Advances in genetic mapping and sequencing techniques : a demonstration using the domestic dog model

• Main Author: Forman, Oliver
• Published: University of Glasgow 2013
• Subjects: Q Science (General)
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.586756

Over the past ten years huge advances have been made in the field of genetics and genomics. Genetic mapping has evolved from laborious linkage and homozygosity based approaches to high-throughput genome-wide association studies using whole genome SNP array technology. Through massively parallel sequencing technology, gigabases of sequencing data can now be produced in a single experiment. The domestic dog has been increasingly recognised as a model for human disease and mapping of inherited disease in the domestic dog is facilitated by fixed and genetically isolated populations. The aims of this thesis were to demonstrate advances in mapping and sequencing techniques by investigating the genetics of five inherited disorders, representing significant welfare issues in the purebred dog. An additional aim was to develop diagnostic DNA tests to identify affected individuals and asymptomatic carriers. A parallel mapping approach was used to map two autosomal recessive conditions in the Cavalier King Charles Spaniel. The use of a single common set of controls for two independent genome-wide association studies was demonstrated as an efficient mapping strategy when studying two conditions affecting a single breed. Newly available target enrichment and massively parallel sequencing methodology was used to simultaneously sequence both disease-associated loci, with one condition acting as a control for the other. A genome-wide homozygosity mapping approach using microsatellite markers was used to investigate spinocerebellar ataxia in the Italian Spinone. The disorder was successfully mapped to a single chromosome using six cases and six controls, and fine mapped with additional microsatellite markers. Subsequently, a progression of sequencing techniques were used to identify the disease-associated mutation, with the study highlighting the potential difficulties of using massively parallel sequencing technologies. Spinocerebellar ataxia (or late onset ataxia) in the Parson Russell Terrier was investigated using a genome-wide association study followed by a target enriched massively parallel sequencing approach. Further sequencing was performed to reduce the large number of potential causal variants, with the entire workflow achieved in-house. The final experimental chapter describes the use of a genome-wide mRNA sequencing (mRNA-seq) approach as a method of candidate gene sequencing of a single case of neonatal cerebellar cortical degeneration in a Beagle dog. The mRNA-seq approach demonstrates a simple, fast and cost effective method of targeted resequencing of expressed genes when a suitable tissue resource is available. For all five disorders under investigation, disease-associated mutations were identified leading to the development of diagnostic tests. Three of the mutations were in genes not previously associated with similar conditions in humans or other model organisms.