O histórico agronômico da cultura, em geral, explica a comunidade microbiana presente na massa ensilada, entretanto, a diversidade e o grau de contaminação da população microbiana epifítica pode auxiliar na compreensão do padrão de fermentação da silagem e da estabilidade desse produto quando em exposição ao ambiente aeróbio. No presente trabalho, foram avaliados a influência do genótipo, maturidade e período de estocagem na composição da comunidade bacteriana em silagens de grãos de milho. Para isso, dois cultivares de milho AG 1051 (\"dent\") e IAC 8390 (\"flint\") foram colhidos em três estágios de maturidade (ponto de silagem de planta inteira, ponto de silagem de grão úmido e ponto de grão seco), moídos e ensilados por 0, 7 e 120 dias. Atualmente, a aplicação de técnicas de microbiologia molecular permite acessar alterações causadas nestas comunidades de maneira independente do cultivo bacteriano, por esse motivo, a comunidade bacteriana foi avaliada por meio da técnica de Eletroforese em Gel de Gradiente Desnaturante (DGGE) e sequenciamento dos produtos de PCR via sistema MiSeqTM Illumina. Foi demonstrado que em silagens de grãos de milho contendo alta umidade, os diferentes estágios de desenvolvimento da cultura, e por conseguinte, do grão, são os principais determinantes da composição da comunidade bacteriana deste, sendo menos importantes o genótipo das plantas e os tempos de estocagem das silagens. Aos 120 dias de estocagem nas amostras de grão seco reconstituído, as sequências afiliadas ao gênero Clostridium representaram total de aproximadamente 40% das sequências afiliadas aos gêneros encontrados, enquanto o gênero Lactobacillus representou menos de 7% das sequências afiliadas a este gênero. Provavelmente, grãos secos sofrem mais estresse a campo, o que consequentemente, pode interferir na qualidade higiênico sanitária das silagens desses grãos. Com base nestes resultados fica evidenciada a possibilidade de realização de recomendações potenciais de aditivos específicos para ensilagem de grãos de milho, direcionados para cada ponto de maturidade da cultura. / The agronomic background of crops in general, explains the microbial community present in silage, however, diversity and contamination status may help on understanding the silage fermentation profile and aerobic stability. In the present work, the influence of factors such as different genotypes, different stages of plants development and storage time in the composition of bacterial communities were evaluated. On this way, maize cultivars AG 1051 (\"dent\") and IAC 8390 (\"flint\") were harvested in three physiological stages (whole plant silage, wet grain silage and dry grain), the grain were grounded and ensiled for 0, 7 and 120 days. Nowadays, the applications of techniques of molecular microbiology allow assessing the shifts caused on these communities by a culture independent approach, therefore, bacterial community were evaluated by Denaturing Gradient Gel Electrophoresis technique (DGGE), and PCR products were sequenced by Illumina MiSeqTM System. It was demonstrated that in high moisture corn silage, different stages of plants development are main determinants of bacterial community composition rather than the plants genotypes and storage time. In addition in the samples of reconstituted dry grain, it was demonstrated that after 120 days of storage, sequences affiliated to the gender Clostridium accounted for a total of approximately 40% of total sequences affiliated to genera found, while the genus Lactobacillus represented less than 7% of sequences affiliated to this gender. Probably dried grains suffer more stress at field conditions, which in turn can interfere with the sanitary hygienic quality of silages obtained from these grains. At least, based on these results it is clear the possibility of performing potential specific additives recommendations, unique at each stage of maize plants development.

