MAGeCK and BAGEL are unable to identify essential genes in our screen using Cas9 positive read data.

<p>(A) We analyzed our screen data using Cas9-positive replicates from T<sub>0</sub> and T<sub>f</sub> using the MAGeCK-RRA (robust rank aggregation) method with a controlled FDR of 10%. The dashed horizontal line represents <i>p</i> < 0.05; any genes above this line are significant. Genes to left of the dashed vertical line have log<sub>2</sub>-fold-change (LFC) < 0 indicating their sgRNA abundances decrease from T<sub>0</sub> to T<sub>f</sub>. We did not find any genes to be significant using these typical parameters for MAGeCK-RRA. (B) Removal of FDR control with MAGeCK-RRA revealed 932 genes (highlighted in purple) that had LFC < 0 and unadjusted <i>p</i> value < 0.05. Genes shown in grey did not meet these criteria. (C) We analyzed our screen data using Cas9-positive replicates from T<sub>0</sub> and T<sub>f</sub> using the MAGeCK-MLE (maximum likelihood estimation) method with a controlled FDR of 10%. Genes above the dashed horizontal line have <i>p</i> < 0.05 and are significant. Genes to the left of the dashed vertical line have LFC < -1, the typically used cutoff for gene essentiality using this method. We did not find any genes that met both of these criteria. (D) Removal of FDR control with MAGeCK-MLE and increasing the LFC cutoff to < 0 revealed 1,918 genes (highlighted in green) that had LFC < 0 and <i>p</i> value < 0.05. Genes shown in grey did not meet these criteria. (E) Venn diagram showing overlap of the 932 genes (in purple) identified by MAGeCK-RRA with the genes identified by TRACS as having low Library ES (5,424 genes total). 259 genes overlap between the two sets (27.8%). (F) Venn diagram showing overlap of the 1,918 genes (in green) identified by MAGeCK-MLE with the genes identified by TRACS as having low Library ES. 499 genes overlap between the two sets (26%). (G) We analyzed our screen data using Cas9-positive replicates from T<sub>0</sub> and T<sub>f</sub> using BAGEL and plotted the Bayes factor output for each gene in relation to the gene ranking. The Bayes factors for all genes were negative, indicating BAGEL did not discover any perturbations in sgRNA abundances between T<sub>0</sub> and T<sub>f</sub>. (H) A graphical representation of Bayes factors calculated by BAGEL for the top 15 genes with the highest integer value Bayes factors. All Bayes factors are < 0 indicating gene essentiality was not detected. Error bars show standard deviation for each gene as calculated by BAGEL.</p> <p>(PDF)</p>