(A) A cartoon of PPiSeq yeast library construction. Strains from the protein interactome collection are individually mated to strains from the double barcoder collection and sporulated to recover haploids that contain a mDHFR-tagged protein and a barcode. Haploids are mated as pools. In diploids, expression of Cre recombinase causes recombination between homologous chromosomes at the loxP locus, resulting in a contiguous double barcode that marks the mDHFR-tagged protein pair. (B) Representative double barcode frequency trajectories over twelve generations of competitive growth. Trajectories are used to calculate a quantitative fitness for each double barcoded strain. (C) Standard error of fitness estimates of protein pairs. The blue and red lines represent the median standard error for a sliding window (width = 0.05) of all fitness-ranked protein pairs and of only the positive protein-protein interactions, respectively. (D) Estimated fitness of strains with different double barcodes representing the same protein pair in the same pooled growth. Positive protein pairs are randomly selected within a fitness window. ORF x Null is a violin plot of the fitness distribution of all interactions with a mDHFR fragment that is not tethered to a yeast protein. DHFR(-) is yeast strains that lack any mDHFR fragment. DHFR(+) is yeast strains that contain a full length mDHFR under a strong promoter. (E) Density plot of the fitness of double barcodes that represent the same putative PPI in the same pooled growth. In (B–E), the data in SD environment are used. (F) Density plot of the normalized mean fitness of the same PPI between two pooled growth cultures in SD environment. PPIs detected in either one growth culture are included. (G) Venn diagram of the number of PPIs identified within our search space by PPiSeq in nine environments (magenta), PPiSeq in SD environment (pink), the interactome-scale protein-fragment complementation screen (PCA, yellow), and the BioGRID database excluding any PPIs previously detected by PCA (blue).