Le poisson est un produit très périssable dont l’altération résulte essentiellement de la croissance bactérienne. Comparé aux régions tempérées, peu d’études portent sur le microbiote d’altération des poissons tropicaux. En Martinique, le thon jaune (Thunnus albacares) et l’ombrine ocellée (Sciaenops ocellatus) représentent des poissons d’intérêt pour les filières pêche et aquaculture. Dans le but de mieux connaître le microbiote d’altération de ces poissons, des analyses culturales et aculturales (séquençage de nouvelle génération des amplicons d’ARNr 16S, Illumina MiSeq) ont été réalisées.Une grande diversité d’espèces bactériennes a été retrouvée dans le thon et l’ombrine fraîchement pêchés (104 et 887 OTUs, respectivement) et la plupart d’entre elles sont communément isolées des poissons (Chryseobacterium, Burkholderia, Flavobacterium, Psychrobacter, Arthrobacter, Staphylococcus). Certaines, comme Ralstonia sp. et Rhodanobacter terrae, en quantité importante dans le thon frais, sont plus atypiques. Au cours de l’entreposage du thon sous-glace, Pseudomonas et Brochothrix deviennent dominants. L’emballage sous atmosphère modifiée (MAP) ou sous vide (VP) entraine clairement la sélection de Brochothrix dans un cas et d’un mélange de Brochothrix, bactéries lactiques (Lactococcus piscium, Carnobacterium maltaromaticum) et d’entérobactéries (Hafnia paralvei) dans l’autre, et ne permet pas une augmentation significative de la durée de conservation. Pour les filets d’ombrine, peu de différences sont observées entre MAP et VP dont le microbiote se compose essentiellement de bactéries lactiques (Carnobacterium spp., Vagococcus spp., Lactococcus spp., Leuconostoc spp.). La durée de conservation est étendue de 15 jours par rapport au poisson entier sous air.L’inoculation de différentes espèces bactériennes dans de la chair pauci-microbienne de thon ou d’ombrine a montré que Hafnia paralvei et Serratia spp. sont les espèces les plus altérantes. Brochothrix thermosphacta et Carnobacterium spp. produisent aussi des odeurs indésirables mais de façon plus modérée. Chez Pseudomonas, les espèces ne sont pas toutes altérantes et présentent même parfois des capacités à empêcher le développement des mauvaises odeurs induites par d’autres bactéries (Pseudomonas psychrophila/fragi) et à dégrader l’histamine (Pseudomonas cedrina, Pseudomonas plecoglossicida/monteilii). En parallèle, des tests sensoriels et des dosages physico-chimiques ont également été réalisés pour comprendre les conséquences de la croissance bactérienne et identifier des indicateurs fiables pour l’évaluation du degré d’altération des produits. / Fish is a highly perishable product and spoilage is mainly due to the bacterial growth. Compared to temperate regions, few studies examined the spoilage microbiota of tropical fish. In Martinique, yellowfin tuna (Thunnus albacares) and red drum (Sciaenops ocellatus) are essential fish of fisheries and aquaculture sectors. For a better characterization of the microbial ecosystem, culture-dependent and culture-independent (next-generation sequencing of 16S rRNA amplicons, Illumina MiSeq) methods were carried out.A wide diversity of species was found in freshly caught tuna and red drum (104 and 887 OTUs, respectively) and most of them are commonly isolated from fish (Chryseobacterium, Burkholderia, Flavobacterium, Psychrobacter, Arthrobacter, Staphylococcus). Others, such as Ralstonia sp. and Rhodanobacter terrae, largely present in fresh tuna, are less familiar. During the ice-storage of tuna, Pseudomonas and Brochothrix became dominant. The modified atmosphere packaging (MAP) and vacuum packaging (VP) clearly leaded to the selection of Brochothrix in one case and to a mixture of Brochothrix, lactic acid bacteria (Lactococcus piscium, Carnobacterium maltaromaticum) and enterobacteria (Hafnia paralvei) in the other case, and not conduct to a significant increase of the shelf-life. For red drum fillets, few differences were observed between MAP and VP with a microbiota essentially composed by lactic acid bacteria (Carnobacterium spp., Vagococcus spp., Lactococcus spp., Leuconostoc spp.). The shelf-life was extended by 15 days compared to the whole fish ice-stored.The inoculation of different bacterial species into the pauci-microbial flesh of tuna or red drum showed that Hafnia paralvei and Serratia spp. were the most spoiling bacteria. Brochothrix thermosphacta and Carnobacterium spp. produced more moderate undesirable odors. Among the Pseudomonas genus, not all species induced spoiling effects and some of them are even able to prevent the development of unpleasant odors from other bacteria (Pseudomonas psychrophila/fragi) and to degrade histamine (Pseudomonas cedrina, Pseudomonas plecoglossicida/monteilii).At the same time, sensory tests and physico-chemical assays were performed to understand the consequences of the bacterial growth and to identify reliable indices for the evaluation of the spoilage degree of the products.

