The rapid and reliable detection of pathogens is of utmost importance in healthcare settings to ensure the appropriate treatment thereby reducing morbidity and mortality for the patient. Current culturing, PCR based and NGS species detection methods are time consuming (Opota et al., 2015), limited in their detection (Buckley et al., 2015), or require specialist skills and are expensive (Basho and Eterovic., 2015). Oxford Technologies Nanopore devices could provide detailed genomic sequencing at a fraction of the cost and without the need for technical bioinformatic skills. This study evaluates the MinION device and analysis tools to suggest best practice. Classification and genotyping of 12 Klebsiella isolates were performed using EPI2ME automated workflows and manual de novo assembly.  Automated workflows using raw MinION reads provided clinically relevant information identified in ~6hrs. Manual de novo assembly and analysis used hybrid, and single source data took >24hrs. The inclusion of MinION long reads overcome problems assembling short reads. Hybrid genomes provided the most contiguous and highly detailed contigs. MinION only read assemblies contained more errors but still identified similar genotypic findings. Automated workflows are rapid and require minimal bioinformatic know-how. There should be a dialogue between clinicians and bioinformaticians to develop bespoke analysis tools.  Although challenges remain around compatible kits and vulnerable flowcells long read sequencing can be an effective tool for species detection and pathogen typing. Furthermore, hybrid assemblies have the potential to advance our genome detailing and discovery.

Within the collections of the Museu de Angra do Heroismo (Terceira Island, Azores, Portugal) are nine cast bronze guns from the 16th century. Most were raised from the seafloor between the 1960s and 1990s, but this study comprises the first in-depth research into their design and manufacture. The importance of this kind of study lies in the fact that ordnance is commonly found on shipwrecks of this time. A greater knowledge of guns will help provide information about the ships from which they came. Careful documentation and study of the Museu de Angra cannon will add greatly to their value as museum exhibits, by allowing museum patrons to better understand where the guns came from, how they were cast, and why they were important. This documentation adds to our knowledge of Western European gunfounding technology during the sixteenth century, as four different countries commissioned the guns: Portugal, Spain, France, and England. With detailed documentation and publication, the Museu de Angra bronze guns can be added to the bibliography of ordnance of this period, which will aid future researchers who encounter similar pieces. The Museu de Angra bronze guns, as symbols of the military and naval power of the countries that commissioned them, were sent aboard ships, into the field, and mounted on fortress walls. Bronze guns of this time period are particularly important, as bronze was an expensive commodity, and the demand for ordnance was increasing rapidly. Countries developed more effective ways to make use of iron for the founding of guns, and the use of bronze became more symbolic of wealth. The information that each gun contains includes both the cutting-edge military technology of the time and the artistic statement of the founder. Some of the finest metalwork of the period was displayed in cast bronze guns, and due to the founding techniques, no two are the same, making each an important piece of history.

Methicillin-resistant Staphylococcus aureus (MRSA) is a variant of the more common Methicillin-Susceptible Staphylococcus aureus (MSSA), an opportunistic pathogen a portion of the human population carries as normal bacterial flora. When an outbreak of MRSA occurs, it is often important to determine if and how these strains are related to each other. In this report two different types of epidemiological methods were combined (namely Multi-Locus Sequence Typing and staphylococcal protein A-typing), in order to reduce workload, costs and time. A panel of resistance and virulence markers was also added to gather as much information about the culture as possible in a single analysis. To test the viability of the method extracted DNA and heat-treated bacterial cultures of both MRSA and MSSA were amplified with a curated panel of primers. These products were later sequenced with Nanopore’s MinION using the Flongle flow-cell. The method showed promise and worked as intended regarding the staphylococcal protein A-typing and the panel of resistance and virulence markers. However, the Multi-Locus Sequence Typing did still require optimization in order to be used clinically. In summary the project can be viewed as a success since it succeeded in being more time, cost and work efficient than many of its predecessors, when the problems with the Multi-Locus Sequence Typing are solved.

