Cancer the second most common causes of death in the Czech Republic. Carriers of mutations in genes predisposing to hereditary cancers represent a small but clinically significant group of high risk individuals. Today, dozens of predisposing genes for hereditary tumor syndromes are known and targeted next generation sequencing (NGS) has become a standard approach for their analysis. NGS allows rapid acceleration diagnostics of causal mutation in high-risk individuals. To identify mutations in genes predisposing to hereditary cancers, we designed a panel NGS analysis including subsequent bioinformatics analysis allowing a reliable identification of single nucleotide variants, insertions/deletions, and large intragenic rearrangements. The bioinformatics procedures described in this thesis were used for panel NGS validation, but also for identification of alterations associating with so far undescribed hereditary tumor types. Bioinformatics analyzes have become the basis for the unified processing of large datasets from the CZECANCA consortium and enable the construction of a population-specific database of genotypes that serve to improve clinical diagnostics of cancer predisposition in Czech patients. The versatility of NGS also allows its use for RNA (cDNA-based) analyzes of splicing variants in the...

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>Magister Scientiae - MSc / Rooibos (Aspalathus linearis) is endemic to the Cederberg region of South Africa, and one of the few indigenous medicinal plants commercially cultivated in the country. International interest in rooibos is growing, and currently most of the rooibos harvest is exported overseas to more than 30 countries. Various problems hamper the growth of the rooibos industry, including insect pests, diseases, drought and a decreasing lifespan of the plants. The availability of whole-genome data for rooibos can contribute to the selection of genetically superior plants, facilitating not only the identification of important genes and metabolic pathways in rooibos, but also the establishment of breeding programs.

The application of Y-chromosome analysis is expanding in fields such as forensic science and genealogy. By researching the potential polymorphisms this chromosome can present, we can further our ability to assess DNA profiles for these disciplines to avoid erroneous exclusions of paternal linkage, wrongful convictions based on forensic evidence, and other misinformed genetic conclusions. The conservation of Y-haplotypes during transmission occurs due to a relative lack of genetic recombination events in the inheritance of the Y-chromosome [1]. However, random mutation events can occur in a paternal line resulting in haplotype changes. These changes can include allele duplications and deletions that occur at the STR and SNP loci used in forensic DNA analysis. This can become important in cases of sexual assault where male-female mixture samples have low amounts of male DNA such that the male signal is not amplified in currently used STR multiplexes [7]. In this study, we analyzed a father and his eleven sons using two different methodologies for genetic analysis; next generation sequencing and capillary electrophoresis. The samples were obtained from the Coriell Institute for Medical Research located in Hamden, NJ, in the form of frozen DNA extracts isolated from a blood-sourced lymphocyte cell culture [22]. DNA from these samples was tested with the ForenSeqTM DNA Signature Prep Kit [14] (Verogen, San Diego, CA) primer set A and the YFilerTM Plus PCR Amplification Kit [24] (Thermo Fisher Scientific, Waltham, MA). Using these two platforms, three Y-STR loci were identified as discordant between the father and all of his eleven sons. In all three instances, the father possessed the same allele as the sons as well as one additional allele. At two of these loci (DYS449 and DYS635), the additional allele was one repeat (4bp) longer than that of the shared allele. At the other locus (DYS458), the additional allele was three repeats (12bp) longer than that of the shared allele. Following read count and peak height analysis, it was concluded that these double allele loci are not the product of stutter and are potentially the product of a non-inheritable mutation. With the knowledge that the DNA was extracted from a blood lymphocyte cell culture, it is believed that a somatic mutation may be present in the cell line. We are not able to determine whether the mutations exist in the blood of the father (true somatic mutations) or occurred as a result of the cell culture process. Throughout the study, details concerning the position of these loci on the Y-chromosome, the repeat motifs of the alleles, and the potential for duplication and/or stutter as the originating event are discussed in an effort to further understand this phenomenon. Potential locus duplications were compared to those reported on the National Institute of Standards and Technology STRBase [21] list of allele variations and also to information found in literature. The observed DYS635 locus had an allele designation of 21,22 which is reported on STRBase. The DYS449 and DYS458 loci showed potential allele-specific locus duplications that were not found on STRBase. The implications of potentially undocumented non-inheritable allele patterns in the Y-chromosome, such as this, are significant when considering comparisons between DNA obtained from germline cells (sperm) versus a known casework sample which is usually obtained from blood or saliva [7].