Mon projet de doctorat était de créer un pipeline pour taxono-génomique pour la comparaison de plusieurs génomes bactériens. Deuxièmement, je automatisé le processus d'assemblage (NGS) et annotation à l'aide de divers logiciels open source ainsi que la création de scripts de maison pour le laboratoire. Enfin, nous avons intégré le pipeline dans la description de plusieurs espèces bactériennes de laboratoire sur. Cette thèse est divisée principalement en Taxono- génomique et Microbiogenomics. Les avis de la section taxono-génomique, décrit sur les avancées technologiques en génomique et métagénomique pertinentes dans le domaine de la microbiologie médicale et décrit la stratégie taxono-génomique en détail et comment la stratégie polyphasique avec des approches génomiques sont reformatage de la définition de la taxonomie bactérienne. Les articles décrivent les bactéries cliniquement importantes, leur séquençage complet du génome et les études génomiques comparatives, génomiques et taxono-génomique de ces bactéries. Dans cette thèse, j'ai inclus les articles décrivant ces organismes: Megasphaera massiliensis, Corynebacterium ihumii, Collinsella massiliensis, Clostridium dakarense. Bacillus dielmoensis, jeddahense, Occidentia Massiliensis, Necropsobacter rosorum et Pantoea septica. Oceanobacillus / My PhD project was to create a pipeline for taxono-genomics for the comparison of multiple bacterial genomes. Secondly I automated the process of assembly (NGS) and annotation using various open source softwares as well as creating in house scripts for the lab. Finally we incorporated the pipeline in describing several bacterial species from out lab. This thesis is subdivided mainly into Taxono-genomics and Microbiogenomics. The reviews in taxono-genomics section, describes about the technological advances in genomics and metagenomics relevant to the field of medical microbiology and describes the strategy taxono-genomics in detail and how polyphasic strategy along with genomic approaches are reformatting the definition of bacterial taxonomy. The articles describes clinically important bacteria, their whole genome sequencing and the genomic, comparative genomic and taxono-genomic studies of these bacteria.

The release of the Illumina BovineSNP50 BeadChip in late 2007 has drawn attention from cattle breeders around the world to develop breeding programs that leverage association of these single nucleotide polymorphism (SNP) with economically important quantitative trait loci (QTL). In that context this project has come to study applications of the SNP panel in beef cattle. Analysis of linkage disequilibrium (LD) existing in Angus, Charolais, and crossbred animals revealed the pattern of LD within each breed group, as well as the persistence of LD phase between pairs of the breed groups. This is important for genomic selection where SNP are trained in one population and used to predict breeding value for animals in another population. Detection of chromosome regions potentially carrying QTL or causative mutations affecting the phenotypic variation in economically important traits was presented at individual SNP and haplotype levels. There were 269 SNP associated (P<0.001) with birth weight (BWT), weaning weight (WWT), average daily gain (ADG), dry matter intake (DMI), mid-test metabolic weight (MMWT), residual feed intake (RFI). They explained 1.64% - 8.06% of the phenotypic variation in these traits. There were 520 SNP associated (P<0.001) with carcass quality traits, namely hot carcass weight, back fat thickness, ribeye area, marbling scores, lean yield grade by Beef Improvement Federation, steak tenderness, and six rib dissection traits. These SNP explained 1.90 - 5.89% of the phenotypic variance of the traits. Many of the significant SNP were located on chromosome 6. Six haplotypes were found associated (P<0.05) with ADG, DMI, and RFI. In order for genomic selection to happen in beef cattle, higher density SNP panels should be made available at low genotyping cost. However, the cost of genotyping animals for high density SNP chip is still high, thus genotype imputation has come to practice. The last chapter of this thesis compared two approaches presently used in genotype imputation, investigated factors affecting imputation accuracy, as well as the impact of imputation accuracy on genomic estimated breeding value (GEBV). It proved that the highest possible accuracy of GEBV is attainable with sufficiently large groups of reference animals. / Ontario Ministry of Agriculture, Food and Rural Affairs. Ontario Cattlemen’s Association. Ontario Farm Innovation Program. Agriculture and Agri-Food Canada’s Growing Forward Program. Agriculture Adaptation Council. Ontario Research and Development Program. MITACS Accelerate. Beef Improvement Opportunities.

