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. 2018 Apr 11;7:e32472. doi: 10.7554/eLife.32472

Figure 1. deepPCA.

(A) Leucine zipper domains (colored) and heptad positions of human Fos and Jun. (B) Overview of the assay. Single amino acid variants of Fos and Jun were constructed by overlap-extension PCR using NNS primers and cloned in a head-tail orientation. In PCA, interacting proteins expressed in yeast lead to complementation between their fused DHFR fragments, which is resistant to methotrexate and produces tetrahydrofolate (THF) from dihydrofolate (DHF) to promote growth. Paired-end deep sequencing then allows the frequencies of each variant in the input and output populations to be measured and a PPI score that represents the number of generation of each variant relative to the wild-type interaction to be computed. (C) Scatter plot of PPI scores between biological replicates 1 and 2. (D) Confirmation of single mutants by individual PCA growth assays. Single mutants were reconstructed, sequence confirmed and their PPI scores were derived from their growth curves measured in a plate reader (see Materials and methods). Error bars represent 95% confidence intervals.

Figure 1.

Figure 1—figure supplement 1. Quality control of the PPI scores measured by deepPCA.

Figure 1—figure supplement 1.

(A) Density plots showing the high correlation between PPI scores calculated from sequencing data filtered at different quality cut-offs. (B) Density plot showing the relationship between PPI scores and input read counts (right) and histogram of PPI scores (left). The data corresponds to replicate 1, including variants with stop codons and prior to filtering based on read count. This plot highlights the PPI score boundary (oblique boundary on the left side of the plot) due to low output read count (see Materials and methods). The vertical line represents the input read count filter. (C) Correlations between deepPCA PPI scores from three biological replicate experiments. Contains both single (n = 1,215) and double mutations (n = 107,625) from the trans Fos and Jun library. Variants with less than 10 UMIs in any of the three replicate input samples or without any UMI in any of the replicate output samples were filtered out.