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Abstract

The development of high-throughput sequencing (HTS) technologies has enabled us to gain a much deeper insight into the mysterious and wonderful world of bacteria. These exciting technologies can be used in a variety of ways, ranging from the study of entire bacterial communities to more specific studies of individual bacterial isolates. In this thesis, we examine different HTS-based approaches to studying bacteria, applying insights from our own case studies. These include targeted HTS for metagenomic studies, whole genome sequencing (WGS) to study plant-pathogenic bacteria, and a genome-informed approach for the development of new molecular methods to detect target bacteria with high specificity.
Targeted HTS is a common approach in metagenomics, where it can be used to determine microbiomes. However, the results are often affected by the way samples are processed before sequencing, with the DNA extraction method being especially important. In our study, we examined the impact of three different DNA extraction methods on the results of microbiome and resistome determination using sputum samples as an example. We found that the DNA extraction method significantly affects the results for the diversity and composition of the sputum microbiome and resistome. Despite these differences, the results obtained with targeted HTS were repeatable. This highlights the importance of careful experimental design in microbiome research, where selecting the appropriate DNA extraction method is of crucial importance. WGS is often used to perform a deeper analysis of selected bacteria. The data generated in such an experiment is then used to perform comparative genomics studies. The main focus of our study was the plant pathogen Pantoea stewartii subsp. stewartii, the causative agent of Stewart's wilt in maize. In recent years, this quarantine bacterium has been found in Slovenia and elsewhere in Europe. To shed light on these findings, Slovenian isolates were sequenced alongside historical isolates from collections and compared to publicly available genomes. The results revealed all Slovenian isolates except one group together and are not related to other European isolates, suggesting multiple introduction events. The data generated by both metagenomic sequencing and WGS can be used to develop new molecular tests for the specific detection of target bacteria. Molecular detection methods are particularly well-suited for slow-growing bacteria. In our study, we used available genomic data to design multiple new quantitative real-time PCR (qPCR) tests to detect one such bacterium: Xylophilus ampelinus, the causative agent of bacterial blight in grapevines. In laboratory experiments, three of the newly designed qPCR tests were proven to be suitable for detecting X. ampelinus in different grapevine samples with high efficiency, specificity, and sensitivity.
The examples of HTS applications described in this thesis clearly demonstrate the versatility of this method. These include basic research, in which HTS can be used to develop and test new hypotheses regarding bacterial genomics, biology, and ecology. Other, more practical applications of HTS often include diagnostics, where it can be used to either directly analyse samples or to develop new diagnostic tests. HTS technologies will undoubtedly continue to evolve and remain an indispensable tool in the field of bacteriology.