Rsubread is a Bioconductor R package that encompasses multiple tools for fast and accurate analysis of Next-Generation Sequencing (NGS) data. Major functionalities of this toolbox include mapping of RNA and DNA sequencing reads to a reference genome, counting reads to genomic features such as genes, exons, junctions and genomic intervals (quantification) and discovery of genomic mutations. Challenges in existing pipelines will be highlighted and the basic ideas behind Rsubread to overcome these challenges will be explained. Functionalities of Rsubread and a demonstration of how to use it in the NGS data analysis will be shown. Results comparing Rsubread to other software tools for the mapping and quantification of sequence reads will be shown. The results show that Rsubread achieves a superior computational efficiency than the other software tools without compromising specificity and sensitivity.