Statens veterinärmedicinska anstalt, SVA, is a government agency that works for better animal and human health with a primary focus on infectious animal diseases. One of their projects involves tracking the spread of Campylobacter infection in broilers and the occurrence of antimicrobial resistance in these bacteria. A pipeline was developed to contribute to making the analysis of Whole Genome Sequencing (WGS) data from Campylobacter more effective. This was done by changing the currently used pipeline’s programming language from Perl to Python and adding the possibility to run multiple analyses in parallel. With parallelization, the time for running multiple analyses was reduced compared to running them sequentially, even if it was not as fast in practice as in theory. It also did not work as well when running parallel analyses of different strains compared to identical strains. Furthermore, different attributes of the pipeline were changed or added to improve the pipeline and a database comparison was performed in order to suggest the best ones for future use. VFDB, CARD, and MEGARes were suggested as appropriate databases to use in future WGS analysis of Campylobacter. Due to a lack of resources and technical difficulties, some of the requested attributes for the pipeline could not be implemented, such as the tool Pilon and the inclusion of MLST and cgMLST analysis. Nonetheless, the pipeline is well structured, has most of the requested tools, and is easy to run. With some minor improvements, the pipeline will be a useful tool for SVA and their project.

[ES] Las estaciones depuradoras de aguas (EDAR) tienen un papel fundamental en la protección del medio ambiente, evitan la llegada de nutrientes (nitrógeno y fósforo) y otros contaminantes a los ecosistemas acuáticos. El sistema más utilizado para la eliminación de nitrógeno en las EDAR es el proceso biológico de nitrificación-desnitrificación vía nitrato. El rendimiento de los sistemas biológicos está directamente relacionado con la estructura de la comunidad bacteriana y su metabolismo. En este trabajo se monitorizaron las variaciones temporales de las características fisicoquímicas del afluente, los parámetros operacionales y los rendimientos de eliminación del amonio en 6 reactores biológicos con sistemas de fangos activos. Para la caracterización de comunidades involucradas en el proceso de nitrificación se utilizaron diferentes técnicas de biología molecular: hibridación in situ con sondas marcadas con fluoróforos (FISH), secuenciación de segunda generación Illumina y secuenciación de tercera generación SMRT de PacBio, la cual no había sido utilizada hasta la fecha para el análisis de la microbiota de sistemas convencionales de eliminación de nutrientes. Para valorar la actividad de la biomasa nitrificante y de la biomasa heterótrofa se utilizaron técnicas respirométricas y técnicas para la cuantificación del ATP de última generación. Para analizar el gran volumen de datos generado tras la aplicación de las diferentes técnicas, se utilizaron técnicas estadísticas de análisis multivariante, como los modelos de regresión lineal multivariante basados en la distancia (DISTLM). Estas técnicas estadísticas permitieron valorar la contribución de las variables ambientales a la variabilidad observada en la estructura de las comunidades de bacterias nitrificantes, los rendimientos de eliminación del nitrógeno y su actividad. Las técnicas moleculares empleadas permitieron determinar que Nitrosomonas oligotropha, Nitrospira spp. y Nitrotoga sp. fueron las especies responsable de los procesos de nitrificación en las EDAR analizadas. Los resultados alcanzados con las técnicas FISH e Illumina sobre la estructura de la población de bacterias nitrificantes fueron similares, y permitieron detectar los sesgos de la secuenciación SMRT de PacBio. Los modelos de regresión permitieron valorar la contribución de las bacterias nitrificantes a la eliminación del amonio y cuáles fueron los factores que influían en su abundancia en cada una de las EDAR. La carga orgánica, la concentración de sólidos volátiles, la concentración de oxígeno y la temperatura fueron las variables de mayor influencia en la abundancia de estas especies. Mientras que la carga de fósforo influenció significativamente en su actividad. Este estudio reveló que la aplicación de ozono disminuye significativamente a los rendimientos de