose. However, the volume of data grows rapidly with evolvement of sequencing techniques and the alignment-based methods became inconvenient due to computationally demanding alignment. A new technique is introduced in this master’s thesis, which allows alignment-free metagenomic data classification. The method is based on transformation of sequences to genomic signals in form of phase representation, from which feature vectors are extracted. These features are three Hjorth descriptors, which are then subjected expectation maximization for Gaussian mixture model method allowing reliable binning of metagenomic data.

Viruses in the North Sea: viromics and prophage genomics

Despite their small size, viruses have an enormous influence on microbial population dynamics, due to lysis and horizontal gene transfer. Due the high abundance of their hosts, bacteriophages or phages comprise the majority of viruses and provide the largest reservoirs of unexplored genetic diversity in marine environments. The rise of Next Generation Sequencing (NGS) techniques brings new opportunities to investigate the marine virus community. However, there is no current statutory pipeline applied in marine phage ecology. Therefore, this thesis proposes a virus-specific pipeline based on the integration of existing tools and state of the art techniques. The developed pipeline was applied to accomplish the two research aims of this thesis: (1) to analyze the virus community in the North Sea with viromics(virus metagenomics), and (2) to characterize lysogenic phages from potentially pathogenic Vibrio species. The results of the first part of this thesis show that the virus community is dominated by phages and they are not evenly distributed throughout the North Sea. In general, the coastal virus community was genetically more diverse than the open sea community. The influence of riverine inflow and currents affects the genetic virus diversity with the community carrying genes from a variety of metabolic pathways and other functions. In the second part, lysogenic Vibrio phages from the North Sea were induced (ca. 40 % of tested isolates) and four phage genomes were characterized. The phages from V. parahaemolyticus (2 tailed phages, 1 filamentous phage) and V. cholerae (1 tailed phage) can integrate into their host genome and might have a role in pathogenicity. This thesis represents an exemplary study of the virus community in the North Sea, with special emphasis on the marine phages. The settled virus-specific pipeline the obtained insights will contribute to extend the study of the virus diversity dynamics in other marine areas to characterize novel phage groups

Metagenomics and functional genomics of bacterial symbionts of Spongia (Porifera, Dictyoceratida) specimens from the Algarvian shore (South Portugal)

Sponges are early-branched, filter-feeding metazoans that usually harbor complex microbial communities comprised of diverse “uncultivable” symbiotic bacteria. In this thesis, the functional and taxonomic features of the marine sponge microbiome are determined, using Spongia officinalis as model host organism. Emphasis is given to adaptive and functional traits of the profuse and biotechnologically-relevant alphaproteobacterial symbionts of sponges. A metagenomics-centred approach was employed to reveal microbial taxa and genomic signatures enriched in the Spongia officinalis endosymbiotic consortium, and thus likely to play pivotal roles in holobiont functioning. Further, a comparative genomics study is presented unveiling the common and specific traits of ten Alphaproteobacteria genera isolated from S. officinalis with alternative symbiont cultivation methodology. Finally, a sequence composition-dependent binning approach is employed to assemble, from metagenomic sequences, the genome of an uncultured alphaproteobacterial symbiont of S. officinalis belonging to the family Rhodospirillaceae. High abundance of polyketide and terpene synthase-, eukaryotic-like protein- (ELPs), type IV secretion system-, plasmid- and ABC transporter-encoding genes, among others, characterized the sponge microbial metagenomes. In contrast, motility and chemotaxis genes were abundant in seawater and sediment microbiomes, but nearly absent in the S. officinalis symbiotic consortium. Much higher frequencies of anti-viral CRISPR-Cas and restrictionmodification systems, along with much lower viral abundances, were observed in the spongeassociated metagenomes than in the environment and interpreted as true hallmarks of this symbiotic consortium. In line with outcomes retrieved for the whole symbiotic community, alphaproteobacterial symbionts of marine sponges likely contribute the most to host fitness through nutritional exchange, cell detoxification processes and chemical defense, the latter being theoretically promoted by both polyketide and terpenoid biosynthesis. The several alphaproteobacterial cultures retrieved in this thesis, displaying high natural product biosynthesis capacities, can now be explored in studies aiming at revealing novel biological activities and chemical structures from these symbionts.As esponjas marinhas (filo Porifera) são consideradas um dos mais simples grupos entre os metazoários em função de sua falta de organização em tecidos e órgãos verdadeiros. Porém, estes animais relativamente simples em termos de plano corporal normalmente abrigam comunidades muito complexas de microorganismos. Em função de seu surgimento basal na história evolutiva do planeta, o conhecimento a respeito deste “holobionte”, isto é, o consórcio de organismos formado pela esponja marinha hospedeira e todos os seus simbiontes microbianos, possui grande relavância ao avanço da nossa compreensão sobre as interações hospedeiro-microorganismos. Nesta tese de doutoramento, tive como objetivo a determinação das características funcionais e taxonómicas do microbioma das esponjas marinhas no contexto de seu ambiente circundante (água e sedimentos marinhos, e suas respecticvas microbiotas), dando ênfase aos traços adaptativos e funcionais de alfaproteobactérias associadas ao organismo modelo Spongia officinalis (“bath sponge”)

