Abstract
Bulk RNA-sequencing (RNA-seq) is commonly used for identifying and characterizing genes through annotation, phylogeny, and differential expression analysis. For organisms without a reference genome, the need to generate a de novo transcriptome assembly for analyzing these data can prove challenging, as it is a multi-step process requiring many tools and computational resources. Therefore, a standardized and reproducible workflow using current best practices is required. We introduce the nf-core/denovotranscript workflow, an open-source solution built using Nextflow and the nf-core framework. The protocols here describe how the workflow can be used to perform pre-processing, de novo transcriptome assembly, redundancy reduction, assembly quality assessment, and quantification using RNA-seq data. This workflow offers simple installation and thorough documentation. The use of Docker, Singularity, and Podman container technologies also makes it portable across various computing environments. © 2025 Wiley Periodicals LLC.
