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The importance of different sources and sequencing protocols in increasing the accuracy of NGS analysis in diagnostic applications
Kvapilová, Kateřina ; Kozmik, Zbyněk (advisor) ; Piherová, Lenka (referee) ; Slabý, Ondřej (referee)
1 ABSTRACT Continued advances in next-generation sequencing (NGS) technology, such as capacity, speed, and reduced cost per sequenced base, revolutionize personalized medicine and bring genomics into routine clinical practice. Nevertheless, NGS is still under rapid development, and the variability of sequencing protocols and validation procedures is one of its current bottlenecks. This thesis aimed to study the influence of different sample sources and NGS protocols (NGS library construction-sequencing-data analysis) on the accuracy of NGS analysis in diagnostic applications. In the first study, performed during the COVID-19 pandemic outbreak, we developed NGS protocols suitable for a whole genome analysis of the SARS-CoV-2 virus. Subsequently, in the second study, we examined the suitability of human saliva-derived gDNA for genomic/genetic analysis of selected variant types compared to traditional blood-derived gDNAusing validated NGS protocol and statistical comparison of the obtained data. Whole genome analysis of the SARS-CoV-2 genome was performed using two captured- based approaches and one amplicon-based approach to study the quality and effectivity of the respective NGS protocols. Synthetic controls were employed to verify the accuracy and specificity of the developed NGS protocols. We proved that...
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Profiling of T-cell receptor rearrangements in type 1 diabetes and celiac disease research
Obermaierová, Anna ; Froňková, Eva (advisor) ; Volejníková, Jana (referee)
This thesis discusses the detection of T-lymphocyte receptor rearrangements (TCR) with NGS methods and its significance in autoimmune disorders such as type 1 diabetes (T1D) and celiac disease. The first part introduces sequencing methods of TCRs and highlights the breakthrough that came with the advent of NGS. The second part then describes various approaches to utilizing these methods in medicine, with special attention dedicated to the endeavour of finding biomarkers for two closely related autoimmune disorders: T1D and celiac disease.
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Optimization of mycovirus detection methods obtained from NGS data analysis
PÍCHALOVÁ, Barbora
One well-known pathogenic fungus Armillaria causing Armillaria root disease which is characterised by infecting the root system of woody plants, resulting in basal decay and the development of a white syrrocium under the bark of trees. Several genome sequences of mycoviruses have already been published in research on this disease, including viruses from the Mymonoviridae, Botourmiaviridae, Partitiviridae, Virgaviridae as well as a newly described group of viruses known as ambi-like viruses. In this study, alongside ambi-like viruses and partitiviruses, by in silico analysis of virus sequences obtained via NGS methods unveiles the identification of previously unknown beny-like viruses within Armillaria spp. from the Czech Republic. To confirm the presence of beny-like viruses in vitro, reverse transcription with PCR was used. Total RNA was isolated using a commercial kit and its presence in the samples was confirmed electrophoretically. The sequence obtained using the Illumina system's NovaSeq 6000 sequencing platform was used to design the triple pairs of primers Beny F1 and Beny R1, Beny F2 and Beny R2, and BV1 F and BV1 R, in which potential ORFs were designed. The primers were designed to target the longest ORF encoding RdRp, the most conserved gene in RNA viruses. The DNA fragment obtained by reverse transcription was amplified by PCR followed by reamplification. Finally the samples were evaluated electrophoretically for the presence of viral single-stranded RNA.
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Analysis of NGS data for study of transposon activity in cancer cells
Hrazdilová, Ivana ; Čegan,, Radim (referee) ; Eduard, Kejnovský (advisor)
Theoretical part of this diploma thesis gives a brief characteristic of human mobile elements (transposons), which represents nearly 50% of human genome. It provides basic transposon clasification and describes types of transposons present in hunam genome, as well as mobilization, activation and regulation mechanisms. The work also deals with the domestication of transposons, describes the ways in which TE contribute to DNA damage and summarizes the diseases caused by mutagenic activity of transposons in the human genome. Conclusion of theoretical part describes next-generation sequencing technologies (NGS). As practical part, data from RNA-seq experimet were analyzed in order to compare differen transposon activity in normal and cancer cells from prostate and colorectal tissues. As like as publicly available sophisticated tools (TopHat), new scripts were created to analyze these data. The results show that cancer cells exhibit overexpression of transposons. This corresponds with the published results and suggests a connection of transposon activation with cancer development.
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Mapping techniques for reference-based genome assembly
Petrovský, Jan ; Sedlář, Karel (referee) ; Bartoň, Vojtěch (advisor)
The bachelor thesis deals with the tools used to map the genome to the reference sequence. The theoretical part describes the currently used sequencing technologies, whether the first generation or the latest technologies of the third generation, an overview of the tools used for de novo genome construction, approaches and tools used for mapping to the reference, the ART program and related data formats. The practical part consists of creating a test dataset of NGS data and creating appropriate metrics to evaluate the quality of the mapping and their subsequent usage on the chosen mapping tools.
