The yeast deletion collection: a decade of functional genomics

Abstract

The yeast deletion collections comprise >21,000 mutant strains that carry precise start-to-stop deletions of ∼6000 open reading frames. This collection includes heterozygous and homozygous diploids, and haploids of both MAT A: and MATα mating types. The yeast deletion collection, or yeast knockout (YKO) set, represents the first and only complete, systematically constructed deletion collection available for any organism. Conceived during the Saccharomyces cerevisiae sequencing project, work on the project began in 1998 and was completed in 2002. The YKO strains have been used in numerous laboratories in >1000 genome-wide screens. This landmark genome project has inspired development of numerous genome-wide technologies in organisms from yeast to man. Notable spinoff technologies include synthetic genetic array and HIPHOP chemogenomics. In this retrospective, we briefly describe the yeast deletion project and some of its most noteworthy biological contributions and the impact that these collections have had on the yeast research community and on genomics in general.

Keywords: fitness profiling; functional genomics; mutant phenotype; yeast deletion.

Figure 1

(A) Saccharomyces cerevisiae Genome Deletion Project overview. The Stanford Genome Technology Center (SGTC) (yellow boxes) served as the resource for: (1) The 20-bp unique molecular barcode or tag sequence (UPTAG and DNTAG) assigned to each ORF (in collaboration with Affymetrix, peach box). (2) Automated primer picking for deletion cassette construction and deletion strain confirmation oligonucleotides (oligos) using sequencing data from the SGD (Cherry et al. 2012). (3) Primer-picking scripts were formatted for use with the automated multiplex oligonucleotide synthesizer (AMOS). (4) Resulting PCR-amplified deletion cassette modules (purple) and five premixed oligonucleotides pairs for the PCR confirmations of each strain (yellow) (A–B, A–kanB, C–D, kanC–D, and A–D) were arrayed into 96-well “6-pks” and sent to consortium members. (5) Successful deletion phenotypes and results of PCR confirmations were logged into the deletion database at the SGTC and directly made available to the yeast community by Research Genetics, SGTC, and ATCC. (6) Strains that failed to be deleted in the first round of strain construction were sent back to the SGTC for primer redesign. (B) Deletion strain strategy. Each deletion “cassette” was constructed using two sequential PCR reactions. In the first amplification, 74-bp UPTAG and 74-bp DNTAG primers amplify the KanMX gene from pFA6-kanMX4 DNA, whose KanMX expression confers dominant selection of geneticin (G418) to yeast (Wach et al. 1994). The primers consist of (5′–3′): 18 bp of genomic sequence that flank either the 5′ or 3′ end of the ORF (directly proximal and distal to the start and stop codons, respectively); 18 and 17 bp of sequence common to all gene disruptions (for amplifying the “molecular barcodes” in a PCR; (U1: 5′-GATGTCCACGAGGTCTCT-3′ or D1: 5′-CGGTGTCGGTCTCGTAG-3′); a 20-bp unique sequence (the molecular barcode TAG); and 18 and 19 bp of sequence, respectively, homologous to the KanMX4 cassette (U2: 5′- CGTACGCTGCAGGTCGAC-3′ or D2: 5′-ATCGATGAATTCGAGCTCG-3′) the other priming site for amplifying the molecular barcodes. In the second PCR reaction, two ORF-specific 45-mer oligonucleotides (UP_45 and DOWN_45) are used to extend the ORF-specific homology to 45 bp, increasing the targeting specificity during mitotic recombination of the gene disruption cassette. The presence of two tags (UPTAG and DNTAG) increases the quality of the hybridization data from the oligonucleotide arrays by adding redundancy (∼3.2% of the strains harbor only one unique UPTAG sequence). Note that in version 2.0 and subsequent collections, the two-step PCR was replaced with a single, longer primer PCR. The original length constraint was due to high error rates in longer primers, a problem that was significantly reduced by the time the V 2.0 strains were constructed. (C) Deletion strain confirmation. The correct genomic replacement of the gene with the KanMX cassette was verified in the mutants by the presence of PCR products of the expected size, using primers that span the left and right junctions of the deletion module within the genome. Four ORF-specific confirmation primers (A, B, C, and D primers) were selected for each ORF disruption. The A and D primers were positioned 200–400 bp from the start and stop codons of the gene, respectively. The B and C primers were located within the coding region of the ORF and, when used with the A or D primers, gave product sizes between 250 and 1000 bp. The KanB and KanC primers are internal to the KanMX4 module. For haploid or homozygous isolates, the junctions of the disruption were verified by amplification of genomic DNA using primers A and KanB and primers KanC and D. Deletion of the ORF was verified by the absence of a PCR product using primers A with B and C with D. In the case of heterozygous strains a successful deletion was indicated by the appearance of an additional, wild-type-sized PCR product in reactions A with B, C with D, and A with D. Each deletion mutant was checked for a PCR product of the proper size using the primers flanking the gene. In addition, each strain background was checked for the appropriate auxotrophic markers and mating type. The rigorous strain verification used in the deletion project is unfortunately not the norm. Formally, the five confirmations were required for confirmation; this was reduced to three when the long A–D PCR product proved problematic, with many groups verifying only the upstream and downstream KanMX-genomic junctions, omitting the A and D reactions that verify both the presence of the deletion and, equally importantly, that confirm the absence of the wild-type allele.

Figure 2

Pooled chemogenomic screens of the yeast deletion collection. Fitness profiling of pooled deletion strains involves six main steps: (1) Strains are first pooled at approximately equal abundance. (2) The pool is grown competitively in the condition of choice. If a gene is required for growth under this condition, the strain carrying this deletion will grow more slowly and become underrepresented in the culture (red strain). Resistant strains will grow faster and become overrepresented (blue strain). (3) Genomic DNA is isolated from cells harvested at the end of pooled growth. (4) Barcodes are amplified from the genomic DNA with universal primers in two PCRs, one for the uptags and one for the downtags. (5) PCR products are then hybridized to an array that detects the tag sequences. (6) Tag intensities for the treatment sample are compared to tag intensities for a control sample to determine the relative fitness of each strain. Here, the starting pool shown in step 1 is used as a control; steps 3–5 are not shown for this control sample.

Figure 3

(A) Network of citations of the Winzeler et al. (1999) and Giaever et al. (2002) deletion project publications. A total of 428 publications cite both publications (nodes with two edges). Of the ∼2200 (2231) total unique citations, ∼900 (864) cite Giaever et al., ∼1600 (1584) cite Winzeler et al., and ∼400 (428) cite both. Node size reflects number of citations per publication. Triangular node shape, self-citations; 126 publications. (B) Citations per year classified by article or review. (C) Distribution of 205 large-scale phenotypic assays using the deletion collection. The top pie chart depicts the six primary categories of yeast deletion collection screen by type; regardless of the particular method used (e.g., colony size, pooled screen). The three most common screen types are those that interrogate biological processes (39%, blue), drug/small molecule screens (31%, red), and environmental screes (19%, yellow). These three categories are further subdivided in the three bottom pie charts.