For this second assay, Lex-A Y2H was run in a "reverse" orientation in which the two proteins cloned as bait and prey, respectively, were exchanged as prey and bait. We tested 251 of the 470 predictions for which sequence-verified clones were available in the opposite orientation. This resulted in 35 positives, elevating these interactions to probability >99.9% and adding them to the map. The pairs that tested negative in the reverse orientation were downgraded to 88.1% probability. Overall, after performing Y2H in both forward and reverse orientations, 171 new interactions were identified out of 606 protein pairs for a success rate of 28%. Although we ended our experimentation at this point, the Prediction strategy could be continued by next testing the "double negatives" (pairs testing negative in both orientations of Lex-A Y2H) using a third type of assay such as Gal4-based Y2H.