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A simple answer to this question would be: 'That depends on what you want to simulate.' The basic operation of the Flux Simulator requires transcript annotations in the form of a GTF file. Then, if read sequences–and potential biases–are to be simulated, also a set of genomic reference sequences (one per chromosome) is required. Finally, some parameters allow to specify empiric data, i.e., insert size distributions, sequence biases, etc. deduced from experimental evidence.