Scalable Feature Selection Applications for Genome-Wide Association Studies of Complex Diseases

Personalized medicine will revolutionize our capabilities to combat disease. Working toward this goal, a fundamental task is the deciphering of geneticvariants that are predictive of complex diseases. Modern studies, in the formof genome-wide association studies (GWAS) have afforded researchers with the opportunity to reveal new genotype-phenotype relationships through the extensive scanning of genetic variants. These studies typically contain over half a million genetic features for thousands of individuals. Examining this with methods other than univariate statistics is a challenging task requiring advanced algorithms that are scalable to the genome-wide level. In the future, next-generation sequencing studies (NGS) will contain an even larger number of common and rare variants. Machine learning-based feature selection algorithms have been shown to have the ability to effectively create predictive models for various genotype-phenotype relationships. This work explores the problem of selecting genetic variant subsets that are the most predictive of complex disease phenotypes through various feature selection methodologies, including filter, wrapper and embedded algorithms. The examined machine learning algorithms were demonstrated to not only be effective at predicting the disease phenotypes, but also doing so efficiently through the use of computational shortcuts. While much of the work was able to be run on high-end desktops, some work was further extended so that it could be implemented on parallel computers helping to assure that they will also scale to the NGS data sets. Further, these studies analyzed the relationships between various feature selection methods and demonstrated the need for careful testing when selecting an algorithm. It was shown that there is no universally optimal algorithm for variant selection in GWAS, but rather methodologies need to be selected based on the desired outcome, such as the number of features to be included in the prediction model. It was also demonstrated that without proper model validation, for example using nested cross-validation, the models can result in overly-optimistic prediction accuracies and decreased generalization ability. It is through the implementation and application of machine learning methods that one can extract predictive genotype–phenotype relationships and biological insights from genetic data sets.

Characterisation of Cladonia rangiferina transcriptome and genome, and the effects of dehydration and rehydration on its gene expression

Lichens are symbiotic organisms, which consist of the fungal partner and the photosynthetic partner, which can be either an alga or a cyanobacterium. In some lichen species the symbiosis is tripartite, where the relationship includes both an alga and a cyanobacterium alongside the primary symbiont, fungus. The lichen symbiosis is an evolutionarily old adaptation to life on land and many extant fungal species have evolved from lichenised ancestors. Lichens inhabit a wide range of habitats and are capable of living in harsh environments and on nutrient poor substrates, such as bare rocks, often enduring frequent cycles of drying and wetting. Most lichen species are desiccation tolerant, and they can survive long periods of dehydration, but can rapidly resume photosynthesis upon rehydration. The molecular mechanisms behind lichen desiccation tolerance are still largely uncharacterised and little information is available for any lichen species at the genomic or transcriptomic level. The emergence of the high-throughput next generation sequencing (NGS) technologies and the subsequent decrease in the cost of sequencing new genomes and transcriptomes has enabled non-model organism research on the whole genome level. In this doctoral work the transcriptome and genome of the grey reindeer lichen, Cladonia rangiferina, were sequenced, de novo assembled and characterised using NGS and traditional expressed sequence tag (EST) technologies. RNA extraction methods were optimised to improve the yield and quality of RNA extracted from lichen tissue. The effects of rehydration and desiccation on C. rangiferina gene expression on whole transcriptome level were studied and the most differentially expressed genes were identified. The secondary metabolites present in C. rangiferina decreased the quality – integrity, optical characteristics and utility for sensitive molecular biological applications – of the extracted RNA requiring an optimised RNA extraction method for isolating sufficient quantities of high-quality RNA from lichen tissue in a time- and cost-efficient manner. The de novo assembly of the transcriptome of C. rangiferina was used to produce a set of contiguous unigene sequences that were used to investigate the biological functions and pathways active in a hydrated lichen thallus. The de novo assembly of the genome yielded an assembly containing mostly genes derived from the fungal partner. The assembly was of sufficient quality, in size similar to other lichen-forming fungal genomes and included most of the core eukaryotic genes. Differences in gene expression were detected in all studied stages of desiccation and rehydration, but the largest changes occurred during the early stages of rehydration. The most differentially expressed genes did not have any annotations, making them potentially lichen-specific genes, but several genes known to participate in environmental stress tolerance in other organisms were also identified as differentially expressed.

