Molecular-epidemiological analysis of Mycobacterium tuberculosis strains isolated in the West-Bohemian Region of the Czech Republic including detailed characterisation of anti-tuberculosis drugs - resistant strains MUDr. Jana Amlerová Abstract Tuberculosis is contagious infectious disease that embodies significant epidemiological and clinical problem worldwide. Tuberculosis incidence differs considerably in various regions of the world but even the countries with low incidence engage strongly in epidemiology of tuberculosis. Tuberculosis belongs to one of the priorities of WHO, cooperation in this matter takes place on a global scale. Tuberculosis is a social illness; accordingly, the countries with high occurrence of tuberculosis are classified as developing countries. Mainly in Africa, there is the situation being complicated by coexistence of HIV. Generally, Europe represents a region with low incidence of tuberculosis. The Czech Republic is a country with the lowest incidence in the world with less than five new cases per 100 000 inhabitants every year. This situation is among others result of high-level tuberculosis surveillance and effective application of epidemiological arrangements based in legislation. This dissertation thesis examines several fields of tuberculosis, mainly focused on modern...

Advances in sequencing technologies constantly improves the throughput andaccuracy of sequencing instruments. Together with this development comes newdemands and opportunities to fully take advantage of the massive amounts of dataproduced within a sequence run. One way of doing this is by analyzing a large set ofsamples in parallel by pooling them together prior to sequencing and associating thereads to the corresponding samples using DNA sequence tags. Amplicon sequencingis a common application for this technique, enabling ultra deep sequencing andidentification of rare allelic variants. However, a common problem for ampliconsequencing projects is formation of unspecific PCR products and primer dimersoccupying large portions of the data sets. This thesis is based on two papers exploring these new kinds of possibilities andissues. In the first paper, a method for including thousands of samples in the samesequencing run without dramatically increasing the cost or sample handlingcomplexity is presented. The second paper presents how the amount of high qualitydata from an amplicon sequencing run can be maximized. The findings from the first paper shows that a two-tagging system, where the first tagis introduced by PCR and the second tag is introduced by ligation, can be used foreffectively sequence a cohort of 3500 samples using the 454 GS FLX Titaniumchemistry. The tagging procedure allows for simple and easy scalable samplehandling during sequence library preparation. The first PCR introduced tags, that arepresent in both ends of the fragments, enables detection of chimeric formation andhence, avoiding false typing in the data set. In the second paper, a FACS-machine is used to sort and enrich target DNA covered emPCR beads. This is facilitated by tagging quality beads using hybridization of afluorescently labeled target specific DNA probe prior to sorting. The system wasevaluated by sequencing two amplicon libraries, one FACS sorted and one standardenriched, on the 454 showing a three-fold increase of quality data obtained. / QC20100907

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Using computer systems for probabilistic genotyping on DNA evidence in forensic casework is beneficial as it allows a complete analysis of the data available for a wide range of profiles, a range that is limited when analyzed manually. One such software, Bulletproof, uses the exact method as the statistical foundation of its web-based interface to estimate the likelihood ratio of two hypotheses that explain the given evidence. In this investigation, the capability of Bulletproof was examined by analyzing the effects of evidence and reference sample template amount, injection time, and stutter filter utilization on likelihood ratio. In terms of likelihood ratio, deconvolution by the software is more efficient in cases in which evidence samples of high contrast ratios (such as 1:9 vs. 1:1) and low contributor count have high template, and when sample injection times are low. Reference sample template amount and injection time are less impactful than that of evidentiary samples. As with unknown samples, reference samples should be analyzed beforehand and artifacts removed for better deconvolution.

Giardia intestinalis is a single-celled intestinal parasite infecting humans and animals. The species is divided into eight genetic groups, assemblages, with different host specificity. Stool samples from 99 dogs, 61 cats and 22 chinchillas were examined for the presence of Giardia using microscopy and PCR diagnostics. The found populations were assigned to assemblages using a multi-locus genotyping scheme, with the goal of mapping the occurrence of zoonotic assemblages A and B and evaluating the risk of transmission of Giardia from pets to humans. The Giardia prevalence in examined dogs was 36,4%. The majority of dog infections was caused by dog-specific assemblages D and C. Individual cases of infection with assemblage F, or a mix of assemblages A+D, A+F, B+D, C+D and D+F were also detected. The prevalence in cats was 14.8%, and the dog assemblages C and D prevailed as well. In individual cases, cats were infected with assemblages A or F, which is specific for cats. The highest prevalence, 85.7%, was detected in chinchillas. The majority of chinchilla infections was caused by the zoonotic assemblage B (88.9%). The found sequences were compared to those obtained from animals with clinical giardiasis, but no identical matches were found between these two pools. The nature of mixed infections was studied by...

Introduction: The development of molecular genetic methods has in many fields necessitated their inclusion in routine clinical practice, including ophthalmology. The main aim of this thesis was detailed clinical characterization of Czech patients with suspected inherited corneal disorders, followed by genetic testing to determine or specify their clinical diagnosis and subsequently to use the knowledge gained in clinical and genetic counselling and to apply preventive measures in order to avoid loss of vision. Material and Methods: Individuals included in this research were either followed up or newly referred to the Cornea clinic of the Department of Ophthalmology, First Faculty of Medicine, Charles University and General University Hospital in Prague. Detailed clinical examination included corneal tomography, specular microscopy, spectral domain optical coherence tomography, biometry and genealogical analysis. DNA was extracted from peripheral blood leucocytes or buccal cells. Disease-causing variants were searched for using Sanger or massively parallel sequencing, variant pathogenicity was assessed in silico using various algorithms and by segregation analyses within the families. In some cases assessment of the functional impact on the pre-mRNA splicing process was performed. In patients with...

