| 1. |
- Faust, Ellika
(författare)
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Genetic Identification of Corkwing Wrasse Cleaner Fish Escaping from Norwegian Aquaculture
- 2020
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The genetic impact of farmed fish escaping aquaculture is a highly debated issue. However, non-target species, such as cleaner fish that are used in fish farms to remove parasitic sea lice, are rarely considered. Here, we report that wild corkwing wrasse (Symphodus melops), which are transported long distances to be used as cleaner fish in salmon farms, escape and hybridize with local wrasse populations. Recently, increasing numbers of corkwing wrasse have been reported north of its described distribution range, in Flatanger in Trøndelag in Norway, an area heavily relying on the import of cleaner fish from Skagerrak. Using a high number of nuclear genetic markers identified with 2bRAD sequencing, we show that, although the Flatanger population is largely a result of a northward range expansion, there is also evidence of considerable gene flow from southern populations in Skagerrak. Of the 40 corkwing wrasses first sampled in Flatanger, we discovered two individuals with clear southern genotypes, one first-generation hybrid, and 12 potential second-generation hybrids. Thus, we found clear evidence of gene flow from source populations of translocated cleaner fish at the edge of an ongoing northwards range expansion. To better understand the extent of gene flow we then greatly expanded our sampling. Based on patterns of genetic divergence and homogeneity, we identified a smaller battery of 84 SNPs which is able to detect escapees with a Skagerrak origin as well as first and secondgeneration hybrids with high accuracy and power. We then used these SNPs to investigate the magnitude and geographic extent of escaping and hybridizing cleaner fish along the Norwegian coast. We found that escapees and hybrids may constitute up to 20 % of the local populations at the northern edge of the species distribution. In other parts of the Norwegian coast where salmon farming is also common, we found surprisingly few escapees and hybrids. Possible causes for few escapees and hybrids found in these areas are difficult to evaluate with the current lack of reporting of translocations by aquaculture operators. Overall, these findings provide critical information both for aquaculture management and conservation of wild populations of non-target species, and have implications for the increasing use of cleaner fish as parasite control in fish farms, that is both poorly documented and regulated. Moving genetic material between isolated populations could drastically alter the genetic composition and erode population structure, potentially resulting in loss of local adaptation and hampering natural range expansion. Although the ecological and evolutionary significance of escapees warrant further investigation, these results should be taken into consideration in the use of translocated cleaner fish.
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| 2. |
- Lund, David, 1994
(författare)
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Computational discovery of antibiotic resistance genes and their horizontal transfer
- 2022
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Antibiotic resistance is increasing among clinical infections and represents one of the most serious threats to public health. Pathogens often become resistant by acquiring mobile antibiotic resistance genes (ARGs) via horizontal gene transfer (HGT). To limit the spread of new ARGs, it is important that we identify emerging threats early, and that we improve our understanding of what drives the HGT of ARGs. The three papers encompassing this thesis aim to increase our knowledge about ARGs and their mobility. In paper I, computational screening of large genomic datasets was used to identify new resistance genes for macrolide antibiotics, and to clarify their evolution. A large diversity of new erm and mph genes was identified, including six new families of mobile ARGs carried by pathogens, that showed varied phylogenetic origins. Of the tested genes, 70% induced resistance in Escherichia coli . In paper II, we identified previously undiscovered mobile genes giving resistance to aminoglycoside antibiotics in pathogens, further demonstrating how computational methods can discover potential emerging ARGs. Close to one million bacterial genomes were screened for aac and aph genes, and the mobility of each predicted gene was evaluated. A total of 50 families of new mobile ARGs were identified in pathogens. When new ARGs were tested in E. coli . 86% were functional, with 39% giving clinical resistance. In paper III, the factors influencing the HGT of ARGs were investigated. Phylogenetic analysis was used to identify HGT events from a large set of ARGs. For each event, the genetic compatibility of the involved gene(s) and genomes, as well as the co-occurrence of donor and recipient in different environments, were computed and used as input to train random forest classifiers. The resulting models suggested that the most important factor for determining if a mobile ARG successfully undergoes horizontal transfer is the genetic compatibility between the gene and the recipient genome. The findings presented in this thesis increase our knowledge about new genes giving resistance to two important classes of antibiotics. Furthermore, the results provide new insights into the horizontal transfer of resistance genes.
