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Biases in RNA Sequencing: RNA sequencing (RNA-Seq) workflows comprise RNA purification, library generation, the sequencing itself, and the evaluation of the sequenced fragments. The initial steps impose biases for which the data processing algorithms try to compensate afterwards. Key tasks for data evaluation algorithms are the concordant assignment of fragments to the transcript variants, robustness towards annotation flaws and the subsequent deduction of the corresponding abundance values. Unless the quality of all individual processing steps can be unequivocally determined, subsequent comparisons of experimental data remain ambiguous.