One of the first step in RNA-Sequencing (RNA-Seq) data analysis consists of aligning (Next Generation Sequencing) reads to a reference genome. In literature, there are several tools implemented by practitioners and researchers for the alignment step. However, two tools are the de-facto-standard used by bioinformatics researchers in their pipelines: HISAT (version 2) and STAR (version 2). The aim of this study is to determine the impact of the alignment tool on the RNA-Seq analysis in terms of biological relevance of the results and computational time. The two implemented pipelines return different results on the biological side. This is due to assumptions the used tools made and to the specific characteristics of the underlying (statistical) models. The study provides valuable insights for researchers interested in optimizing their RNA-Seq pipelines and making informed decisions about which pipeline to use. As lesson learned, we suggest bioinformatics researchers to use more pipelines when make experiments to reduce the prediction errors induced by assumption of a specific tool or method.
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