This work summarizes the results of a research inquiring into relatively rare neuroendocrine tumors - pheochromocytomas and paragangliomas (PHEO/PGL) These tumors may arise on a hereditary genetic predisposition basis. On that account we primarily focused on a genetic examination of patients with PHEO/PGL. Methods for diagnostics of changes in SDHD, SDHB and RET genes were implemented. The number of examined genes has been (and is still being) extended. Currently we are investigating these genes: ATRX, BRAF, CDH1, CDKN2A, CDKN2B, FGFR1, FH, FHIT, GNAS, HIF2A (EPAS1), H-RAS, IDH1, IDH2, KIF1Bß, KMT2D, K-RAS, MAML3, MAX, MDH2, MET, NF1, NGFR, N-RAS, PHD2/EGLN1, RET, SDHA, SDHAF2, SDHB, SDHC, SDHD, TERT, TMEM 127, TP53 and VHL, using next generation sequencing. The number of variations of the above mentioned genes is different (23%) in Czech patients with PHEO/PGL in comparison with some foreign studies (27%, 40%). This may be caused by geographical influences or selection of patients. PHEO/PGL occur mainly (75%) in a benign form. A malignant form may be indicated by the presence of chromaffin tissue in locations where these tumors do not usually occur - liver, lungs, bones. In our study we focused on characteristics indicating the malignancy, for example, the lower age of patients with the first manifestation...

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

During the last decade, the use of Next-Generation Sequencing(NGS) technologies has sky-rocketed. The vast amount of data produced by these platforms require processing and analysis. This is usually performed at locations remote from the sequencing facilities thereby introducing the need for data-transportation to the place of analysis. The use of internet transfer would greatly facilitate the process, however since NGS data is considered to be personal sensitive information the handling of the data is highly regulated by the General Data Protection Regulation(GDPR). During this project, a web-based application was developed for the privacy-protecting transfer of personal sensitive data, implementing an in-motion encryption scheme which ensures data integrity and authenticity. The application consists of three scripts: the HTML web page with JavaScript functionality, a PHP script responsible for connection establishment and integrity verification, and a Python script executing the majority of the server-side operations. The resulting application uses the symmetric encryption algorithm AES in GCM mode, using a key size of 128 bits and transfers 60 Kibibytes of the file at a time. The key is established by using the asymmetric RSA encryption scheme with a 4096 bit key pair. SHA-256 is used for verifying the integrity of the transferred files. The JavaScript encryption speed is 584 MB/s and the Python decryption speed 251 MB/s. While the focus of the project was to optimize the application for NGS data, it is not limited to this type of file and can transfer different formats, enabling the use in multiple different fields.

Hepatitis B Virus (HBV) is a DNA virus and belongs to the genus Orthohepadnavirus of the Hepadnaviridae family which represents one of two animal viruses with a DNA genome which replicates by reverse transcription of a viral RNA intermediate. Nucleotide variation led to further sub-classification into 8 genotypes (A to H). The reverse transcription step within its life cycle is prone to the introduction of errors and recombination when dually infected. This leads to a viral quasispecies which forms during the course of infection with many minor population variants; such variants can however only be detected by means of ultra-deep sequencing. A recent study in the Department of Medical Virology (UP) by Mayaphi et al. identified a number of the specimens that partitioned away from the typical subgenotype A1 clades with high bootstrap values and longer branch lengths. Thus, the main objective of the current study was to characterize the full genome of all variants for the outliers observed in the aforementioned study, inclusive of potential recombination, dual infection and minor populations. Twenty samples were selected from a previous cohort for purposes of the present study. The viral DNA was extracted and amplified by PCR according to the methods described by Günther et al. with modified primer sets. Nineteen of the samples were successfully amplified and 15 of these were sequenced. Specimens were sequenced by NGS on the Illumina MiSeq™ sequencer and sequence data used to reconstruct the viral quasispecies of each specimen. Further analyses of the reconstructed variants included molecular characterization as well as phylogenetic analysis and screening for recombination and drug resistance mutations. Full genome coverage was obtained for twelve of the fifteen samples and full genome variants reconstructed, generating nearly 40 full genomes. Phylogenetic analysis showed that the majority of the samples are of genotype A, more specifically of subgenotype A1, differing by less than 4% from known sequences. The phylogenetic analysis revealed a similar clade of outliers, where four samples clustered together with significant bootstrap support (75%) and a fifth sample partitioned separate from, yet close to, this clade, away from the typical African A1 clade. This clade was assigned to genogroup III. Three samples were of the Asian A1 clade (genogroup I) with remaining specimens grouping within genotype D and E. The variants showed low diversity within each specimen with some differing at but a few positions across the genome while even the most diverse quasispecies differed by less than a percentage (32 positions). Several unique and atypical positional variations were observed amongst study samples of which some were present in but one of the variants for that sample. Twenty-six lead to shared amino acid changes. Some observed changes, such as A1762T/G1764A and G1896A, could explain the serological patterns such as HBeAg negativity while others, such as C2002T, were previously implicated in disease progression and severity. Sample N199 presented a longer branch length and revealed short regions within the genome that display evidence of recombination between HBV/A1 and HBV/A2. The results illustrate the utility of NGS technology in characterizing viral variants. / Dissertation (MSc)--University of Pretoria, 2014. / lk2014 / Medical Virology / MSc / Unrestricted