eliminación del amonio. Los resultados obtenidos ayudaron a mejorar la comprensión sobre el proceso de nitrificación en cada una de las EDAR y manifiestan la importancia de la dinámica poblacional de las bacterias nitrificantes en los rendimientos de eliminación del amonio en las EDAR. Estos resultados establecen que las técnicas moleculares combinadas con respirometría y los modelos de ordenación multivariante empleados en esta tesis, son una herramienta fiable para la monitorización y el control del proceso de nitrificación en sistemas de eliminación biológica de nitrógeno. Los resultados de esta tesis sugieren que la medida de los sólidos suspendidos volátiles activos mediante técnicas de determinación de ATP de segunda generación puede mejorar el cálculo de las variables de diseño y control más habituales de las EDAR. / [CA] Les estacions depuradores d'aigües residuals (EDAR) tenen un paper fonamental en la protecció del medi ambient, eviten l'arribada de nutrients (nitrogen i fòsfor) i altres substàncies contaminants als ecosistemes aquàtics. El sistema més utilitzat per a l'eliminació de nitrogen en les EDAR és el procés biològic de nitrificació-desnitrificació via nitrat. El rendiment dels sistemes biològics està directament relacionat amb l'estructura de la comunitat bacteriana i el seu metabolisme. En aquest treball es van monitorar les variacions temporals de les característiques fisicoquímiques de l'afluent, els paràmetres operacionals i els rendiments d'eliminació de l'amoni en 6 reactors biològics amb sistemes de fangs actius. Per a caracteritzar les comunitats involucrades en el procés de nitrificació s'han utilitzat diferents tècniques de biologia molecular: hibridació in situ amb sondes marcades amb fluoròfors (FISH), seqüenciació de segona generació Illumina i seqüenciació de tercera generació SMRT de PacBio, la qual no havia estat utilitzada fins avui per a l'anàlisi de la microbiota dels sistemes convencionals d'eliminació de nutrients. Per a valorar l'activitat de la biomassa nitrificant i de la biomassa heteròtrofa es van utilitzar tècniques respiromètriques i tècniques per a la quantificació de l'ATP d'última generació. Per a analitzar el gran volum de dades generat després de l'aplicació de les diferents tècniques, es van utilitzar tècniques estadístiques d'anàlisi multivariant, com els models de regressió lineal multivariant basats en la distància (DISTLM). Aquestes tècniques estadístiques van permetre valorar la contribució de les variables ambientals a la variabilitat observada en l'estructura de les comunitats de bacteris nitrificants, els rendiments d'eliminació del nitrogen i la seua activitat. Les tècniques moleculars emprades van permetre determinar que Nitrosomonas oligotropha, Nitrospira spp. i Nitrotoga sp resultaren les espècies responsables del procés de nitrificació en les EDAR analitzades. Les tècniques FISH i Illumnina van mostrar resultats molt similars sobre l'estructura de la població de bacteris nitrificants i van permetre detectar els biaixos de la seqüenciació SMRT de PacBio. Els models de regressió van permetre valorar la contribució dels bacteris nitrificants a l'eliminació de l'amoni i quins van ser els factors d'influència en la seua abundància en cadascuna de les EDAR. La concentració de matèria orgànica, sòlids volàtils en suspensió, la concentració d 'oxigen i la temperatura foren les variables amb més influència en l'abundància d'aquestes especies. Així mateix la càrrega de fòsfor va influir en la seua activitat. Aquest estudi va determinar que l 'aplicació ozó va causar una disminució significativa dels rendiments d 'eliminació del amoni. Aquests models van ajudar a millorar la comprensió sobre el procés de nitrificació en cadascuna de les EDAR i ressalten la importància de la dinàmica poblacional dels bacteris nitrificants en el rendiments de l 'eliminació de l 'amoni. Els resultats estableixen que les tècniques moleculars combinades amb respirometría i els models d'ordenació multivariant emprats en aquesta tesi, són una eina fiable per al monitoratge i el control del procés de nitrificació en sistemes d'eliminació biològica de nitrogen. Els resultats d'aquesta tesi suggereixen que la mesura dels sòlids suspesos volàtils actius mitjançant tècniques de determinació d'ATP de segona generació pot millorar el càlcul de les variables de disseny i control més habituals de les EDAR. / [EN] Wastewater