A current trend in biological science is the increased use of computational tools for both the production and analysis of experimental data. This is especially true in the field of genomics, where advancements in DNA sequencing technology have dramatically decreased the time and cost associated with DNA sequencing resulting in increased pressure on the time required to prepare and analyze data generated during these experiments. As a result, the role of computational science in such biological research is increasing. This thesis seeks to address several major questions with respect to the development and application of single nucleotide polymorphism (SNP) resources in non-model organisms. Traditional SNP discovery using polymerase chain reaction (PCR) amplification and low-throughput DNA sequencing is a time consuming and laborious process, which is often limited by the time required to design intron-spanning PCR primers. While next-generation DNA sequencing (NGS) has largely supplanted low-throughput sequencing for SNP discovery applications, the PCR based SNP discovery method remains in use for cost effective, targeted SNP discovery. This thesis seeks to develop an automated method for intron-spanning PCR design which would remove a significant bottleneck in this process. This work develops algorithms for combining SNP data from multiple individuals, independent of the DNA sequencing platforms, for the purpose of developing SNP genotyping arrays. Additionally, tools for the filtering and selection of SNPs will be developed, providing start to finish support for the development of SNP genotyping arrays in complex polyploids using NGS. The result of this work includes two automated pipelines for the design of intron-spanning PCR primers, one which designs a single primer pair per target and another that designs multiple primer pairs per target. These automated pipelines are shown to reduce the time required to design primers from one hour per primer pair using the semi-automated method to 10 minutes per 100 primer pairs while maintaining a very high efficacy. Efficacy is tested by comparing the number of successful PCR amplifications of the semi- automated method with that of the automated pipelines. Using the Chi-squared test, the semi-automated and automated approaches are determined not to differ in efficacy. Three algorithms for combining SNP output from NGS data from multiple individuals are developed and evaluated for their time and space complexities. These algorithms were found to be computationally efficient, requiring time and space linear to the size of the input. These algorithms are then implemented in the Perl language and their time and memory performance profiled using experimental data. Profiling results are evaluated by applying linear models, which allow for predictions of resource requirements for various input sizes. Additional tools for the filtering of SNPs and selection of SNPs for a SNP array are developed and applied to the creation of two SNP arrays in the polyploid crop Brassica napus. These arrays, when compared to arrays in similar species, show higher numbers of polymorphic markers and better 3-cluster genotype separation, a viable method for determining the efficacy of design in complex genomes.

abstract: Although the Caribbean has been continuously inhabited for the last 7,000 years, European contact in the last 500 years dramatically reshaped the cultural and genetic makeup of island populations. Several recent studies have explored the genetic diversity of Caribbean Latinos and have characterized Native American variation present within their genomes. However, the difficulty of obtaining ancient DNA from pre-contact populations and the underrepresentation of non-Latino Caribbean islanders in current research have prevented a complete understanding of genetic variation over time and space in the Caribbean basin. This dissertation uses two approaches to characterize the role of migration and admixture in the demographic history of Caribbean islanders. First, autosomal variants were genotyped in a sample of 55 Afro-Caribbeans from five islands in the Lesser Antilles: Grenada, St. Kitts, St. Lucia, Trinidad, and St. Vincent. These data were used to characterize genetic structure, ancestry and signatures of selection in these populations. The results demonstrate a complex pattern of admixture since European contact, including a strong signature of sex-biased mating and inputs from at least five continental populations to the autosomal ancestry of Afro-Caribbean peoples. Second, ancient mitochondrial and nuclear DNA were obtained from 60 skeletal remains, dated between A.D. 500–1300, from three archaeological sites in Puerto Rico: Paso del Indio, Punta Candelero and Tibes. The ancient data were used to reassesses existing models for the peopling of Puerto Rico and the Caribbean and to examine the extent of genetic continuity between ancient and modern populations. Project findings support a largely South American origin for Ceramic Age Caribbean populations and identify some genetic continuity between pre and post contact islanders. The above study was aided by development and testing of extraction methods optimized for recovery of ancient DNA from tropical contexts. Overall, project findings characterize how ancient indigenous groups, European colonial regimes, the African Slave Trade and modern labor movements have shaped the genomic diversity of Caribbean islanders. In addition to its anthropological and historical importance, such knowledge is also essential for informing the identification of medically relevant genetic variation in these populations. / Dissertation/Thesis / Zipped file contains Appendices A-K. Supplemental tables, figures, protocols and spreadsheets associated with dissertation. / Doctoral Dissertation Anthropology 2017

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.