Tuberculosis (TB) is an airborne disease caused by Mycobacterium tuberculosis, with an incidence in a quarter of the world population. Despite the scientific and technological advances, an effective diagnostic method has not yet been found that allows an early diagnosis and, also, to detect the strain present in the patient. The combination of nanotechnology with molecular diagnostics has shown promising advances offering new possibilities, such as the development of nanoflares.  Nanoflares represent a new class of molecular probes, composed of gold nanoparticles functionalized with a recognition sequence that can be amplified by rolling circle amplification (RCA) technique, producing a fluorescence signal.  This thesis focuses in the synthesis of gold nanoparticles, with different coatings and sizes, as well as their subsequent application in the preparation and optimization of nanoflares for the genotyping of synthetic M. tuberculosis targets using RCA technique. The different preparations of nanoflares have an impact in the assay sensitivity, showing two times increase in sensitivity for citrate-coated nanoparticles with respect to those coated with PEG. Furthermore, it was observed that the sensitivity is directly related to the synthesized particle size.  Sensitivity is also affected by the application of a purification post-treatment of the synthesis product. This post-treatment reduces the sensitivity of nanoflares by up to 37% but, by contrast, extends its useful life.  The results obtained are shown as a proof of concept for a future cost-effective, rapid and robust in situ diagnostic method that identifies the strain of tuberculosis present in the patient.

The grain amaranths (Amaranthus sp.) and quinoa (Chenopodium quinoa Willd.) are important seed crops in South America. These crops have gained international attention in recent years for their nutritional quality and tolerance to abiotic stress. We report the identification and development of functional single nucleotide polymorphism (SNP) assays for both amaranth and quinoa. SNPs were identified using a genome reduction protocol and next generation sequencing. SNP assays are based on KASPar genotyping chemistry and were detected using the Fluidigm dynamic array platform. A diversity screen consisting of 41 amaranth accessions showed that the minor allele frequency (MAF) of the amaranth markers ranged from 0.05 to 0.5 with an average MAF of 0.27. A diversity screen of 113 quinoa accessions showed that the MAF of the quinoa markers ranged from 0.02 to 0.5 with an average MAF of 0.28. Linkage mapping in amaranth produced a linkage map consisting of 16 linkage groups, presumably corresponding to each of the 16 amaranth haploid chromosomes. This map spans 1288 cM with an average marker density of 3.1 cM per marker. Linkage mapping in quinoa resulted in a linkage map consisting of 29 linkage groups with 20 large linkage groups, spanning 1,404 cM with a marker density of 3.1 cM per SNP marker. The SNPs identified here represent important genomic tools needed for genetic dissection of agronomically important characteristics and advanced genetic analysis of agronomic traits in amaranth and quinoa. We also describe in detail the scalable and cost effective SNP genotyping method used in this research. This method is based on KBioscience's competitive allele specific PCR amplification of target sequences and endpoint fluorescence genotyping (KASPar) using a FRET capable plate reader or Fluidigm's dynamic array high throughput platform.

Next-generation sequencing (NGS) has revolutionized the field of genetics by providing a means for fast and relatively affordable sequencing. With the advancement of NGS, whole- genome sequencing (WGS) has become more commonplace. However, sequencing an entire genome is still not cost effective or even beneficial in all cases. In studies that do not require a whole-genome survey, WGS yields lower sequencing depth and sequencing of uninformative loci. Targeted sequencing utilizes the speed and low cost of NGS while providing deeper coverage for desired loci. This thesis applies targeted sequencing to the genomes of two different, non-model plants, Artemisia tridentate (sagebrush) and Lupinus luteus (yellow lupine). We first targeted the transcriptomes of three species of sagebrush (Artemisia) using RNA-seq. By targeting the transcriptome of sagebrush we have built a resource of transcripts previously unmatched in sagebrush and identify transcripts related to terpenes. Terpenes are of growing interest in sagebrush because of their ability to identify certain species of sagebrush and because they play a role in the feeding habits of the threatened sage-grouse. Lastly, using paralogs with synonymous mutations we reconstructed an evolutionary time line of ancient genome duplications. Second, we targeted the flanking loci of recognition sites of two endorestriction enzymes in genome of L. luteus genome through genotyping-by-sequencing (GBS). GBS of yellow lupine provided enough single-nucleotide polymorphic loci for the construction of a genetic map of yellow lupine. Additionally we compare GBS strategies for plant species without a reference genome sequence.

Ruminants are considered the commonest animal reservoir for human infection of Coxiella burnetii, the Q fever causative agent. Considering the recently described importance of human Q fever in Greece, we aimed at providing the first comprehensive direct evidence of C. burnetii in dairy cows in Greece, including the genetic characterization of strains. The 462 examined dairy farms represented all geographical areas of Greece. One bulk tank milk sample was collected from every farm and tested for the presence of C. burnetii. Molecular genotyping of strains, performed directly on samples, revealed the existence of two separate clades characterized by single nucleotide polymorphism (SNP) genotypes of type 1 and type 2. The two clades were clearly distinguished in multiple locus variable-number tandem repeat analysis (MLVA) by two discriminative loci: MS30 and MS28. Whereas MLVA profiles of SNP-type 2 clade were closely related to strains described in other European cattle populations, the MLVA profile observed within the SNP type 1 clade highlighted a peculiar genetic signature for Greece, related to genotypes found in sheep and goats in Europe. The shedding of C. burnetii bearing this genotype might have yet undefined human epidemiological consequences. Surveillance of the genetic distribution of C. burnetii from different sources is needed to fully understand the epidemiology of Q fever in Greece.