Massively parallel analysis of the functional effects of mutations

Abstract

Massively parallel assays can dramatically advance our understanding of biological processes. Coupling them with modern mutagenesis techniques allows for fine mapping of the link between genotype and phenotype. In this dissertation, I first discuss the current state of the field of using massively parallel assays and analysis to study the functional effects of mutations on both coding and non-coding sequences, and I describe many methods for creating large libraries of variants for using these assays. I then describe two studies applying massively parallel assays to address questions in evolution and cancer. In Chapter 2 I measured the effects of mutations in the promoter of the yeast gene SUL1, and showed that no mutation (or combination of high-fitness single mutations) was capable of increasing fitness to the extent of amplification of the gene. In Chapter 3 I measured the effects of synonymous mutations in an exon of the tumor suppressor TP53, and showed that multiple synonymous mutation had detrimental effects on protein expression due to changes in splicing patterns. In Chapter 4, I discuss initial work developing a new method for linking genotypes found in the same cell by using trans-splicing ribozymes, a technology that could enable massively parallel all-by-all screening. In Chapter 5 I developed a fine-mapping approach based on creating large libraries of chimeric sequences using DNA shuffling, and applied the method to yeast flocculation. Finally, in Chapter 6 I discuss some of the outstanding questions for massively parallel assays, including research directions that I believe will be important in the future.