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Single-cell RNA sequencing in leukemia
Brodská, Johana ; Froňková, Eva (advisor) ; Obr, Adam (referee)
Leukemia is a cancer of hematopoietic cells affecting the whole organism. Currently, there are many treatment options for all disease types, but it is still not always possible to fully cure the patient. The single-cell RNA sequencing method offers a new insight into the heterogeneity of both cancerous and non-cancerous cells in the leukemic environment. This thesis aims to briefly present the method and its history and to highlight current findings about leukemia obtained with the help of it. Keywords leukemia (AML, CML, ALL, CLL), sequencing, scRNA-seq, cells, transcriptome, treatment Okomentoval(a): [o1]: To the reader se v odborné literatuře moc nepoužívá
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Changes in biological monitoring of running waters using benthos with respect to diatoms
Fiedler, Jan ; Kulichová, Jana (advisor) ; Tichá, Anna (referee)
This work focuses on the development of a methodological approach to biomonitoring of running waters using benthic organisms. It discusses the development from methods focused on determining organisms by their external appearance to modern biomonitoring based on the determination of organisms by eDNA metabarcoding. Modern metagenomic methods (genomics, transcriptomics, metabolomics and proteomics) are also mentioned. This thesis compares the development of ecological status assessment of freshwater ecosystems using diatoms and invertebrates. It also describes the common points of their methodological development. Another outcome of the comparison is information on which hydrochemical and geochemical environmental factors can be easily monitored by diatoms and which by macroscopic invertebrates. Furthermore, a prognosis for the further development of biomonitoring is mentioned in the conclusion. It is formulated as a synthesis of traditional methods based on morphological species determination, biomonitoring 2.0 (using eDNA metabarcoding) and metagenomic methods. Key words: autecology, morphospecies, sequencing, biotic index, pollution
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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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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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Contribution of Next Generation Sequencing for Laboratory Diagnostics
Votýpka, Pavel ; Beránek, Martin (advisor) ; Macháček, Miloslav (referee)
5 ABSTRACT Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Pavel Votýpka Supervisor: Doc. PharmDr. Martin Beránek, Ph.D. Consultant: Mgr. Nikola Ptáková Title of diploma thesis: Contribution of Next Generation Sequencing for Laboratory Diagnostics The endeavor to sequence the whole human genome lead not only to the knowledge acquisition regarding the human genetic information but as well to the development of new sequencing methods and technologies. In order to keep up with progress in genetic field in many clinical and research laboratories the new massive parallel sequencing equipment is being utilized. On the market are currently established four leading platforms - Illumina, Solid, Ion Torrent and 454 Life Technologies. The process of sequencing analysis can be summarized into three main steps - the sequencing library preparation, sequencing itself, variant calling and data analysis. Each part of the sequencing analysis exhibits certain specifics, we need to count with and as well its pitfalls, we need to avoid or to minimalize their impact on the analysis final result. Recently new methods termed sequencing of the 3rd generation are being developed, enabling sequence of a single DNA molecule to be determined without previous...
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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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Influence of second and third generation sequencers on cgMLST analysis of closely-related bacterial strains.
Slavíček, David ; Sedlář, Karel (referee) ; Nykrýnová, Markéta (advisor)
This bachelor thesis is about influence of sequncers of second and third generationon cgMLST analysis of bacterial genome. In the theoretical section were described selected sequencers of second and third generation and genome assembly principles. In the practical part were assembled six genomes of Klebsiella pneumoniae bacteria from University Hospital Brno. The genomes were sequenced each on two different sequencers, Illumina Miseq and Oxford Nanopore Technologies Minion. The genomes were assembled. Suitable genes were selected and insufficient quality genomes removed for the cgMLST analysis. The cgMLST analysis was performed and the results are shownin graphs.
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Pregnancy proteins - molecular biological and biochemical analysis
Muravská, Alexandra ; Kalousová, Marta (advisor) ; Černá, Marie (referee) ; Průša, Richard (referee)
The aim of this thesis was to establish methods for selected PAPP-A (Pregnancy- Associated Plasma Protein A) gene polymorphisms analysis and to study genetic background of PAPP-A and biochemical background of PAPP-A and PlGF (Placental Growth Factor) in relation to risk pregnancy. Secondly, the aim was to establish method for two-dimensional (2D) electrophoresis of amniotic fluid. Methods for analysis of ten PAPP-A gene polymorphisms were established. These polymorphisms, PAPP-A and PlGF levels were studied in together 165 women in third trimester pregnancies complicated with threatening preterm labor (n=98), preeclampsia (n=35), IUGR (Intrauterine Growth Restriction) (n=34) and ICP (Intrahepatic Cholestasis of Pregnancy) (n=15). 114 healthy pregnant women served as controls. The method for 2D electrophoresis of amniotic fluid was established. Preeclamptic patients had significantly higher frequency of TT genotype of Cys327Cys (C/T) PAPP-A gene polymorphism compared to controls. Patients with ICP had increased serum levels of PAPP-A compared to controls, in patients with threatening preterm labor PAPP-A levels were rather decreased. PlGF levels did not differ from control group in patients with ICP and threatening preterm labor. Positive correlation was found between PAPP-A and PlGF in group of...
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The Role of Microbiota in the Pathogenesis of Psoriasis
Stehlíková, Zuzana ; Jirásková Zákostelská, Zuzana (advisor) ; Demnerová, Kateřina (referee) ; Hrdý, Jiří (referee)
Psoriasis is a chronic, immune-mediated inflammatory skin disease. Its pathogenesis is associated with dysregulated cooperation among keratinocytes, innate and adaptive immune cells, coupled with environmental triggers, including microbiota. The aim of our study was to describe the microbiota composition in psoriasis and explore the role of bacteria and fungi in the pathogenesis of this disease. We used a mouse model of psoriasis induced by topical application of imiquimod (IISI) in both germ-free (GF) mice and conventional (CV) mice with microbiota manipulated by administration of a mixture of broad-spectrum antibiotics (ATB). ATB treatment markedly changed the intestinal but not the skin bacterial diversity and led to higher resistance to IISI in CV mice. Metronidazole was the most effective antibiotic, alleviating IISI symptoms in CV, but not in GF mice. This confirms that the effect of metronidazole on IISI was microbiota- dependent. Additionally, we characterized the microbiota composition of psoriatic lesions and unaffected skin in psoriatic patients compared to healthy controls, as well as the impact of different sampling approaches on uncovering cutaneous microbiota composition. We observed significant differences in α- and β-diversities when comparing identical samples sequenced on V1V2...
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Identification Of Genetic Heterogeneity In Sequencing Data
Bartoň, Vojtěch
This paper describes the preparation of data obtained from Illumina sequencing platform to analyze low frequency genetic changes within population of Treponema strains, i.e. DNA heterogeneity. A method of characterization of raw sequencing data is proposed. The method includes data filtering to achieve better quality of the collected data and calculation of parameters determining which ones of the DNA changes should be considered as heterogeneity.
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