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| 3. |
- Berglund, Fanny
(författare)
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Identification of novel antibiotic resistance genes through large-scale data analysis
- 2017
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Antibiotic resistance is increasing worldwide, and is considered a serious threat to public health by e.g. the World Health Organization. Antibiotic resistance genes are hypothesized to originate from harmless bacteria in and around us, from where they are horizontally transfered into human pathogens. It is therefore of great importance to explore human-associated and environmental bacterial communities to identify novel antibiotic resistance genes before they reach clinical settings. The three papers presented in this thesis aim to identify novel antibiotic resistance genes in large genomic and metagenomic datasets. In paper I, the aim was to identify novel genes of the clinically important subclass B1 metallo-β-lactamases. By analyzing whole bacterial genomes as well as metagenomes from environmental and human-associated bacterial communities, 76 novel putative B1 genes were predicted. Twenty-one of these were selected for experimental validation, whereof 18 expressed the predicted phenotype in E. coli. Phylogentic analysis revealed that the novel genes formed 59 previously undescribed gene families. In paper II, a large volume of genomic and metagenomic data was searched for novel plasmid-mediated quinolone resistance (qnr ) genes. In total, 611 qnr genes were predicted, of which 20 were putative novel. Nine of these were experimentally tested in E. coli, whereof eight expressed the predicted phenotype. In paper III, a new method for identification and reconstruction of novel antibiotic resistance genes from fragmented metagenomic data was presented. The method is based on gene specific models, which are optimized for a high sensitivity and specificity. The method is furthermore computationally efficient and can be applied to any class of resistance genes. The results of this thesis provides a deeper insight to the diversity and evolutionary history of two types of clinically relevant antibiotic resistance genes. It also provides new methods for efficient and reliable identification of novel resistance genes in fragmented metagenomic data.
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| 4. |
- Domingo Prim, Judit, 1989-
(författare)
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The exosome and the maintenance of genome integrity
- 2016
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The RNA exosome acts on different RNA substrates and plays important roles in RNA metabolism. The fact that short non-coding RNAs are involved in the DNA damage response led us to investigate whether the exosome plays a role in DNA repair. We have shown that the exosome catalytic subunit RRP6/EXOSC10 is recruited to DNA double-strand breaks (DSBs) in Drosophila S2 cells and human HeLa cells exposed to either ionizing radiation or I-PpoI endonuclease cleavage. DIS3, the other catalytic subunit of the nuclear exosome, is also recruited to DSBs, whereas the exosome core subunit EXOSC7 is not. Depletion of different exosome subunits does not interfere with the phosphorylation of the histone variants H2Av (Drosophila) or H2AX (humans), but depletion of RRP6/EXOSC10 impairs the recruitment of the homologous recombination factor RAD51 to the damaged sites, without affecting RAD51 levels. The recruitment of RAD51 to DSBs in S2 cells is also inhibited by overexpression of RRP6-Y361A–V5, a catalytically inactive RRP6 mutant. Furthermore, cells depleted of RRP6 or EXOSC10 are more sensitive to radiation, which is consistent with RRP6/EXOSC10 playing a role in DNA repair. RRP6/EXOSC10 can be co-immunoprecipitated with RAD51, which links RRP6/EXOSC10 to the homologous recombination pathway in animal cells. Taken together, our results suggest that a 3’-5’ ribonucleolytic activity is required for efficient DNA repair.
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| 5. |
- Lötstedt, Britta
(författare)
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Towards spatial host-microbiome profiling
- 2021
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Sequencing technologies and applications have pushed the limits and enabled novel studies of biological mechanisms, evolutionary relationships and communication networks between cells. The technical developments leading to single cell RNA-sequencing have enabled detection of rare cell populations while spatial resolution added insights into larger biological environments, like tissues and organs. Massively parallel sequencing has paved the way for integrated high-throughput analyses including that of studying gene expression, protein expression and mapping of microbial communities. This thesis starts with an introduction describing the technical and biological advancements made in recent years with focus on spatially resolved approaches. Then, a summary of recent accomplishments is presented, which enabled ongoing work in a novel field of spatial hostmicrobiome profiling. Lastly, the concluding remarks include both a future perspective and a short reflection on the current developments in the spatial multi-omics field. 16S sequencing is often used for taxonomic classification of bacteria. In Paper I, this sequencing technique was used to study the aerodigestive microbiome in pediatric lung transplant recipients. Many of these patients regretfully reject the organ after transplant, but the underlying cause is, in many cases, unknown. In this paper, multiple factors influencing rejection were examined including that of the aerodigestive microbiome. Pediatric lung transplant recipients often suffer from gastrointestinal dysmotility and the focus of this study was also to analyze changes in the microbiome in relation to irregular gastric muscle movements. The results showed that lung transplant recipients had, in general, lower microbial diversity in the gastric fluid and throat and also that the microbial overlap between lung and gastric sampling sites was significantly less in transplant recipients compared to controls. In addition, gastrointestinal dysmotility was shown to influence the gastric microbiome in lung transplant recipients, but, given the small sample size available in this study, the correlation to patient outcome could not be examined. Integrated analysis of the transcriptome and the antibody-based proteome in the same tissue section was enabled using the method developed in Paper II. Spatial Multi- Omics (SM-Omics) uses a barcoded glass array to capture mRNA and antibody-based expression of selected proteins in the same section. The antibody-based profiling of the tissue section was enabled by either immunofluorescence or DNA-barcoded antibodies that were then decoded by sequencing. The protocol was scaled-up using an automated liquidhandling system. Using this method, simultaneous profiling of the transcriptome and multiplexed protein values was determined in both the mouse brain cortex and mouse spleen. Results showed a high correlation in spatial pattern between gene expression and antibody measurements, independently of the antibody labelling technique. SM-Omics generates a high-plex multi-omics characterization of the tissue in a high throughput manner while exhibiting low technical variation.
