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

Nature Protocols volume 2, pages 2958–2974 (2007)Cite this article

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.

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Figure 1: Deletion cassette used for constructing strains in the yeast deletion collection.
Figure 2: Description of the competitive growth assay.
Figure 3: Comparison of homozygous profiling for studying gene function and compound mode of action, and heterozygous profiling for drug-target identification.
Figure 4: Timeline for a pooled growth experiment.
Figure 5: Measurement of noise generated in each step of the assay.
Figure 6: Modeling the sampling error generated during the pooled growth step.
Figure 7: Increasing culture volume decreases noise.
Figure 8: Correcting array saturation increases the correlation of uptag and downtag ratios.
Figure 9: Comparison of mean normalization and quantile normalization, highlighting benefits and disadvantages of each.
Figure 10: Choosing a tag-intensity threshold using uptag–downtag ratio agreement.
Figure 11: Sample data showing data analysis steps and expected results.

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Author information

Authors and Affiliations

  1. Department of Genetics, Stanford Genome Technology Center, 855 S California Ave, Palo Alto, CA, 94304

    Sarah E Pierce & Ron W Davis

  2. Department of Biochemistry, Stanford Genome Technology Center, 855 S California Ave, Palo Alto, CA, 94304

    Ron W Davis

  3. Banting and Best Department of Medical Research, Department of Medical Genetics & Microbiology, Donnelly Centre for Cellular and Biomolecular Research, 160 College St., University of Toronto, Toronto, M5S 3E1, Ontario

    Corey Nislow

  4. Department of Pharmaceutical Sciences and Department of Medical Genetics, Donnelly Centre for Cellular and Biomolecular Research, 160 College St., University of Toronto, Toronto, M5S 3E1, Ontario

    Guri Giaever

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  1. Sarah E Pierce
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  2. Ron W Davis
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  4. Guri Giaever
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Corresponding author

Correspondence to Guri Giaever.

Supplementary information

Supplementary Data 1

Matlab function for modeling samling error (PDF 10 kb)

Supplementary Data 2

Barcode sequences on the Affymetrix TAG4 array (PDF 1105 kb)

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Pierce, S., Davis, R., Nislow, C. et al. Genome-wide analysis of barcoded Saccharomyces cerevisiae gene-deletion mutants in pooled cultures. Nat Protoc 2, 2958–2974 (2007). https://doi.org/10.1038/nprot.2007.427

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