Metagenômica, nanotecnologia e nutrição animal: alternativas para o uso de antibióticos e mitigação de gases de efeito estufa.

bitstream/item/220975/1/DOC-283-Final-em-alta.pd

Caracterização do viroma fecal de frangos de corte saudáveis e afetados pela síndrome da má absorção

A síndrome da má absorção (SMA) é uma doença de aves jovens caracterizada por retardo no crescimento, desenvolvimento defeituoso das penas e diarreia, resultando em relevante impacto econômico. Estudos prévios têm investigado a participação de uma série de agentes virais na etiologia da SMA. No entanto, o conhecimento limitado da comunidade viral presente no intestino das aves dificulta a identificação precisa do(s) agente(s) causal(is). Nesse sentido, o presente estudo foi conduzido visando ampliar o conhecimento sobre o viroma entérico de frangos de corte jovens (3-5 semanas) e seu envolvimento com a ocorrência da SMA. Para tanto, em quatro granjas, foram feitas coletas de conteúdo intestinal de frangos clinicamente saudáveis. DNA e RNA virais foram extraídos, sequenciados e comparados com dados previamente disponíveis através de análises metagenômicas. Genomas representantivos de uma ampla diversidade de reconhecidas famílias virais (Adenoviridae, Caliciviridae, Circoviridae, Parvoviridae, Picobirnaviridae, Picornaviridae e Reoviridae) foram identificados. Igualmente, foram detectados vários agentes não previamente descritos, com genomas de DNA fita simples circular, presentemente denominados vírus “CRESS-DNA”. Numa etapa seguinte, foi conduzido um estudo visando examinar comparativamente o viroma entérico de frangos de corte afetados pela SMA (n=35) e frangos saudáveis (n=35). Foram detectados genomas de membros das seguintes famílias virais:Picornaviridae, vírus CRESS-DNA, Circoviridae, Anelloviridae, Reoviridae, Picobirnaviridae, Astroviridae, Caliciviridae, Parvoviridae e Adenoviridae. A comparação entre a distribuição do número de reads correspondentes as diferentes espécies de vírus eucarióticos identificados no grupo de aves doentes e de aves saudáveis não apresentou diferença estatística. Esses resultados sugerem que a causa da SMA não está relacionada à infecção por um agente viral específico. Futuros estudos são necessários para elucidar a etiologia desta síndrome em frangos de corte.Malabsorption syndrome (MAS) is a disease of young broilers characterized by growth retardation, defective feather development and diarrhea with undigested food, resulting in relevant economic impact. Previous studies have investigated the involvement of a number of viral agents in the MAS etiology. However, the limited knowledge of the viral community in poultry gut hinders identification of a particular causative agent(s). In this sense, the present study was conducted aiming to increase the knowledge about the enteric virome of young broiler chickens (3-5 weeks) and its potencial involvement in the occurrence of MAS. For this purpose, on four poultry farms, intestinal contents were collected from clinically healthy chickens. Viral DNA and RNA were extracted, sequenced and compared with previously available data through metagenomic analyzes. Genomes representing a wide diversity of recognized viral families (Adenoviridae, Caliciviridae, Circoviridae, Parvoviridae, Picobirnaviridae, Picornaviridae e Reoviridae) were identified. Also, several agents not previously described were detected, with single-stranded circular DNA genomes, presently known as viruses “CRESS-DNA”. In a subsequent step, a study was conducted comparing the enteric virome of broilers affected by MAS (n = 35) and healthy broilers (n = 35). Genomes of members of the following viral families were detected: Picornaviridae, virus CRESS-DNA, Circoviridae, Anelloviridae, Reoviridae, Picobirnaviridae, Astroviridae, Caliciviridae, Parvoviridae e Adenoviridae. Comparison between the distribution of sequences reads matching different species of eukaryotic virus identified in the group of diseased birds and healthy birds did not reach statistical difference. These results suggest the cause of SMA is not related to infection by a specific viral agent. Future studies are needed to elucidate the etiology of this syndrome in broiler chickens