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| 6. |
- Karfunkel, Torbjörn, 1969
(författare)
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Amelioration of Horizontally Transferred Genes
- 2008
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The importance of Horizontal (Lateral) Gene Transfer has attracted much attention during the last few decades. It is a very important factor in bacterial evolution and ecology, and it has great implications on human life as well.In response to selective and mutational pressures, horizontally transferred genes will undergo changes in DNA composition, ameliorate. When subject to these pressures long enough, the genes will exhibit a pattern of GC content, codon usage, and oligonucleotide abundance characteristic to the environment. The mechanisms responsible are not known, but it is commonly assumed that they result from processes specific to the host.In this dissertation I demonstrate that* a method used by Lawrence and Ochman in 1997 to estimate the time since the introduction of horizontally transferred genes is overly optimistic. I have developed a better technique for this purpose.* the mechanisms responsible for the amelioration of horizontally transferred genes can be attributed to the replicator molecule on which it is situated, be it the bacterial chromosome or a plasmid.
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| 7. |
- Boulund, Fredrik, 1985
(författare)
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Analysis of large-scale metagenomic data
- 2013
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The topic of this thesis is the analysis of large data sets of DNA sequence data produced from modern high-throughput DNA sequencing machines. Using such machines to sequence the genetic content of a microbial community produces a metagenome. This thesis comprises three research papers, all connected to the study of large metagenomic data sets. In the first paper, we developed a method for discovering fragments of fluoroquinolone antibiotic resistance genes in short fragments of DNA. The method uses hidden Markov models for identifying qnr genes in short DNA fragments. Cross-validation showed that our method for classifying short fragments has high statistical power even for fragments as short as 100 base pairs, a length commonly encountered in modern next-generation sequencing data. In the second paper, the putative qnr genes identified in the first paper were verified using wet-lab experiments. This was a follow-up study to validate the findings from the first paper. An expression system for qnr genes in Escherichia coli hosts was developed and used to evaluate the resistance phenotype of the novel gene candidates discovered in the first paper. In the third paper, we developed an easy-to-use high performance method for distributed gene quantification in metagenomic sequence data. It leverages high-performance computing resources to provide high throughput while maintaining sensitivity. This enables efficient and accurate gene quantification, suitable for use in comparative metagenomics. Next-generation DNA sequencing has had a big impact on molecular biology. As the size of the produced data sets increases, there is an equally increasing need for methods suited for the analysis of such data sets. This thesis presents several new methods that are well adapted to analysis of modern terabase-sized metagenomic data sets.
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| 8. |
- Ausmees, Kristiina
(författare)
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Efficient computational methods for applications in genomics
- 2019
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- During the last two decades, advances in molecular technology have facilitated the sequencing and analysis of ancient DNA recovered from archaeological finds, contributing to novel insights into human evolutionary history. As more ancient genetic information has become available, the need for specialized methods of analysis has also increased. In this thesis, we investigate statistical and computational models for analysis of genetic data, with a particular focus on the context of ancient DNA.The main focus is on imputation, or the inference of missing genotypes based on observed sequence data. We present results from a systematic evaluation of a common imputation pipeline on empirical ancient samples, and show that imputed data can constitute a realistic option for population-genetic analyses. We also discuss preliminary results from a simulation study comparing two methods of phasing and imputation, which suggest that the parametric Li and Stephens framework may be more robust to extremely low levels of sparsity than the parsimonious Browning and Browning model.An evaluation of methods to handle missing data in the application of PCA for dimensionality reduction of genotype data is also presented. We illustrate that non-overlapping sequence data can lead to artifacts in projected scores, and evaluate different methods for handling unobserved genotypes.In genomics, as in other fields of research, increasing sizes of data sets are placing larger demands on efficient data management and compute infrastructures. The last part of this thesis addresses the use of cloud resources for facilitating such analysis. We present two different cloud-based solutions, and exemplify them on applications from genomics.
