Competitive Genomic Screens of Barcoded Yeast Libraries

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. Video LinkThe video component of this article can be found at https://www.jove.com/video/2864/ Protocol Background informationThere are several ways to generate mutants that carry barcode tags. The current gold standard is the Yeast KnockOut (YKO) collection created by a consortium of labs and completed in 2002 1 . Since the original YKO was introduced, other yeast collections have been generated; in different strain backgrounds, using over-expression constructs, and in other microbes such as E. coli 2 . In parallel, the effort to create barcoded shRNA libraries is proceeding rapidly, and in fact, many of the design principles for these mammalian collections have been adopted from yeast. To demonstrate how barcoded transposons can be a rapid, widely applicable strategy for creating systematic mutant collections, we focus on one collection we recently created in the human fungal pathogen, Candida albicans. Our work on Candida was based on the success of barcode screens in S. cerevisiae, and is used here as an example organism. The sample protocol, can with minor modifications be used to screen any organism that can be grown in suspension culture. Because few organisms have the requisite high rates of transformation and efficient mitotic recombination needed to create perfect deletion mutants, accordingly we developed a protocol that uses transposon mutagenesis in vitro to mutagenize a genom...

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