Typical analysis workflow using TRACS to identify essential genes.

(A) The TRACS workflow is separated into five steps. Step 1: Experiment parameters are entered in the graphical user interface (GUI). Step 2: Library reference file (.csv format) and raw read files (.fastq format) for all Cas9-positive replicates are selected in the GUI. Step 3: Raw read files (.fastq format) for all Cas9-negative replicates are selected in the GUI. Step 4: Raw reads are trimmed and aligned to generate read counts, then the TRACS algorithm runs to calculate Library ES, Initial ES, Final ES and the ER for each gene. Step 5: TRACS saves the results with all scores in an output file which can then be explored using the accompanying VisualizeTRACS data explorer. (B) Screenshot of the easy-to-use TRACS GUI asking user to enter experimental parameters (Step 1). Subsequent displays provide a similar interface for selecting input data files for Steps 2–4. (C-D) Screenshot of the accompanying VisualizeTRACS data explorer that researchers can use to visualize and inspect their TRACS output files and generate publication-ready figures. Researchers can control all aspects of filtering and data manipulation (Library ES, Initial ES, Final ES, ER, p_adj) to fine-tune selection of genes.

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