Rockhopper 2 workflow depicting the various phases of Rockhopper 2’s analyses. As input, Rockhopper 2 requires one or more files of sequencing reads from RNA-seq experiments. In the first analysis stage, Rockhopper 2 determines k-mers from the sequencing reads and builds a de Bruijn graph from the k-mers. The de Bruijn graph is used to assemble candidate transcripts, which are stored in a Burrows-Wheeler index. In the second analysis stage, Rockhopper 2 aligns the sequencing reads to the assembled candidate transcripts to determine a final set of high-quality assembled transcripts. After the second stage, transcriptome assembly is complete and Rockhopper 2 performs several downstream analyses, including normalizing data from different experiments, quantifying transcript abundance, and testing for differential gene expression across multiple conditions.