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Reconstruction of DNA Sequences Containing Repetitive Patterns
Dvořáček, Vojtěch ; Vogel, Ivan (referee) ; Martínek, Tomáš (advisor)
This bachelor thesis focuses on reconstruction of repetitive patterns in DNA sequences. Although it is motivated by need to create automated tool for telomere characterization in plant genomes, its goal is creating general solution of given problem. Suggested solution is based on k-mer analysis, which is alignment free method. Its greatest advantage lies in possibility to reconstruct repetitive patterns from NGS sequencing data. Within the scope of this work was designed and iplemented solution, which is able to automatically estiamte suitable parametres for k-mer analysis from input data and optimize them by local state space search method.
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Eucaryotic Genomes Comparison
Puterová, Janka ; Vogel, Ivan (referee) ; Martínek, Tomáš (advisor)
Main motive of this master thesis was the need of good bioinformatics tools for genome comparison and improvement of one of the existing tools - RepeatExplorer. This work offers an overview of transposable elements in DNA, existing tools for identification and analysis of repetitions in sequenced genomes, summary of currently used genome sequencing methods. This work describes shortcomings of RepeatExplorer tool with focus on comparative analysis of genomes. Two solutions to remove these problems were designed and implemented. The first solution is designed for comparing pairs of genomes. The principle of this solution is based on comparison of similarity of distribution of contigs coverages using Kolmogorov-Smirnov test, thanks to which we are able to determine different parts in the genomes.The second solution, which is used to compare multiple genomes, is based on the method of mapping reads from compared genomes to the reference genome contigs and provides contigs coverage graphs, by which we are able to determine the variability of the repeats.Their functionality was verified on real NGS data of organism Silene latifolia.
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Phylogenomics, genome size evolution and repeat dynamics in the genus Amomum Roxb. (Zingiberaceae)
Hlavatá, Kristýna ; Fér, Tomáš (advisor) ; Haevermans, Thomas (referee) ; Zedek, František (referee)
Amomum Roxb. s.l. (black cardamom) is a complicated genus in the ginger family Zingiberaceae (subfamily Alpinioideae) that according to some definitions includes groups of species recognized as separate genera, e.g. genus Elettariopsis Baker. Phylogenetic studies have so far failed to elucidate both the position of Amomum within Alpinioideae and the relationship between Amomum and other genera like Elettariopsis. In this work, Amomum was subjected to a detailed morphological analysis of the largest possible sampling, together with a phylogenetic analysis. Amomum was recircumscribed, Amomum s.s. and three new genera were established, three genera were resurrected, and genus Elettariopsis was merged into Amomum. Meanwhile, new species were described and others epitypified. A well-resolved nuclear-gene phylogeny of Amomum s.s. was obtained using next-generation sequencing (Hyb-Seq) and showed the existence of four clades (A, B, C, D) within the genus. Clade D, the former genus Elettariopsis, was further divided into three subclades (D1-D3). A chloroplast DNA-based phylogeny supported this structure, but additional phylogenies based on ribosomal DNA were incongruent with it, indicating that the frequently used ITS marker and other rDNA markers may not be suitable for reconstructing deeper phylogenetic...
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The role of somatic mutations in the pathogenesis of myelodysplastic syndrome
Minařík, Ľubomír ; Stopka, Tomáš (advisor) ; Žák, Pavel (referee) ; Beličková, Monika (referee)
9 Abstract: Myelodysplastic syndromes (MDS) are a set of severe hematological diseases characterized by ineffective clonal hematopoiesis in the bone marrow, leading to cytopenia in the peripheral blood, the development of transfusion dependence and a high risk of progression to acute myeloid leukemia (AML). The disease is caused by genetic and epigenetic changes leading to the development of pathological stem cells that are unable to mature sufficiently in the bone marrow into blood elements. These changes vary widely between patients, which is reflected in different clinical manifestations, response to treatment, overall survival and, last but not least, this heterogeneity represents a challenge for the study of this disease. The present dissertation is aimed at studying the pathophysiological manifestations and consequences of selected genetic alterations, especially somatic mutations of key genes and other functional units of the genome, in relation to the clinical course of MDS and transformation to AML. Therapy of high-risk MDS is currently based on hypomethylating drugs including 5- azacytidine (AZA). Treatment leads to prolongation of disease progression to AML, but this fate is irreversible for the vast majority of patients whose prognosis becomes hopeless at this point. Results of genetic analysis...
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