An underlying assumption of our simulations is that different assay types are conditionally independent-i.e., given that a tested protein pair is known to be positive or negative, the result of one assay is uncorrelated with that of another. To examine the extent to which this assumption holds, we compared Y2H data for protein pairs tested in both forward and reverse orientations-the two assay types used in our study. Overall, we obtained Y2H tests in both orientations for 309 conservation-based predictions (including data reported above combined with additional tests; Supplementary Data). Of these, we observed 58 positives in the forward orientation and 50 positives in the reverse orientation, for an average positive rate of 17% [(58 + 50)/(309 * 2)]. Fifteen positives were found in both orientations, representing 4.9% of the tests. Assuming all predictions are true interactions, this percentage is very close to that predicted by conditional independence, for which 3.1% of tests are expected to be positive in both orientations [17% ^ 2]. If some predictions are not true as expected, the percentages come into even better agreement-e.g., a prediction FDR of 20% predicts that 4.8% positives would arise in both orientations. A similar analysis was performed on a set of 1,572 combined-evidence predictions that were tested in both orientations, leading to similar agreement with the conditional independence assumption.