Shifted Transversal Design smart-pooling for high coverage interactome mapping.

"Smart-pooling," in which test reagents are multiplexed in a highly redundant manner, is a promising strategy for achieving high efficiency, sensitivity, and specificity in systems-level projects. However, previous applications relied on low redundancy designs that do not leverage the full potential of smart-pooling, and more powerful theoretical constructions, such as the Shifted Transversal Design (STD), lack experimental validation. Here we evaluate STD smart-pooling in yeast two-hybrid (Y2H) interactome mapping. We employed two STD designs and two established methods to perform ORFeome-wide Y2H screens with 12 baits. We found that STD pooling achieves similar levels of sensitivity and specificity as one-on-one array-based Y2H, while the costs and workloads are divided by three. The screening-sequencing approach is the most cost- and labor-efficient, yet STD identifies about twofold more interactions. Screening-sequencing remains an appropriate method for quickly producing low-coverage interactomes, while STD pooling appears as the method of choice for obtaining maps with higher coverage.

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