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Comparison of ITS nrDNA and alternative markers for fungal metabarcoding in environmental samples
Zelenka, Tomáš ; Kolařík, Miroslav (advisor) ; Mašek, Tomáš (referee)
The study of fungal diversity may lead to many fundamental discoveries and conclusions. Molecular genetics, and particularly high throughput sequencing methods using short DNA fragments as barcodes, has recently experienced a boom. The most frequently used marker for fungal research is the partial region of nuclear ribosomal DNA called ITS (Internal Transcribed Spacer). It occurs in the form of tandem repetitions of up to 200 copies. This fact greatly simplifies its amplification from the environment but also introduces some negatives. One of them can be an existence of intragenomic and intraspecific variability which confounds diversity estimates by exaggerating the real number of species. Using alternative low-copy markers can easily prevent these problems. In this study EF-1α and RPB2 protein- coding genes were compared with traditionally used ITS1 and ITS2 markers. An artificial mock community was created by blending genomic DNA of different fungal lineages. The community was sequenced for all markers and the data were processed according to guidelines commonly used in environmental studies. The results show that ITS2 is unequivocally a more suitable marker for environmental studies than other compared markers. The average coefficient of overestimation was deemed to be approximately two for ITS1, ITS2,...
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Construction and quality assessment of the genome assemblies
Korená, Lucie ; Leontovyč, Roman (advisor) ; Vorel, Jiří (referee)
Detailed information of the genome of the studied organism is crucial for many fields of modern research. Actual sequencing technologies are not able to read the whole DNA molecule at once therefore only fragments of the genetic information are obtained, which are not sufficiently informative on their own. The goal of the genomic-bioinformatic approach is to assemble these fragments into complete original information - genome assembly. The process of the genome assembly is demanding in terms of computational power, software equipment and expert staff. Many assemblers - programs for genome assembly are available differing in performance, size of the analyzed genome or target organism. The quality of final assembly is fully dependent on assembler and setting of inner parameters. In practice, multiple assemblies are constructed and their quality evaluated according to the technical and biological parameters. The presented thesis describes current high throughput sequencing technologies, different approaches and algorithms for genome assembly and methodology for their quality assessment. The practical part is focused on assembly and its quality assessment using Illumina data of the bird fluke Trichobilharzia szidati.
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Comparison of ITS nrDNA and alternative markers for fungal metabarcoding in environmental samples
Zelenka, Tomáš ; Kolařík, Miroslav (advisor) ; Mašek, Tomáš (referee)
The study of fungal diversity may lead to many fundamental discoveries and conclusions. Molecular genetics, and particularly high throughput sequencing methods using short DNA fragments as barcodes, has recently experienced a boom. The most frequently used marker for fungal research is the partial region of nuclear ribosomal DNA called ITS (Internal Transcribed Spacer). It occurs in the form of tandem repetitions of up to 200 copies. This fact greatly simplifies its amplification from the environment but also introduces some negatives. One of them can be an existence of intragenomic and intraspecific variability which confounds diversity estimates by exaggerating the real number of species. Using alternative low-copy markers can easily prevent these problems. In this study EF-1α and RPB2 protein- coding genes were compared with traditionally used ITS1 and ITS2 markers. An artificial mock community was created by blending genomic DNA of different fungal lineages. The community was sequenced for all markers and the data were processed according to guidelines commonly used in environmental studies. The results show that ITS2 is unequivocally a more suitable marker for environmental studies than other compared markers. The average coefficient of overestimation was deemed to be approximately two for ITS1, ITS2,...
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