Genome-wide analysis of barcoded Saccharomyces cerevisiae gene-deletion mutants in pooled cultures

Abstract

The availability of a near-complete (96%) collection of gene-deletion mutants in Saccharomyces cerevisiae greatly facilitates the systematic analyses of gene function in yeast. The unique 20 bp DNA 'barcodes' or 'tags' in each deletion strain enable the individual fitness of thousands of deletion mutants to be resolved from a single pooled culture. Here, we present protocols for the study of pooled cultures of tagged yeast deletion mutants with a tag microarray. This process involves five main steps: pooled growth, isolation of genomic DNA, PCR amplification of the barcodes, array hybridization and data analysis. Pooled deletion screening can be used to study gene function, uncover a compound's mode of action and identify drug targets. In addition to these applications, the general method of studying pooled samples with barcode arrays can also be adapted for use with other types of samples, such as mutant collections in other organisms, short interfering RNA vectors and molecular inversion probes.

Quantitative phenotyping via deep barcode sequencing

“…For our analysis, several different pools of yeast mutants were grown competitively in diverse conditions, and following growth, genomic DNA was extracted, molecular barcodes were amplified by PCR, and barcode amplicons were either labeled and hybridized to a barcode microarray as described (Pierce et al 2007) or sequenced using an Illumina Genome Analyzer (Bennett 2004). For the barcode microarray samples, barcode abundance was inferred based on the normalized fluorescence intensity (Pierce et al 2006(Pierce et al , 2007 following detection with an Affymetrix confocal laser scanner. For Bar-seq, barcode abundance was determined by counting the number of times each unique barcode was sequenced (see Methods).…”

“…Additional constraints, for example, errors introduced by liquid handling and other preparative steps, will, of course, limit the upper level of multiplexing. Nonetheless, the sampling error for Bar-seq (assuming 10 million reads/lane) is 0.03%, well below the bottleneck sampling error introduced by cell-harvesting and liquid-handling steps of the assay, which can be as high as ;5%-10% (Pierce et al 2007). …”

“…By comparing Pool-constant with Poolvariable, we could quantify and compare the abundance of each strain/barcode either by microarray hybridization or sequencing. Microarray signals were transformed to account for the observation that they saturate at high probe/signal levels (Pierce et al 2007). We found that both platforms clearly distinguished all four levels of strain abundance between the two pools ( Fig.…”

“…Log 2 signals for each strain were determined, and the relative abundance across subpools was assessed. For tag-array analysis, the signal refers to the raw intensities corrected for saturation effects as described previously (Pierce et al 2007), whereas for sequencing analysis, the signal refers to the sequencing counts. Data were filtered to remove strains with signal below an arbitrary background level (signal of 40 for sequencing data, 200 for hybridization data).…”

“…Except where indicated, pooled assays were performed as described by Pierce et al (2007). Genomic DNA was isolated from cells grown for 20 generations, and barcodes were amplified and hybridized to barcode microarrays, where each barcode deletion mutant is represented by 10 hybridization signals (the uptag and downtag for each strain are represented on the array five times).…”