treatment plants play an important role in environmental protection. These facilities protect aquatic ecosystems from excessive inputs of nutrients (nitrogen and phosphorous) and other pollutants. The most widespread system of nitrogen removal in wastewater treatment plants is a conventional method involving a biological nitrification-denitrification via nitrate. The efficiency of biological systems is directly related to bacterial community structure and its metabolism. In this work, the temporal variations of influent characteristics, operational parameters and ammonium removal efficiency in 6 bioreactors with activated sludge systems were monitored. To characterize the microbial communities involved in the nitrification process different molecular biology techniques were used: fluorescence in situ hybridization (FISH); second generation sequencing (Illumina), and third generation sequencing (SMRT PacBio). To assess the activity of nitrifying and heterotrophic bacteria, respirometric tests and second-generation ATP determination techniques were used. To analyse the large volume of data generated, statistical multivariate analysis techniques were used, including distance-based multivariate linear regression models (DISTLM). These statistical techniques allowed us to assess the contribution of environmental variables to the variability observed in the nitrifying community structure, in nitrogen removal performance and nitrifying activity. The molecular techniques employed determined that Nitrosomonas oligotropha, Nitrospira spp. and Nitrotoga sp. where the dominant nitrifying bacteria in the monitored WWTP. FISH and Illumnina technique showed very similar results and allowed for the detection of biases in PacBio SMRT sequencing. The regression models determined the contribution of nitrifying bacteria to ammonium oxidation and the factors influencing their abundance and activity. The main factors influencing the nitrifying bacteria abundance were organic load, volatile suspended solids concentration, dissolved oxygen and temperature. The activity of nitrifying bacteria also was influenced by phosphorous loading rate. This study revealed that ozone concentration was the main factor determining the low ammonium removal performance. These models helped to improve our understanding of the nitrification process in each WWTP and highlight the importance of nitrifying bacterial community structure in nitrogen removal performance. The results establish that the molecular techniques combined with respirometry and the multivariate ordering models used in this thesis, are a reliable tool for the monitoring and control of the nitrification process. The results of this thesis suggest that the measurement of active volatile suspended solids by means of second- generation ATP determination techniques can improve calculation of the most common design and control parameters of WWTPs. / A la Entidad de Saneamiento y Depuración de la Región de Murcia (ESAMUR) por la financiación del proyecto “Influencia de las variables operacionales y fisicoquímicas en la dinámica y estructura de la población de bacterias nitrificantes”, al Grupo de Química y Microbiología del Agua del IIAMA. A la Entidad Pública de Saneamiento de Aguas Residuales de la Comunidad Valenciana (EPSAR), por la financiación del proyecto “Estudio integrado del proceso biológico en plantas de tratamiento por fangos activos, análisis de interrelación entre los distintos componentes y optimización de métodos moleculares para la identificación de bacterias formadoras de espumas” al Grupo de Química y Microbiología del Agua del IIAMA / Barbarroja Ortiz, P. (2019). Estudio de la dinámica poblacional y actividad de los organismos nitrificantes en sistemas de depuración de aguas residuales [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/124063 / TESIS

