Overview
This paper reports on two successful applications of high-throughput sequencing.
1) To assess relative strain abundance in parallel growth assays of molecularly barcoded yeast deletion mutants (Bar-seq).
2) To re-characterize the yeast deletion collection by re-sequencing all barcodes and common priming sites and confirming genomic positions of each.
Abstract
Next generation DNA sequencing technologies have revolutionized diverse genomics applications, including de novo genome sequencing, SNP detection, and chromatin immunoprecipitation and transcriptome analysis. Here we apply deep sequencing to genome-scale fitness profiling to evaluate yeast strain collections in parallel. This method, Barcode analysis by Sequencing or “Bar-seq” outperforms the current benchmark, barcode microarray assay, in terms of both dynamic range and throughput. When applied to a complex chemogenomic assay, Bar-seq quantitatively identifies drug targets, with performance superior to the benchmark microarray assay. We also show that Bar-seq is well-suited for a multiplex format. We completely re-sequenced and re-annotated the yeast deletion collection using deep sequencing, found ~20% of the barcodes and common priming sequences varied from expectation and used this revised list of barcode sequences to improve data quality. Together, this new assay and analysis routine provide a deep-sequencing-based toolkit for identifying gene-environment interactions on a genome-wide scale.