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High throughput methods for functional genomics in saccharomyces cerevisiae [electronic resource]

The Saccharomyces cerevisiae deletion collection has revolutionized yeast genetics by allowing genome-wide deletion screens to be performed in pooled cultures. The inclusion of unique, 20-bp DNA sequences known as "tags" or "barcodes" in each strain allows all ~6000 yeast deletion mutants to be analyzed in a single culture by using a microarray to detect changes in tag abundance. Here we discuss improvements and extensions made to the pooled-yeast-deletion assay. This includes a redesign of the barcode array that is used for detecting the tags included in the deletion collection. Our analysis of this barcode array examines whether it is better to use available microarray surface area on a single feature for each probe, or to instead include multiple smaller features. It also covers aspects of data analysis such as the correction of hybridization defects and array saturation, tag-sequence repairs, and estimation of background hybridization. In addition to these microarray changes, we also examine the impact of experimental design on the quality of pooled growth data. Each step in both the experimental protocol and the data-analysis pipeline is examined for possible improvement. Finally, we discuss preliminary experiments testing the application of pooled fitness profiling to the study of epistasis. Data are shown that demonstrate the feasibility of a pooled-double-mutant fitness assay that is compatible with existing barcode array technology. This assay has the potential to become a valuable functional-genomics tool that may provide an improved understanding of pathway relationships in yeast.