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Comparative and phylogenetic analysis of viruses in the University Hospital Brno
Švestková, Tereza ; Sedlář, Karel (referee) ; Nykrýnová, Markéta (advisor)
This thesis is focused on the SARs-CoV-2 coronavirus associated with severe acute respiratory syndrome, which was first identified in 2019. This coronavirus caused a pandemic that affected almost the entire world. Knowledge of the genetic information is needed for vaccine development, to determine infectivity and to predict the evolution of SARs-CoV-2 variants. To obtain genetic information, RNA must be sequenced and these genomic sequences must be assembled. By comparing the assembled genomes, it is possible to find out which part of the organism has mutated. Phylogenetic analysis is performed on the basis of the concordance or divergence in the assembled genomes, which indicates the evolution of the organism and shows the evolutionary relationship with other organisms. The practical part is focused on the assembly of genomes from samples from patients in the University Hospital Brno and evaluation of the quality of the assembly. After the genomes are assembled, the next goal is to evaluate the variability and subsequent phylogenetic analysis.
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Identification of non-coding RNAs of Clostridium beijerinckii NRRL B-598 using RNA-Seq data
Pomykalová, Barbora ; Sedlář, Karel (referee) ; Jurečková, Kateřina (advisor)
This bachelor thesis contains short introduction into bacterial small non-coding RNA problematic. It is oriented on their features and functions in organisms, especially in bacteria Clostridium beijerinckii NRRL B-598. Bachelor thesis also contains description of various laboratory methods for gene expression determination and suggests a detection method for small non-coding RNA in bacteria Clostridium beijerinckii NRRL B-598. Suggested method works with data, which were obtained by RNA-Seq technology. Within the framework of the bachelor thesis was suggested method implemented in programming and numeric computing platform MATLAB and its results were discussed.
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Properties of current signals in nanopore sequencing
Plocková, Veronika ; Nykrýnová, Markéta (referee) ; Sedlář, Karel (advisor)
Oxford Nanopore technologies brought new and revolutionary technology in the field of DNA sequencing. Their sequencing device measures changes in the electric current flowing through pores together with DNA. This work aims to describe differences between raw signals produced by various sequencing kits and sequencing flowcells while sequencing several different bacteria. Two datasets combining five different organisms, two sequencing kits, and two types of flowcells were used to analyze various statistical parameters that would be suitable for the description of current signals gathered from nanopores. Finally, the samples were classified using the k-means algorithm and the results were discussed.
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CNV detection in bacterial genomes
Lacinová, Michaela ; Sedlář, Karel (referee) ; Škutková, Helena (advisor)
This master thesis deals with analysis of structural variation of genome and with methods of its sequencing across all generations. Subsequently it contains a description of copy number variation and methods of its detection. The experimental part focuses on algorithm proposal for CNV detection according analysis and testing of uneven coverage in genome, variable representation of GC content and distance of sequence reads. Finally, the algorithm for detecting copy number variation is tested on genomic data of bacteria Klebsiella pneumoniae.
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Effect of TNF-α inhibitors on microbiota composition and immune response in patients with inflammatory bowel disease
Mihula, Martin ; Jirásková Zákostelská, Zuzana (advisor) ; Grobárová, Valéria (referee)
One of the most common used therapies in inflammatory bowel diseases (IBD) treatment are inhibitors of a cytokine TNF-α. Nevertheless, up to one third of IBD patients stop respond to this therapy for unknown reason. In these days, there are not any ideal biomarkers which could predict patient's long- term response to anti-TNF-α therapy. Because the gut microbiota composition changes are tightly related to the pathogenesis of IBD, my aim in this thesis was to find out if these changes in composition are happening also due the therapy by inhibitors of TNF-α as well. Moreover, I tried to find out if there are changes in production of serum biomarkers related to the gut barrier damage and to the immune response associated with microbial translocation. Also, I focused on the immune response of IBD patients against common gut commensal bacterial antigens during the anti-TNF-α therapy. In our study, we collected for these purposes stool or blood samples from 46 IBD patients before the therapy and at 38th week from the start of the therapy and 39 healthy controls. I found that IBD patients had higher bacterial diversity (α-diversity) as well as different bacterial composition across observed groups (β-diversity) at 38th week of the anti-TNF-α therapy than before the therapy. When I divided IBD patients...