High-throughput next-generation sequencing techniques have hugely decreased the cost and increased the speed of sequencing, resulting in an explosion of sequencing data. This motivates the development of high-efficiency sequence alignment algorithms. In this thesis, I present multiple bit-parallel and Single Instruction Multiple Data (SIMD) algorithms that greatly accelerate the processing of biological sequences. The first chapter describes the BitPAl bit-parallel algorithms for global alignment with general integer scoring, which assigns integer weights for match, mismatch, and insertion/deletion. The bit-parallel approach represents individual cells in an alignment scoring matrix as bits in computer words and emulates the calculation of scores by a series of logic operations. Bit-parallelism has previously been applied to other pattern matching problems, producing fast algorithms. In timed tests, we show that BitPAl runs 7 - 25 times faster than a standard iterative algorithm. The second part involves two approaches to alignment with substitution scoring, which assigns a potentially different substitution weight to every pair of alphabet characters, better representing the relative rates of different mutations. The first approach extends the existing BitPAl method. The second approach is a new SIMD algorithm that uses partial sums of adjacent score differences. I present a simple partial sum method as well as one that uses parallel scan for additional acceleration. Results demonstrate that these algorithms are significantly faster than existing SIMD dynamic programming algorithms. Finally, I describe two extensions to the partial sums algorithm. The first adds support for affine gap penalty scoring. Affine gap scoring represents the biological likelihood that it is more likely for gaps to be continuous than to be distributed throughout a region by introducing a gap opening penalty and a gap extension penalty. The second extension is an algorithm that uses the partial sums method to calculate the tandem alignment of a pattern against a text sequence using a single pattern copy. Next generation sequencing data provides a wealth of information to researchers. Extracting that information in a timely manner increases the utility and practicality of sequence analysis algorithms. This thesis presents a family of algorithms which provide alignment scores in less time than previous algorithms.

The North American genus Speyeria is an especially challenging radiation of butterflies due to ongoing hybridization, incomplete lineage sorting, and similar morphological characters among species. Adaptive radiations often require considerable evidence in order to resolve the evolutionary relationships of closely related individuals. Previous studies of this genus have found paraphyly among species and have been unable to disentangle these taxa due to a lack of data and/or incomplete sampling of the genus. As a result, the interspecific relationships among Speyeria remain unresolved. In an attempt to achieve phylogenetic resolution of the genus, we conducted population genomic and phylogenomic analyses of all North American Speyeria species, as well as several subspecies, based on genome wide markers using the SbfI restriction enzyme and restriction site associated DNA sequencing (RADseq). Together, our analyses recovered 16 species within Speyeria, validating previous taxonomic work. However, consistent with recent molecular analyses, internal relationships have poor support. This lack of resolution indicates Speyeria represent an ongoing adaptive radiation, with incomplete lineage sorting, hybridization, and lack of postzygotic reproductive barriers, supporting this hypothesis.