Research Doctorate - Doctor of Philosophy (PhD) / Endometrial cancer is one of the most common female cancers in industrialized countries. Traditional risk factors associated with endometrial cancer are well understood and include excessive exposure to estrogen or estrogen unopposed by progesterone. However, variations in the genes that influence these hormones and their association with endometrial cancer have not been well investigated. By studying genetic variation in endometrial cancer, novel markers of risk may be discovered that can be used to identify women at high risk and for the implementation of specialised treatments. Polymorphisms in the genes involved in the following pathways; hormone biosynthesis, hormone receptors, estrogen metabolism, DNA repair and cell cycle control, have been suggested to be involved in the initiation and development of endometrial cancer. The focus of this study was to examine genetic variants in these pathways to assess the existence of an association with the risk of endometrial cancer. In the first part of this study, the COMT V158M polymorphism was examined in a hereditary non-polyposis colorectal cancer (HNPCC) cohort to determine its association with disease expression. The heterozygous genotype was over-represented in women with endometrial/ovarian cancer that did not harbour mismatch repair (MMR) gene mutations. This result suggested that the COMT V158M polymorphism may alter the risk of developing HNPCC related endometrial/ovarian cancer in MMR mutation negative women. Since COMT is involved in the metabolism of estrogen and that estrogen is the main risk factor for endometrial cancer development, closer examination was warranted to determine the association of genetic variation involved in hormone-related pathways and endometrial cancer risk, outside of the context of an inherited predisposition to disease. In the second part of this study, a cohort of 191 women with endometrial cancer and 291 healthy control women were genotyped for polymorphisms in genes involved in hormone biosynthesis, hormone receptors, estrogen metabolism, DNA repair and cell cycle control. The results revealed that variations in estrogen receptor alpha (ESR1) and beta (ESR2), and the androgen receptor (AR), were associated with an increase and decrease in endometrial cancer risk, respectively. Additionally, polymorphisms in CYP1A1, CYP1B1, GSTM1 and GSTP1 were related to a decrease in endometrial cancer risk. A trend was observed for the cyclin D1 870 G>A polymorphism and an increase in endometrial cancer risk, however, this result did not reach significance. Taken together, these results revealed that perturbations in the hormone receptors and estrogen metabolism genes, may aid in the identification of women at high risk of developing endometrial cancer. Interestingly, stratification of the women with endometrial cancer revealed that combinations of polymorphisms in TP53 and MDM2 were associated with higher grades of cancer. This finding may possibly have significant implications as women with reduced apoptotic ability, due to combinations of polymorphisms in these genes, have an increased risk of presenting with higher grades of endometrial cancer, that are associated with lower survival rates. In summary, the results of this thesis showed that variation in the estrogen and androgen receptors, and estrogen metabolism genes, may alter the risk of developing endometrial cancer. Moreover, polymorphisms in the cell cycle control genes, TP53 and MDM2, appear to be associated with higher grades of endometrial cancer. This study of polymorphisms may help explain genetic differences in individual susceptibility to endometrial cancer and are markers of risk that aid in the development of effective and personalised strategies to prevent disease development. This study has improved the understanding of genetic variation associated with endometrial cancer risk. It has the potential to enhance our ability to treat women with endometrial cancer through improved identification and treatment strategies, by virtue of the genetic variation identified, that appears to predispose to disease.