Cystic fibrosis (CF) is an autosomal recessive genetic disorder involving a mutation in the CF transmembrane conductance regulator protein (CFTR), which causes dysfunctional transport of chloride ions across cell membranes. CF affects multiple body systems and a few of its symptoms include chronic cough, difficulty breathing, obstructive airway disease, bacterial pulmonary infections, maldigestion, malabsorption, pancreatitis, and male infertility. Until recently, treatment options have been limited to alleviating symptoms, but a new classification of drugs, CFTR modulators, provide an opportunity to slow the progression of the disease and improve clinical outcomes. The effect of CFTR modulators may be attributed to the reduction of persistently colonizing bacteria in CF lungs. Though, the effects of modulators on microbial communities colonizing the CF lung remains unknown, specifically with common respiratory pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus. Particularly, previous CF studies have been limited in scope due to focusing on only one type of modulator and by using low-yield sequencing techniques. To address this gap, we seek to study the changes in CF respiratory pathogens of patients initiating CFTR modulator therapy at Nemours Hospital using long-read metagenomic sequencing (Oxford Nanopore) of longitudinally collected respiratory samples. We have optimized a protocol for host DNA depletion and microbial metagenomic sequencing to characterize the respiratory microbiome. This study focuses on utilizing these sequencing data to compare the microbiome among two healthy controls to pre-CFTR-treatment microbial communities of two recruited pediatric CF patients.

Palynology or the study of pollen, is essential understand the relationship between plants and their pollinators. Traditionally, pollen grains are identified by microscopy. The method has several shortcomings, such as being time-consuming and having low taxonomic resolution. DNA-barcoding-based sequencing can identify pollen at the genus and species levels without specialized paleontological expertise. Aim of this study is to assess which molecular approach can be the most effective tool and is the most cost-effective for the identification of pollen from mixed pollen samples. A DNA metabarcoding study was conducted using the rbcL barcode gene for pollen identification using two sequencing techniques: Sanger and MinION. DNA metabarcoding produced taxonomic data easily. For the analysis of Sanger and MinION sequencing data, BLAST and KRAKEN2 were used respectively. Pavian and KRONA were later used to visualize the MinION sequencing data. Various plant species native to Sweden were identified with this metabarcoding approach. However, the reference database failed to identify a few of them, thus indicating the need to expand the reference database.

No description available.