Development of packaging cell lines for rescuing BAV2 viral vectors

The construction of adenovirus vectors for cloning and foreign gene expression requires packaging cell lines that can complement missing viral functions caused by sequence deletions and/or replacement with foreign DNA sequences. In this study, packaging cell lines were designed to provide in trans the missing bovine adenovirus functions, so that recombinant viruses could be generated. Fetal bovine kidney and lUng cells, acquired at the trimester term from a pregnant cow, were tranfected with both digested wild type BAV2 genomic DNA and pCMV-EI. The plasmid pCMV-EI was specifically constructed to express El of BAV2 under the control of the cytomegalovirus enhancer/promoter (CMV). Selection for "true" transformants by continuous passaging showed no success in isolating immortalised cells, since the cells underwent crisis resulting in complete cell death. Moreover, selection for G418 resistance, using the same cells, also did not result in the isolation of an immortalised cell line and the same culture-collapse event was observed. The lack of success in establishing an immortalised cell line from fetal tissue prompted us to transfect a pre-established cell line. We began by transfecting MDBK (Mardin-Dardy bovine kidney) cells with pCMV-El-neo, which contain the bacterial selectable marker neo gene. A series of MDBK-derived cell lines, that constitutively express bovine adenoviral (BAV) early region 1 (El), were then isolated. Cells selected for resistance to the drug G418 were isolated collectively for full characterisation to assess their suitability as packaging cell lines. Individual colonies were isolated by limiting dilution and further tested for El expression and efficiency of DNA uptake. Two cell lines, L-23 and L-24, out of 48 generated foci tested positive for £1 expression using Northern Blot analysis. DNA uptake studies, using both lipofectamine and calcium phosphate methods, were performed to compare these cells, their parental MDBK cells, 8 and the unrelated human 293 cells as a benchmark. The results revealed that the new MDBKderived clones were no more efficient than MDBK cells in the transient expression of transfected DNA and that they were inferior to 293 cells, when using lacZ as the reporter gene. In view of the inherently poor transfection efficiency of MDBK cells and their derivatives, a number of other bovine cells were investigated for their potential as packaging cells. The cell line CCL40 was chosen for its high efficiency in DNA uptake and subsequently transfected with the plasmid vector pCMV El-neo. By selection with the drug G418, two cell lines were isolated, ProCell 1 and ProCell 2. These cell lines were tested for El expression, permissivity to BAV2 and DNA uptake efficiency, revealing a DNA uptake efficiency of 37 % , comparable to that of CCL40. Attempts to rescue BAV2 mutants carrying the lacZ gene in place of £1 or £3 were carried out by co-transfecting wild type viral DNA with either the plasmid pdlElE-Z (which contains BAV2 sequences from 0% to 40.4% with the lacZ gene in place of the £1 region from 1.1% to 8.25%) or with the plasmid pdlE3-5-Z (which contains BAV2 sequences from 64.8% to 100% with the lacZ gene in place of the E3 region from 75.8% to 81.4%). These cotransfections did not result in the generation of a viral mutant. The lack of mutant generation was thought to be caused by the relative inefficiency ofDNA uptake. Consequently, cosBAV2, a cosmid vector carrying the BAV2 genome, was modified to carry the neo reporter gene in place of the £3 region from 75.8% to 81.4%. The use of a single cosmid vector earring the whole genome would eliminate the need for homologous recombination in order to generate a viral vector. Unfortunately, the transfection of cosBAV2- neo also did not result in the generation of a viral mutant. This may have been caused by the size of the £3 deletion, where excess sequences that are essential to the virus' survival might have been deleted. As an extension to this study, the spontaneous E3 deletion, accidently discovered in our viral stock, could be used as site of foreign gene insertion.

Molecular cloning restriction enzyme analysis, bovine adenovirus type 2 genome

Adenoviruses are non-enveloped icosahedral-shaped particles which possess a double-stranded DNA genome. Currently, nearly 100 serotypes of adenoviruses have been identified, 48 of which are of human origin. Bovine adenoviruses (BAVs), causing both mild respiratory and/or enteral diseases in cattle, have been reported in many countries all over the world. Currently, nine serotypes of SAVs have been isolated which have been placed into two subgroups based on a number of characteristics which include complement fixation tests as well as the ability to replicate in various cell lines. Bovine adenovirus type 2 (BAV2), belonging to subgroup I, is able to cause pneumonia as well as pneumonic-like symptoms in calves. In this study, the genome of BAV2 (strain No. 19) was subcloned into the plasmid vector pUC19. In total, 16 plasmids were constructed; three carry internal San fragments (spanning 3.1 to 65.2% ), and 10 carry internal Pstl fragments (spanning 4.9 to 97.4%), of the viral genome. Each of these plasmids was analyzed using twelve restriction endonucleases; BamHI, CiaI, EcoRl, HiOOlll, Kpnl, Noll, NS(N, Ps~, Pvul, Saj, Xbal, and Xhol. Terminal end fragments were also cloned and analyzed, sUbsequent to the removal of the 5' terminal protein, in the form of 2 BamHI B fragments, cloned in opposite orientations (spanning 0 to 18.1°k), and one Pstll fragment (spanning 97.4 to 1000/0). These cloned fragments, along with two other plasmids previously constructed carrying internal EcoRI fragments (spanning 20.6 to 90.5%), were then used to construct a detailed physical restriction map using the twelve restriction endonucleases, as well as to estimate the size of the genome for BAV2(32.5 Kbp). The DNA sequences of the early region 1 (E1) and hexon-associated gene (protein IX) have also been determined. The amino acid sequences of four open reading frames (ORFs) have been compared to those of the E1 proteins and protein IX from other Ads.