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Gene expression analysis on a subgene level
Kloda, František ; Fišer, Karel (advisor) ; Novotný, Marian (referee)
RNA sequencing allows investigation of expression of singular genes in cells. It is possible to interpret the arisen data on multiple levels, each level providing a different type of information. Apart from measuring expression of whole genes, it is possible to quantify expression of singular exons, or transcripts (gene isoforms), which allows more detailed study of regulatory mechanisms. The main difference between approaches is in determining the origin of short reads. This step is significantly more complex in analysis of expression of transcripts, as transcripts derived from the same gene have typically larger rate of sequential similarity. In this thesis, we describe eleven tools for subgene level expression analysis a as comparison we have tested three of these tools on real patient data. The results provided by all three tools proved to be very similar with the greatest difference being the time needed for the analysis.
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Metagenomic analysis of child gut microbioma
Zourková, Tereza ; Škutková, Helena (referee) ; Bartoň, Vojtěch (advisor)
The thesis is focused on the influence of the metagenome on child growth and the reasons for the usefulness of investigating the consequences of changes in the human intestinal microbiome. It also describes the general problematics of the microbiome and the history of its studies. The composition of the human intestinal microbiome and the connection with the immune system or, for example, hormone production, are also described here. The thesis also contains information on some methods of metagenome analysis, namely microscopic methods using previous cultivation, mass spectrometry and sequencing methods. The data analysis procedure using various bioinformatics software, with which the data were appropriately pre-processed, is also described here. Filtered data in this way are ready for the following metagenome profiling, which is also described here. At the end of the thesis, the profiling results are evaluated, based on which the course of changes in the microbiome during the aging of the child are estimated, which is the main goal of this bachelor's thesis.
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Optimization of DNA amplification isolated from buccal swabs for sequencing purposes
ROŠTÍKOVÁ, Kristýna
The topic of my bachelor thesis was the issue of taking primary DNA samples from the buccal mucosa, determination of the ideal length and optimal conditions and subsequent analysis of these samples. In the theoretical part, I focused on the preanalytical phase of the laboratory examination. It was about, the methods of sampling and the type of material that is most often used used for DNA isolation in the molecular biological laboratory. Subsequently, I focused on the methods themselves, which are used for DNA analysis. These included DNA isolation, DNA amplification methods, nucleic acid electrophoresis and DNA sequencing. In the practical part, I processed 42 samples. First, I isolated DNA from the samples and measured its concentration and purity. Then I performed electrophoresis in these samples and based on its results I decided which samples I would process further by PCR. Subsequently, I determined by electrophoresis whether DNA was amplified in the samples. Finally, I selected samples that I sent for sequencing. The aim of my bachelor thesis was to determine the optimal length and storage conditions of samples from the buccal mucosa for further processing. Then I evaluated how these parameters affect the quality, purity and quantity of isolated DNA as well as the course of the polymerase chain reaction. According to the obtained results, the best option is to store the samples in a refrigerator temperature and perform their analysis in the shortest possible time, or to store samples in a refrigerator temperature and process them within weeks to three months.
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Analysis of the G2964A polymorphism of the STAT6 gene by sequencing
KOTOVA, Valeriia
This bachelor's thesis deals with the polymorphism of the STAT6 gene, which was investigated by the sequencing method and the impact of this polymorphism on the occurrence of atopic diseases, mainly nut allergies. The STAT6 gene is located on chromosome 12 in humans at locus 12q13.3 and contains 23 exons. STAT6 was previously associated with total serum IgE levels and atopy in various populations. One of the most common polymorphisms in that gene is the SNP polymorphism G2964A, which is in the 3-untranslated region (rs324015). The theoretical part contains basic information about DNA sequencing, terms from allergology (including food allergies) and information about the investigated STAT6 gene. The practical part is focused on the detection of the rs324015 polymorphism in the examined group from 20 people of the Caucasian population.
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