Competitive Genomic Screens of Barcoded Yeast Libraries |
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Authors: | Andrew M. Smith Tanja Durbic Julia Oh Malene Urbanus Michael Proctor Lawrence E. Heisler Guri Giaever Corey Nislow |
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Affiliation: | Banting and Best Department of Medical Research and Department of Molecular Genetics, University of Toronto;Donnelly Centre for Cellular and Biomolecular Research, University of Toronto;Donnelly Sequencing Centre, University of Toronto;Genetics and Molecular Biology Branch, National Human Genome Research Institute, NIH;Stanford Genome Technology Center, Stanford School of Medicine, Stanford University ;Department of Pharmaceutical Sciences, University of Toronto |
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Abstract: | By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA ''tags'', or ''barcodes,'' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment. |
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Keywords: | Biochemistry Issue 54 chemical biology chemogenomics chemical probes barcode microarray next generation sequencing |
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