The Falls of Niagara : being a complete guide to all the points of interest around and in the immediate neighbourhood of the great cataract with views taken from sketches by Washington Friend, and from photographs. --

Includes index.

The complete illustrated guide to Niagara Falls and vicinity. -

Attributed to Peter Augustus Porter--National Union Catalog, pre-1956 imprints.

A New guide to Niagara Falls and vicinity : giving a full and complete description of Niagara Falls, suspension bridge, Buffalo, Rochester, Ontario beach, Toronto, Lockport, Tonawanda, Lewiston, Niagara-on-the-Lake, Chautauqua, and other places of interest. --

Cover title: Illustrated guide to Niagara Falls and vicinity.

Transposable Elements Are a Significant Contributor to Tandem Repeats in the Human Genome

Sequence repeats are an important phenomenon in the human genome, playing important roles in genomic alteration often with phenotypic consequences. The two major types of repeat elements in the human genome are tandem repeats (TRs) including microsatellites, minisatellites, and satellites and transposable elements (TEs). So far, very little has been known about the relationship between these two types of repeats. In this study, we identified TRs that are derived from TEs either based on sequence similarity or overlapping genomic positions. We then analyzed the distribution of these TRs among TE families/subfamilies. Our study shows that at least 7,276 TRs or 23% of all minisatellites/satellites is derived from TEs, contributing ∼0.32% of the human genome. TRs seem to be generated more likely from younger/more active TEs, and once initiated they are expanded with time via local duplication of the repeat units. The currently postulated mechanisms for origin of TRs can explain only 6% of all TE-derived TRs, indicating the presence of one or more yet to be identified mechanisms for the initiation of such repeats. Our result suggests that TEs are contributing to genome expansion and alteration not only by transposition but also by generating tandem repeats.

HUMAN GENOME VARIATIONS AND EVOLUTION WITH A FOCUS ON THE ANALYSIS OF TRANSPOSABLE ELEMENTS

Genome sequence varies in numerous ways among individuals although the gross architecture is fixed for all humans. Retrotransposons create one of the most abundant structural variants in the human genome and are divided in many families, with certain members in some families, e.g., L1, Alu, SVA, and HERV-K, remaining active for transposition. Along with other types of genomic variants, retrotransponson-derived variants contribute to the whole spectrum of genome variants in humans. With the advancement of sequencing techniques, many human genomes are being sequenced at the individual level, fueling the comparative research on these variants among individuals. In this thesis, the evolution and functional impact of structural variations is examined primarily focusing on retrotransposons in the context of human evolution. The thesis comprises of three different studies on the topics that are presented in three data chapters. First, the recent evolution of all human specific AluYb members, representing the second most active subfamily of Alus, was tracked to identify their source/master copy using a novel approach. All human-specific AluYb elements from the reference genome were extracted, aligned with one another to construct clusters of similar copies and each cluster was analyzed to generate the evolutionary relationship between the members of the cluster. The approach resulted in identification of one major driver copy of all human specific Yb8 and the source copy of the Yb9 lineage. Three new subfamilies within the AluYb family – Yb8a1, Yb10 and Yb11 were also identified, with Yb11 being the youngest and most polymorphic. Second, an attempt to construct a relation between transposable elements (TEs) and tandem repeats (TRs) was made at a genome-wide scale for the first time. Upon sequence comparison, positional cross-checking and other relevant analyses, it was observed that over 20% of all TRs are derived from TEs. This result established the first connection between these two types of repetitive elements, and extends our appreciation for the impact of TEs on genomes. Furthermore, only 6% of these TE-derived TRs follow the already postulated initiation and expansion mechanisms, suggesting that the others are likely to follow a yet-unidentified mechanism. Third, by taking a combination of multiple computational approaches involving all types of genetic variations published so far including transposable elements, the first whole genome sequence of the most recent common ancestor of all modern human populations that diverged into different populations around 125,000-100,000 years ago was constructed. The study shows that the current reference genome sequence is 8.89 million base pairs larger than our common ancestor’s genome, contributed by a whole spectrum of genetic mechanisms. The use of this ancestral reference genome to facilitate the analysis of personal genomes was demonstrated using an example genome and more insightful recent evolutionary analyses involving the Neanderthal genome. The three data chapters presented in this thesis conclude that the tandem repeats and transposable elements are not two entirely distinctly isolated elements as over 20% TRs are actually derived from TEs. Certain subfamilies of TEs themselves are still evolving with the generation of newer subfamilies. The evolutionary analyses of all TEs along with other genomic variants helped to construct the genome sequence of the most recent common ancestor to all modern human populations which provides a better alternative to human reference genome and can be a useful resource for the study of personal genomics, population genetics, human and primate evolution.

Agreement to undertake construction and complete the road in accordance with the engineer or the Port Robinson and Thorold macadamized road

Agreement to undertake construction and complete the road in accordance with the engineer or the Port Robinson and Thorold macadamized road. This is signed by John Williams, Aug. 11, 1855.