Bien que les sexes séparés (dioecie) soient plus rares que chez les animaux, ∼15 600 espèces dioiques ont évolué chez les angiospermes (∼6% de l'ensemble des espèces). La manière dont le sexe de ces plantes est contrôlé est une question centrale de la biologie végétale, mais également de l'agronomie car de nombreuses plantes cultivées sont des plantes dioiques (∼20% des espèces cultivées) mais dont un seul sexe (généralement les femelles) présente un intérêt agronomique. Pourtant, seulement trois gènes du déterminisme du sexe ont été identifiés à ce jour chez les plantes dioiques, chez le kaki, l'asperge et la fraise. La dioecie a vraisemblablement évolué plusieurs fois chez les angiospermes et il est possible que les gènes du déterminisme du sexe soient divers. Deux voies principales d'évolution vers la dioecie ont été identifiées. Les deux partent d'une espèce dont les fleurs sont hermaphrodites, le régime de reproduction ancestral chez les angiospermes, puis passent soit par un intermédiaire monoique (espèce avec des fleurs unisexuées mâles et femelles sur le même individu), soit par un intermédiaire gynodioique (espèce avec des femelles et des individus avec des fleurs hermaphrodites). Cette thèse a pour objet la comparaison de deux systèmes de plantes représentant ces deux voies. Chez Coccinia grandis, une cucurbitacée ayant également des chromosomes XY, l'évolution de la dioecie est passée par la monoecie. Chez Silene latifolia, une plante dioique bien étudiée avec des chromosomes sexuels XY, l'évolution de la dioecie s'est faite à partir de la gynodioecie. Trois gènes contrôlant la monoecie ont été identifiés chez le melon et il a été proposé que ces gènes soient les gènes du déterminisme dans les espèces dioiques proches du melon comme C. grandis. Nous avons donc opté pour une approche gène candidat dans cette espèce. Très peu de ressources génétiques et génomiques sont disponibles chez C. grandis, et nous avons choisi d'utiliser SEXDETector, une méthode probabiliste qui utilise des données RNA-seq pour génotyper des parents et leurs descendants, et qui infère les gènes lies au sexe sans génome de référence. Cette méthode m'a permis d'identifier 1 364 gènes présents sur les chromosomes sexuels de C. grandis. J'ai établi que les gènes differentiellement exprimés entre les sexes étaient plus abondants sur chromosomes sexuels que sur les autosomes. J'ai également observé des marques de la dégénérescence du chromosome Y chez cette plante, comme des diminutions d'expression ou des pertes de gènes. Enfin, mes résultats démontrent la présence de compensation de dosage chez C. grandis. Le test des gènes candidats est en cours. Chez S. latifolia, 3 grandes régions liées au déterminisme ont déjà été identifiées sur le chromosome Y. Pour identifier les gènes du déterminisme, nous avons choisi de séquencer ce chromosome. Le séquençage des chromosomes Y est encore un défi pour la génomique. La phase d'assemblage est très difficile à cause des répétitions présentes en grand nombre sur ces chromosomes. En conséquence, les séquences complètes de chromosome Y sont très rares, et principalement disponibles chez les animaux. Afin de minimiser les problèmes d'assemblage dus aux répétitions, nous avons utilisé des techniques dites de 3eme génération (avec de grandes lectures). J'ai moi-même généré des données MinION (Oxford Nanopore) à partir d'ADN de chromosome Y. L'assemblage a été réalisé en combinant des données Illumina, PacBio et MinION. Notre assemblage final fait une taille de 563 Mb pour un N50 de 6 114 pb, et contient 16 219 gènes annotés de novo / Although rarer than in animals, separate sexes (dioecy) have evolved in ∼15,600 angiosperm species (∼6% of all angiosperm species). How sex is controlled is a central question in plant sciences and also in agronomy as many crops are dioecious (∼20% of crops) with only one useful sex (usually female). Only three master sex-determining genes have been identified in dioecious plants so far, namely in persimmons, asparagus and strawberry. Dioecy likely evolved several times independently in angiosperms, suggesting that sex-determining genes are of diverse origins. Hermaphroditism is the predicted ancestral state of the angiosperm flower. Two main pathways have been identified that explain the evolution of hermaphroditism towards dioecy: either through a monoecious state (with both unisexual male and female flowers on the same individual) or a gynodioecious state (with females and individuals having hermaphroditic flowers). My aim is to compare two plant systems representing each one of these two pathways. In Coccinia grandis, a Cucurbitaceae with an XY chromosome system, dioecy evolved through monoecy. In Silene latifolia, a well-studied dioecious plant with XY sex chromosomes, dioecy evolved through gynodioecy. Three genes controlling monoecy have been identified in melon, and it was suggested that these genes act as sex-determining genes in closely related dioecious species such as C. grandis. I therefore chose a candidate gene approach in this species. Very few genetic and genomic data are available in C. grandis, and we chose to use SEX-DETector, a probabilistic method that uses RNA-seq data to genotype parents and their offspring, and infers sex-linked genes with no need for a reference genome. This method allowed me to identify 1,364 genes that are present on the sex chromosomes of C. grandis. I found that the sex chromosomes are enriched in sex-biasedgenes when compared to autosomes and I characterized Y chromosome degeneration in terms of decreased expression and gene loss. Finally, I showed that dosage compensation occurs in C. grandis. Testing for the three candidates genes is ongoing. In S. latifolia 3 regions involved in sex determination have already been identified on the Y chromosome. We chose to sequence this chromosome to identify sex-determining genes. The sequencing of Y chromosomes remains one of the greatest challenges of current genomics. The assembly step is very difficult because of their highly repeated content. Consequently, fully sequenced Y chromosomes are rare and mainly available for research in animals. To overcome the difficulty of assembling reads with many repeats, I used third generation sequencing (TGS, producing long reads). I produced a dataset using the Oxford Nanopore MinION sequencer with Y chromosome DNA. Assembling was performed using a combination of Illumina, MinION and PacBio sequencing data. The final assembly had a total length of 563 Mb with a scaffold N50 of 6,114 bp, and contained 16,219 de novo annotated genes

Detta arbete beskriver en undersökning som har utvärderat två navigeringstekniker: flockbeteenden och potential fields. I undersökningen har minioner i ett MOBA-scenario simulerat dessa två navigeringstekniker för att avgöra vilken av dem som ger den mjukaste navigeringen. Mjukhet i detta sammanhang innebär att minionerna kontinuerligt ska kunna navigera utan att fastna, samt minimera förändringar av riktningen mellan bilduppdateringar. En prototyp av ett MOBA-scenario utvecklades för att genomföra två experiment där mätningar togs när minionerna navigerade med de två navigeringsteknikerna. Resultatet visade att algoritmen för flockbeteendena medförde den mjukaste navigeringen. Dock konstaterades det att minionerna fastnade alltför ofta, vilket indikerar att framtida utveckling bör fokusera på att eliminera detta oönskade fenomen. / <p>Det finns övrigt digitalt material (t.ex. film-, bild- eller ljudfiler) eller modeller/artefakter tillhörande examensarbetet som ska skickas till arkivet.</p><p>There are other digital material (eg film, image or audio files) or models/artifacts that belongs to the thesis and need to be archived.</p>