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| 9. |
- Khan Mirzaei, Mohammadali, 1981-
(författare)
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Study of phages for phage therapy : An Escherichia coli experience
- 2014
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The world is rapidly moving toward a post-antibiotic era as effective antibiotics are becoming fewer. phage therapy is a potentially possible method for treating infectious diseases and counts as an alternative method for antibiotic therapy. Phages' number on earth is estimated to be 1031 which ensures a great variety for these entities, and a never ending source for new phages. However, phages vary in their morphology, life cycle and virulent ability, and thus phage therapy will be unsuccessful without enough knowledge in phage biology. Only phages with a good virulence and broad host ranges are fitted for phage therapy applications, host range being one the most important factors that needs a careful study. There is no common method for studying the host range of phages and host range measurements are consequently dependent on the method that was used to study it. In our study, six polyvalent phages were selected from thirty virulent phages isolated using strains from E. coli reference collection (ECOR) as hostes. The selected phages were tested for their host range across 234 strains of E.coli (both sensetive and resistant to antibiotics) and Salmonella (reference collection SARA and SARB) using both spot test and efficiency of plating (EOP) method. Spot test results show no correlation to results of EOP analyses. One way to explain why spot test results are uncorrelated to EOP is the effects of resistant mechanisms of the host. In spot test, phages of a high concentration lystae might absorb, degrade the cell wall and kill the bacteria but the intrinsic resistant mechanisms of the host can recognize and degrade the genome, and stop phage replication and reproduction, which is visualized in the EOP analysis. Based on our studies, it is possible that prophages can contribute to bacterial defence against phages. Phages cannot in most cases produce a high EOP on those E. coli strains that have a P2 prophage in their genome.The phage SU10 had the highest EOP/spot test ratio among six selected phages. Transmission electron microscopy images revealed a very rare morphology for SU10, a short tail of 19 nm long and a 137 elongated head, and it was classified as a member of Podoviridae family. Genomic analysis of the phage show a genome size of 77,327 kb, 123 ORF encodes 38 proteins with known function. Twenty-two of theses proteins were identified using a mass-spectrometry based proteomics. The size of the tail was measured to be longer, 41 nm, in ultra-thin sectioning transmission electron microscopy images. A honeycomb structure is formed by the phage capsids during structural assembly, which is very rare for bacteriophages. The honeycomb structure of SU10 capsids might work as an aid or and external scaffolding protein during morphogenesis. The SU10 capsid is elongated before its genome is packed into the head according to the ultra-thin sectioning transmission electrographs which indicates that the genome packaging process has no role in elongation of the head. Based on our phylogenetic analysis, the scaffolding protein and major head protein have coevolved and the scaffolding protein in SU10 is not a recent attachment to the major head protein. According to the phylogenetic analysis of two proteins, the C1 and C3 morphotypes diverged 280 million years.
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| 10. |
- Neiman, Mårten, 1982-
(författare)
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Tagging systems for sequencing large cohorts
- 2010
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Advances in sequencing technologies constantly improves the throughput andaccuracy of sequencing instruments. Together with this development comes newdemands and opportunities to fully take advantage of the massive amounts of dataproduced within a sequence run. One way of doing this is by analyzing a large set ofsamples in parallel by pooling them together prior to sequencing and associating thereads to the corresponding samples using DNA sequence tags. Amplicon sequencingis a common application for this technique, enabling ultra deep sequencing andidentification of rare allelic variants. However, a common problem for ampliconsequencing projects is formation of unspecific PCR products and primer dimersoccupying large portions of the data sets. This thesis is based on two papers exploring these new kinds of possibilities andissues. In the first paper, a method for including thousands of samples in the samesequencing run without dramatically increasing the cost or sample handlingcomplexity is presented. The second paper presents how the amount of high qualitydata from an amplicon sequencing run can be maximized. The findings from the first paper shows that a two-tagging system, where the first tagis introduced by PCR and the second tag is introduced by ligation, can be used foreffectively sequence a cohort of 3500 samples using the 454 GS FLX Titaniumchemistry. The tagging procedure allows for simple and easy scalable samplehandling during sequence library preparation. The first PCR introduced tags, that arepresent in both ends of the fragments, enables detection of chimeric formation andhence, avoiding false typing in the data set. In the second paper, a FACS-machine is used to sort and enrich target DNA covered emPCR beads. This is facilitated by tagging quality beads using hybridization of afluorescently labeled target specific DNA probe prior to sorting. The system wasevaluated by sequencing two amplicon libraries, one FACS sorted and one standardenriched, on the 454 showing a three-fold increase of quality data obtained.
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