Genes with low Library ES tend to have extreme Initial ES and/or Final ES.

<p>(A) TRACS 3D plot illustrating the distribution of Library ES, Initial ES, Final ES in an extreme case example screen that had very poor representation of sgRNAs at T<sub>0</sub> in Cas9-negative cells. Genes that have low Library ES (genes that fall into the first quartile of all Library ES across all genes) are shown in dark blue. These genes also tend to have extreme values for Initial ES and/or Final ES which can lead to potential false positives. This extreme example demonstrates how initial sgRNA abundances can be low due to non-gene-editing events and skew gene scores at T<sub>0</sub> (Initial ES) and T<sub>f</sub> (Final ES). (B) Histogram illustrating the distribution of Library ES across all genes in our GO-CRISPR experiment. To diminish the effects of poorly represented sgRNAs, TRACS determines the distribution of the Library ES across all genes and computes the cutoff value for the first quartile (the bottom 25% of all Library ES; highlighted in dark blue). TRACS then discards genes that have Library ES below this threshold (< 985 for our iOvCa147 screen), however researchers can increase or decrease the threshold within the TRACS software suite for further fine-tuning.</p> <p>(PDF)</p>