Global fitness profiling of fission yeast deletion strains by barcode

A genome-wide deletion library is a powerful tool for probing gene functions and it has recently become available for the fission yeast Schizosaccharomyces pombe. Here we present a high-throughput method for quantitatively measuring the fitness of fission yeast deletion strains by deep sequencing of the DNA barcodes built into each strain. In this method, thousands of strains are pooled together in one culture and then subject to various treatments. The fitness phenotypes of pooled strains are analyzed in parallel in one sequencing reaction. The throughput and cost-effectiveness of the assay are further improved by a multiplexed sequencing strategy. The barcode sequencing data showed good reproducibility and linearity, and we validated the use of barcode sequencing for fitness analysis by detecting auxotrophic mutants that failed to grow in a minimal medium. We applied barcode sequencing to profile the fitness changes of mutants upon treatment with three types of genotoxins and an anti-microtubule compound thiabendazole (TBZ). More than two hundred mutants hypersensitive to at least one treatment were identified. Genes with known functions in DNA damage response and mitosis were highly enriched among the hypersensitive hits. Unexpectedly, besides sensitive mutants, fitness profiling also revealed mutants resistant to drug treatments, including several mutants resistant to anticancer drug camptothecin (CPT). Finally, as a demonstration of the use of barcode sequencing in revealing new gene functions, we report the identification of a previously uncharacterized gene required for centromere silencing.