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The necessary first step in order to simulate the experiment is the loading of a reference annotation. Input data has to be in GTF format at the path "Gene Expression" step employs the annotation specified by REF_FILE_NAME . Each transcript has to have "exon" features, LOAD_CODING takes into account the ones that have additionally "CDS" features, LOAD_NONCODING those which don't. Initiating the reading of the reference annotation (button "Run" in the toolbar) first causes a check whether the GTF structure is well sorted for efficiency of the subsequent operations. In case, the FLUX SIMULATOR will sort your file in the temporary directory, and subsequently a sorted copy of the file (with the suffix "_sorted" before the extension) should appear in the folder containing the project. Make sure to use sorted files instead of the original files in future runs, because file sorting can contribute a substantial part of the running time.Upon termination of reading and parsing the annotation, you see several statistics including a histogram of spliced transcript lengths (upper panel) and a zoom-in onto the first 3 quartiles (lower panel). This step also initiates the pro file by writing the first 4 columns, i.e., splice locus ID, transcript ID, CDS/NC and spliced transcript lengthand creates an artificial expression profile of the described transcripts. By the flag LOAD_CODING transcripts with an annotated coding sequence are taken into account, by LOAD_NONCODING correspondingly those which don't. Results from in silico gene expression are stored in the files specified by the parameters PRO_FILE respectively LIB_FILE; if no explicit values are provided for these parameters, then the corresponding files are created in the folder of the parameter file.