Following its introduction to North America in the late nineteenth century, Bromus tectorum L., an inbreeding invasive winter annual grass, has become dominant on millions of hectares of sagebrush steppe habitat throughout Intermountain Western North America. It appears that within the last 30-40 years, B. tectorum has expanded its range southward into the Mojave Desert and also into more climatically extreme salt desert environments. Previous research using microsatellite markers and experimental studies has suggested that lineages found in desert habitats are genetically distinct from those found in the sagebrush-steppe habitat and possess suites of traits that pre-adapt them to these environments. To provide additional support for our hypothesis that desert habitat-specific haplotypes dominate and are widely distributed across warm and salt desert habitats, we genotyped approximately 20 individuals from each of 39 B. tectorum populations from these habitats and adjacent sagebrush steppe habitats using 71 single nucleotide polymorphic (SNP) markers. Our data clearly demonstrate that populations throughout the Mojave Desert region, as well as in salt desert habitats further north, are dominated by a small number of closely related SNP haplotypes that belong to the desert clade. In contrast, populations from adjacent environments are largely dominated by haplotypes of the common clade, which is widely distributed throughout the North American sagebrush steppe. Populations across all habitats were usually dominated by 1-2 SNP haplotypes. This suggests that inbreeding B. tectorum lineages can often maintain their genetic integrity. It also explains the strong association between marker fingerprints and suites of adaptive traits in this species.

Many countries in the global south - which harbour the majority of the world’s biodiversity - face serious resource limitations and a lack of access to affordable sequencing services. Furthermore, biodiversity research and monitoring of non-model, threatened and/or cryptic species often relies on low-quality non-invasive genetic samples. In situ conservation genomics approaches optimised for field conditions and low-quality DNA can help empower local researchers and meet their needs. To do so, however, accessible and reproducible sequencing and genotyping alternatives are needed. I designed a SNP panel as a field-friendly genotyping approach for two species of Amazonian primates using both high- and low-quality DNA samples, and two different sequencing platforms, Illumina and Nanopore. I used 14 high-quality genomes to identify a set of 210 SNPs that allow for identification of species (twelve SNPs), sex (twelve SNPs) and individual identity (186 SNPs) in two species of tamarins, Leontocebus weddelli and Saguinus imperator. Primers, adapters and indexes were designed in a Genotyping-in-Thousands by sequencing approach that is compatible with both sequencing platforms. This approach is based on sequencing multiplexed PCR products of a few hundred target SNPs to genotype thousands of individuals in a single sequencing run. In an effort to make conservation genomics more accessible, the reproducible pipeline to obtain the informative SNPs is being modulated with Snakemake, a workflow management system. / Muchos países en el sur global - los cuales poseen la mayoría de la biodiversidad mundial - enfrentan serias limitaciones de recursos y una falta de acceso a servicios económicos de secuenciación. Con frecuencia, la investigación y el monitoreo de biodiversidad y especies no-modelo, amenazadas y/o crípticas, dependen de muestras genéticas no-invasivas de baja calidad. La genómica de la conservación in situ optimizada para condiciones de campo y ADN de baja calidad puede empoderar a investigadorxs locales y ayudarles a responder a sus necesidades. Para ello, sin embargo, se requieren alternativas accesibles y reproducibles de secuenciación y genotipado. Diseñé un panel de SNPs como una aproximación de genotipado apta para el campo y dirigida a dos especies de primates amazónicos con el uso de ADN de baja y alta calidad, y dos plataformas de secuenciación (Illumina y Nanopore). Usé 14 genomas de alta calidad para encontrar 210 SNPs que permiten la identificación de la especie (doce SNPs), del sexo (doce SNPs) y de la identidad individual (186 SNPs) en dos especies de pichicos, Leontocebus weddelli y Saguinus imperator. Los cebadores, adaptadores e índices fueron diseñados con un enfoque de Genotyping-in-Thousands by sequencing (Genotipado en los miles por secuenciación) que es compatible con ambas plataformas de secuenciación. Este método está basado en la secuenciación de productos de PCR multiplexados de unos cientos de SNPs para genotipar miles de individuos en una sola corrida de secuenciación. En un intento de mejorar la accesibilidad de la genómica de la conservación, el proceso reproducible para obtener a los SNPs informativos está siendo modulado con Snakemake, un sistema de manejo de flujos de trabajo.

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