Implementación de una plataforma digital para el registro, procesamiento y categorización de datos relacionados a los perfiles de los sujetos de prueba, para estudios de metagenómica intestinal humana

La metagenómica es la ciencia que emplea el análisis genético directo de una población de microorganismos contenidos en una muestra ambiental, mediante la extracción directa y clonación de ADN (Thomas, Gilbert & Meyer, 2012; Singh, et. al., 2009). Uno de los focos de la metagenómica es el microbioma intestinal humano, debido a que desempeña un papel clave en la salud (Davenport et. al., 2017; Sekirov, 2010). En los estudios de metagenómica intestinal, se realiza un muestreo de las heces de los sujetos de prueba (Aagaard et. al., 2013), se secuencian los microorganismos que se encuentran en esta, se procesa esta información mediante herramientas bioinformáticas y finalmente los investigadores analizan los resultados obtenidos (Lloyd-Price et. al., 2016). Previamente al proceso de muestreo, se requiere recopilar los metadatos de la muestra (Kunin et. al., 2008), los cuales son datos de los sujetos de prueba que influyen en su microbioma intestinal. Actualmente, estos metadatos se recopilan y procesan de una forma manual, a modo de formulario físico, se almacenan de forma incompleta y no estandarizada, y requieren mucho tiempo para ser procesados y categorizados. Es por ello que, en el presente trabajo de fin de carrera, se busca proponer una herramienta digital que permita la recopilación, procesamiento y categorización de los datos de los sujetos de prueba. Estos datos, los cuales son de distintos tipos, serán recopilados de una manera uniforme en una base de datos, de tal manera que se preserven en el tiempo y los investigadores puedan reutilizar esta información en futuros estudios, sin tener que recurrir a volver a realizar el costoso proceso de secuenciación. Con el fin de resolver este problema, se diseñó una base de datos que almacene los datos de los sujetos de prueba, de una manera estandarizada. Utilizando las entidades y las relaciones identificadas en la revisión de la literatura, se pudo plantear un diseño de base de datos que permita la recopilación de los datos de los participantes. En ese mismo sentido, usando la base de datos planteada, se implementó una plataforma digital que permite gestionar estudios de metagenómica y recopilar los datos de sus participantes. De esta manera, se pueden almacenar los metadatos de las muestras a secuenciar de una manera digital, permitiendo a los investigadores revisar estos datos en un futuro. Finalmente, se identificó las funcionalidades necesarias para el procesamiento de los datos de los sujetos de prueba. Estas funcionalidades fueron implementadas en la plataforma digital, para poder permitir a los investigadores analizar estos datos de una manera rápida y sencilla

Web Resources for Metagenomics Studies

The development of next-generation sequencing (NGS) platforms spawned an enormous volume of data. This explosion in data has unearthed new scalability challenges for existing bioinformatics tools. The analysis of metagenomic sequences using bioinformatics pipelines is complicated by the substantial complexity of these data. In this article, we review several commonly-used online tools for metagenomics data analysis with respect to their quality and detail of analysis using simulated metagenomics data. There are at least a dozen such software tools presently available in the public domain. Among them, MGRAST, IMG/M, and METAVIR are the most well-known tools according to the number of citations by peer-reviewed scientific media up to mid-2015. Here, we describe 12 online tools with respect to their web link, annotation pipelines, clustering methods, online user support, and availability of data storage. We have also done the rating for each tool to screen more potential and preferential tools and evaluated five best tools using synthetic metagenome. The article comprehensively deals with the contemporary problems and the prospects of metagenomics from a bioinformatics viewpoint