UCSF

Understanding DNA sequence variation is the first step to understanding the underlying genetic architecture of a complex trait, often a manifestation of joint contributions from multiple genes. While instrumental to the successful completion of the Human Genome Project and still considered to be the gold standard, Sanger sequencing is rapidly being phased out for discovery as newer technologies that deliver sequence data with higher throughput and at lower costs become available. With the rise of these newer technologies, generation of sequencing data is no longer the bottleneck. However, one is faced with having to decide between competing technologies, and interpretation of sequencing data remains a significant challenge. In this dissertation, I will present my research on three sequencing projects and one genome mapping project. The first sequencing project involves the use of an array-based resequencing platform, the Affymetrix MitoChip to resequence the mitochondrial DNA for a pancreatic cancer case-control study and for a subset of samples from the Health ABC study. Array-based resequencing represents a high-throughput option for resequencing selected, well-defined regions. We uncovered many novel, rare variants in the mitochondrial sequence, some of which are highly conserved, protein-altering, and predicted to be damaging. While their functional consequences are not clear, we show that more rare variants remain to be found, and the abundance of them suggests that mitochondrial variants could contribute to disease phenotypes. Then, two targeted sequencing studies will be described. The first used an emulsion PCR based approach to study variants in genes involved in drug pathways; the second used a solution-phase target capture approach to fine map causative variants in a region in canine chromosome 6 associated with adult-onset deafness in Border collies. Finally, I will describe our use of nanochannel arrays for genome mapping, an improved version of optical mapping, in structural variation analysis and sequence assembly. Sequence motif maps were constructed 95 BACs previously sequenced for the MHC Sequencing Consortium. The BAC set was sequenced and assembled de novo with assistance from the sequence motif scaffold. We show that the sequence motif map could serve as a scaffold, facilitating gap closing and assembly finishing. Together, our work highlights the potential roles these new technologies play in genetic studies and the importance of taking a multigene approach while considering polygenic traits.