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omosomal regions.Benchmarking RNA-seq differential expression analysis methods using spike-in and simulated RNA-seq data has often yielded inconsistent results. The spike-in data, which were generated from the same bulk RNA sample, only represent technical variability, making the test results less reliable. We compared the performance of 12 differential expression analysis methods for RNA-seq data, including recent variants in widely used software packages, using both RNA spike-in and simulation data for negative binomial (N model. Pe