Interpreting results from the array-based yeast two-hybrid screens:

How is the screen performed?
The genome-wide two-hybrid array consists of approximately 6000 yeast haploid strains each containing a plasmid (pOAD) with an ORF fused to the Gal4 activation domain (these are the prey strains). Another yeast strain, the bait strain, is of the opposite mating type and contains a plasmid (pOBD2) with an ORF of interest fused to the Gal4 DNA-binding domain. A single bait transformant is mated against the genome-wide activation domain array and subsequent diploids containing both plasmids are selected on auxotrophic media (SD lacking leucine and tryptophan). Two-hybrid positives are diploids with the ability to activate the HIS3 reporter gene and are identified by selection on media lacking histidine. A duplicate screen is performed with a separate bait transformant containing the same ORF of interest. Colonies that grow above the background level of growth of the rest of the array are scored as positives.

Double vs Single positives
Two-hybrid screening in the array format allows the facile classification of two-hybrid positives. Two-hybrid screens result in many non-reproducible two-hybrid positives through ill-defined mechanisms that may include mutations to the yeast strains or the bait vector. We can circumvent this problem using the array method, by considering true positives as those that appear twice in duplicate screens (i.e. bait-prey combinations that are labeled with 2 hits). It is unlikely that a mutation will appear twice in the same array position and this is confirmed by the fact that double positives are usually reproducible upon re-testing. We consider the positives that have arisen twice in the duplicate screens as confirmed high confidence two-hybrid positives that can be published. Some reproducible positives are observed at a high frequency in separate screens with baits of unrelated function and we consider these to be false positives. Presumably, these positives have a tendency to activate the two-hybrid reporter gene so we omit these prey strains from our published data.

Another class of false positives, are the single positives that are not reproducible in duplicate screens using the same bait. These non-reproducible positives are the main source of false positives in two-hybrid screens and are efficiently identified in our array screens. It is important to note that a fraction of the single positives may be reproducible due to technical reasons such as a pinning error on one set of the duplicate plates or insufficient transfer of cells by the robot. For this reason, we provide the identity of the single positives from each screen in case researchers observe an interesting candidate.