(A–E) Processing of PR and PL libraries. (A) All reads in the PR and PL libraries (gray), from which we take only those reads that are ±4 bp away in length from the wildtype sequence (dark gray). (B) Inverse cumulative distribution function (normalized to the total number of sequences), with shaded indicating the sequences we removed due to having less than 10× coverage. (C) We removed sequences that had 20 or more single point mutations compared to their respective wildtype sequence. Note that this mainly affected the PL library (orange), as the original plasmid from which the libraries were cloned contained the wildtype PR sequence. (D) Cumulative distribution function (CDF) of standard deviation of expression bin numbers, with shaded sequences the ones we removed from subsequent analyses. (E) Box plots indicating the distributions of mean values (in bin units) for a given mode (in bin units), before (left) and after (right) selecting for only those where mean, mode, and median are within 0.5. (F–J) Processing of the 36N library. (F) Average histogram of alignment similarity for the 1000 most covered sequences (shaded area indicates 95% confidence interval). We used the similarity threshold of 0.7 between low- and high-scoring modes to select for unique sequences and eliminate sequencing errors. (G) Histogram of coverage (black line), with highlighted contributions of the noise cloud around the reference sequence (dark red), and the clouds around the 10, 100, and 1,000 most abundant sequences (from darkest to lightest shade of red, respectively). (H) Histogram of counts for the reference sequence per bin, used to debias all other distributions. (I) Template probability distribution functions (PDFs) obtained as averages of PDFs that have the same mode (indicated by color). The inferred fluorescence activated cell sorting (FACS) noise background is shown as a thick gray line. Given a distribution, we only accepted values in the bins in which the appropriate reference was three times above the inferred background. Such filter is shown in (J).