Introduction: RNA viruses are economically important pathogens of fish, and among these viruses, infectious pancreatic necrosis virus (IPNV) is of particular concern for the aquaculture industry, especially for farmed rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). This non-enveloped aquatic virus, which was first isolated in the UK in 1971, belongs to the family of Birnaviridae and has a bi-segmented dsRNA genome of about 6kb. IPNV is classified in 6 genogroups with correspondence to 10 known serotypes and an additional proposed genogroup of marine aquabirnaviruses (MABV). IPNV causes high mortality in fry and a reduced mortality in adult fish, respectively. Fish, which survive, can become carriers and this can lead to a clinical outbreak by releasing infective material into water or by vertical transmission via oocytes, milt and seminal fluids. Methods: This project aimed at determining the phylogeny and genomic changes of IPNV in Scotland by whole genome sequence analysis of IPNV isolates (diagnostic TCID50 supernatants) spanning 3 decades since 1982, using next generation sequencing technology. Viral RNA of IPNV culture supernatant (CHSE-214 and TO cell culture) was processed for next generation sequencing on an Illumina MiSeq platform. Library preparation was performed using the Nextera XT DNA Library Kit, prior to sequencing according to the manufacturer's MiSeq Reagent Kit v3 (150cycles) protocol. To optimize whole genome next generation sequencing for IPNV, we compared two RNA processing protocols, the Glasgow (GLAP) and the Goettingen protocol (GOEP) with focus on missing terminal nucleotides after a de novo genome assembly. Sequences were used to determine the phylogeny and selection pressure on the genome as well as a possible virus-host adaptation. Results: The results showed that both protocols were able to give full length genomes as well as genomes with missing terminal nucleotides. The phylogenetic analysis of 57 sequenced IPVN isolates shows that 78.95 % of the isolates group within genogroup V, which includes serogroup Sp and 5.26 % within genogroup I which includes serogroup Ja. Segment A of 15.79 % of the isolate grouped within genogroup III, which includes serotype Ca1 and Te but only 7.02 % of the segment B isolates grouped in the genogroup III. The remaining 8.77 % of segment B groups within genogroup II, containing the Ab serotype. Previous research has shown that residue substitutions at positions 217 and 221 in the major capsid protein VP2 have an impact on the virulence of the virus, leading to different virulence types: virulent (T217, A221), low virulence (P217, A221), avirulent (T217, T221) and persistent (P217, T221). Whole genome sequence results show that 58.93 % of the sequenced isolates belong to the persistent, 32.14 % to the low virulent type, only one isolate was of a virulent type and 7.15 % had not virulence assigned amino acid compositions in positions 217 and 221. The selection pressure analysis showed that especially VP2 is experiencing selection pressure in the variable region. In the VP1 protein we see two sites under positive selection pressure within specific motifs. VP5 showed positive selected sites mostly within the truncated region of the protein. Other proteins showed no particular interesting sites of selection. The codon adaptation analysis showed highest adaptation index for VP2. Besides VP5, which had an CAI index below one, therefore showing negative adaptation, other IPNV proteins had an CAI of barely above the value of 1. The dinucleotide abundance, focussing on CpG, showed that CpG is underrepresented in segment A and B. Discussion Phylogenetic analysis of the sequenced IPNV strains shows separate clustering of different genogroups. Genetic reassortment is observed in segment B showing a grouping within genogroup III and II although the segment A of these isolates was grouping exclusively within III. We found that over 50 % of the isolates belong to the persistent and over 30 % to the low virulent type, assuming that due to not sterilising vaccination these types were selected in the vaccinated population. The results from the CAI calculations indicate an adaptation of IPNV to its host. Together with the findings that CpG is underrepresented in IPNV it suggests that this leads to an immune escape. Especially since the selection pressure analysis showed positive selection in VP2 within the virulence determination sites of the protein, indicating that IPNV "tries" to downregulate immune recognition. The prevalence of mostly persistent type of isolates indicates together with the assumption of adaptation and immune escape that IPNV is evolving with the host in order to ensure survival.