We next considered an interaction mapping strategy that, rather than treat all protein pairs equally, maintains a rank-ordered list of pairs according to their probabilities of interaction (Thresholding strategy, Table 1 ). All probabilities start at the background frequency of interaction for random protein pairs (as for Basic-serial and Pooling). Protein interactions are initially tested using pooled screening, and after each two-phase pooled experiment the probabilities increase for interactions that are observed and decrease for interactions that are tested but not observed. Unlike previous strategies, however, protein pairs with probability greater than an upper threshold (i.e., 95%) are declared to be definite "interactors" and are removed from subsequent testing (Fig. 1b) . Likewise, interactions with probability beneath a lower threshold are declared to be "non-interactors" and are also removed from further consideration. The motivation for thresholding is to more quickly exclude the overwhelming number of non-interacting protein pairs. Finally, candidate interactions are defined as those with probabilities between the upper threshold and background. When candidates are available they are always tested immediately using pairwise assays, before resorting to pooling, until their probabilities are pushed above the upper threshold or below background. The motivation for prioritizing candidate interactions is to more quickly cover the interactions likely to be positive. Overall, Thresholding resulted in more than a two-fold cost reduction compared to Pooling (Table 1 and Fig. 3a ).