Quinoa has been utilized for millennia in the Andes region of South America as a nutritious and hardy food crop. In recent years interest in quinoa has grown as need increases for an alternative to traditional cereal crops that can tolerate marginal environments while offering superior nutrition. Growers outside the Andes have experienced several complications adopting quinoa, including undesirable secondary metabolites, poor yield, lodging, and height inconsistency. Unfortunately, access to native ecotypes for crop improvement is limited, and desirable traits are difficult to introduce into available quinoa cultivars because of its allotetraploid genome and tendency to self-pollinate. A genome-wide survey of induced mutations in 244 sequenced M2 families was created from a bank of EMS-treated quinoa seeds and assembled into a library of mutant lineages with predicted variants and their effects on genes to assist in identifying agronomically valuable mutations in target genes as a supplement to crop improvement efforts. Using this library, eight families containing mutations in genes associated with reduced height "GAI1, GA20OX, GID1, and L " were identified. Several individuals exhibited a shorter than average phenotype; however, because each family contains thousands of EMS-induced mutations, the causative mutation of the reduced height phenotype in each family could not be definitively identified. In one family, absence of the GAI1 mutant allele, but the presence of a mutant CKX3 allele, provided a correlation between a mutation and the short phenotype. Genotyping each generation would be required for a targeted mutant allele to be tracked through selection.

Human noroviruses are a major cause of acute gastroenteritis worldwide and can betransmitted through consumption of contaminated raw food. Shellfish like oysters can becontaminated by human sewage during production and accumulate multiple Norovirus strainsin low concentrations. Here we developed a simplified and cost effective targetedmetagenomic approach by sequencing PCR amplicons with next generation sequencing(NGS) of the capsid (VP1) viral gene. New design of reverse primers using the codehopstrategy and direct addition of illumina adapter with one step RT-PCR and sequencing onnano chip reduced hand on time and cost of the analysis. A mix of faecal samples and oystersamples associated with outbreaks were used to evaluate the ability and limitations in theidentification of strains from Norovirus genogroup I (GI) and genogroup II (GII). Withsamples containing only one genotype the method was able to identify all strains. Usingartificially mixed samples the method was able to identify almost all strains except a few GIIat low concentrations. Oyster samples showed more limitations for the method and it waswere only able to identify the strain in some of the samples but did find multiple GI strains inmore than one sample. Despite some limitations, the simplified method for VP1-targetedmetagenomics is a sensitive approach allowing the study of norovirus diversity incontaminated oysters and the identification of norovirus strains implicated in outbreaks. Thisat a lower cost and hands on time compared to published methods.

Recent advances in high-throughput sequencing (HTS) technologies have revolutionized our understanding of microbial diversity and composition in relation to their environment. HTS-based characterization of metabolically active (RNA-derived) and total (DNA-derived) fungal communities in different terrestrial habitats has revealed profound differences in both richness and community compositions. However, such DNA- and RNA-based HTS comparisons are widely missing for fungal communities of groundwater aquifers in the terrestrial biogeosphere. Therefore, in this study, we extracted DNA and RNA from groundwater samples of two pristine aquifers in the Hainich CZE and employed paired-end Illumina sequencing of the fungal nuclear ribosomal internal transcribed spacer 2 (ITS2) region to comprehensively test difference/similarities in the “total” and “active” fungal communities. We found no significant differences in the species richness between the DNA- and RNA-derived fungal communities, but the relative abundances of various fungal operational taxonomic units (OTUs) appeared to differ. We also found the same set of environmental parameters to shape the “total” and “active” fungal communities in the targeted aquifers. Furthermore, our comparison also underlined that about 30%–40% of the fungal OTUs were only detected in RNA-derived communities. This implies that the active fungal communities analyzed by HTS methods in the subsurface aquifers are actually not a subset of supposedly total fungal communities. In general, our study highlights the importance of differentiating the potential (DNA-derived) and expressed (RNA-derived) members of the fungal communities in aquatic ecosystems.

No description available.