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Metagenomic analysis of animal gut microbioma based on diet
Spanakis, Martin ; Čejková, Darina (referee) ; Bartoň, Vojtěch (advisor)
This bachelor’s thesis deals with the sequencing of the microbiome and its composi- tion. It focuses on the gut microbiome of animals, which differs among animals with different diets. The work describes theoretical knowledge about metagenomic analysis of the microbiome, such as sampling procedures, various sequencing methods and data processing. The practical part of the work includes the preparation of the dataset, which includes the collection of data and their preparation for the following metagenomic anal- ysis. The result of the work is the taxonomic classification of bacterial species in the samples and the analysis of their diversity according to the type of diet of individual animals.
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Analysis of horizontal transfer of genetic components using static network analysis
Labanava, Anastasiya ; Jurečková, Kateřina (referee) ; Schwarzerová, Jana (advisor)
The bachelor's thesis focuses on the issue of horizontal genetic elements transfer between bacteria of different strains and the software analysis implementation that enables horizontally transferred genes identification. The packages and tools used were tested on a dataset of bacterial genomes from several strains. The thesis’ theoretical part provides a detailed description of the genetic components transfer between bacteria and describes modern laboratory techniques that enable genome sequencing in various ways. In the practical part, the thesis deals with the preprocessing of genomic files to obtain suitable data for annotation. To detect the horizontal transfer of genetic elements between bacteria, a script is introduced, which organizes annotated bacteria to tables and searches for the same genes in their genomes that, under theoretical assumptions, were horizontally transferred. Furthermore, the gene transfer is visualized using tools that graphically represent phylogenetic relations between bacteria. In the final step, bacterial genomes are connected into networks, and based on their static analysis, a discussion is conducted on the results accuracy and the success of the proposed 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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Properties of Current Signals in Nanopore Sequencing
Plocková, V. ; Sedlář, K.
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.
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Examination of polymorphisms in the IFITM3 gene using sequences and their clinical significance for the course of viral diseases.
TROJÁKOVÁ, Simona
Interferon Induced Transmembrane Protein 3 is an anti-inflammatory cytokine that belongs to the group of interferon-stimulated genes. The topology of the IFITM3 gene was clarified by the analysis of electron paramagnetic and nuclear magnetic resonance. The protein encoded by this gene induces immunity against influenza A virus and other viral diseases. It also disturbs the homeostasis of intracelular cholesterol, inhibits the entry of viruses into the cytoplasm of the host cells and inactivates new enveloped viruses originating from an infected cell. IFITM proteins reduce virus replication by regulating the expression of viral protein and by reducing the infectivity of developing viruses. The analysis of the single-nucleotid polymorphisms in the IFITM3 gene is very significant to clarify the mechanism of the effect of the IFITM3 protein and its influence on the severity of the course of the viral diseases. The theoretical part deals with the description of Interferon Induced Transmembrane Proteins, particularly the description and function of the IFITM3 gene, including its polymorphisms. This gene is located on chromosome 11 and its size is approximately 18 Kb. The gene variability of the IFITM3 can fundamentally affect the course of influenza and other viral diseases. The practical part of the thesis focuses on the detection of polymorphisms rs12252, rs34481144 and rs1136853 in the IFITM3 gene using the PCR method and Sanger sequencing method. It was necessary to master some basic laboratory methods such as DNA isolation from primary samples, PCR method, preparation of the PCR product for sequencing, sequencing data analysis, processing of results and determination of specific genotypes in tested individuals.
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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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Detection of human adenoviruses from the respiratory samples from the patients of Motol University Hospital
NOVÁKOVÁ, Veronika
Human adenoviruses are world-wide pathogens causing endemic and epidemic outbre-aks of disease. Adenoviral infections are related with high morbidity and mortality especially among the paediatric allogeneic haematopoietic stem cell recipients. Presented work is comparing the commercial and in-house assay for detection of the adenoviruses to improve the diagnostics of these viruses in Motol University Hospital, especially among the immunocompromissed patients, in which is the rapid and precise detection highly important. Theoretical part of work presents the characteristics of adenoviruses, clinical symptoms and diseases of the adenoviral infection, their use in the experimental therapy. Practical part is aiming the screening of the respiratory tract samples with quantitative in-house test and comparing of the results with previously performed detection by commercial Anyplex TM II RV16 test. Detection of adenoviruses was performed in 1,881 samples from 1,420 patients with symptoms of respiratory tract infection by real-time PCR. In total, adenovirus was detected in 187 samples (9.9%) from 169 patients (11.9%). One hundred samples (53.5%) was positive by both methods, 26 (13.9%) with commercial detection only and 61 (32.6%) was positive only with in-house assay. Most frequently, adenoviruses were detected among the patients from Dept. of Paediatrics, Dept. of Paediatric Haematology and Oncology and from adult patients from 3rd Dept. of Surgery and Dept. of Anaestesiology, resuscitation and intensive medicine. In all positive samples, subsequent sequence analysis of hypervariable part of 1-6 hexon gene was performed and representative sequence for identification was obtained in 70 samples. Most frequently detected adenovirus genotypes were C2 (25.7 %), A31 (24.3 %), B3 (15.7 %) a C1 (11.4 %). From 26 samples positive by Anyplex TM II RV16, only 3 samples were identified (all as genotype B3). Contrary, among 61 positive samples detected only with in-house assay, 15 samples were identified as genotype A31 and 3 as AdV from group C. Our results shows the lower sensitivity of the commercial test in detection of adenoviru-ses.
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Molecular diagnostics of hepatitis C in a selected group of patients
KALINAYOVÁ, Daniela
This bachelor thesis deals with the occurrence of hepatitis C and its detection focused on direct evidence of the virus. The theoretical part describes and divides hepatitis C into acute and chronic, focuses on its genetic variability and explains how this virus behaves, is transmitted and mutated. Mutation creates other variants of it, which must be distinguished from each other if we want to set up an effective treatment. The practical part gives us information about the incidence of HCV among men and women, which are clearly divided in tables according to the year of birth, positivity and negativity. It also deals with the determination of a given genotype / subtype.
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Monitoring of DWV, BQCV and CBPV viruses in colonies in the Czech Republic
ZAHRADNÍK, Václav
This doplima thesis deals with the determination of the degree of infection by selec-ted diseases among hives in Czech republic. The literature search mentions viral diseases, problems and methods of isolating total RNA from a sample of bees. The methodology is further focused on the isolation of total RNA, amplification of viral RNA in the sample by RT-PCR with specific primers. Evaluation of the results is performed by electrophoresis and subsequent sequencing of the positive sample and sequence comparison in the NCBI database.
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