Ragnarok: a flexible and RApid GeNe Annotation (ROcKs) pipeline deployed through Nextflow

MotivationHigh-quality genome assemblies and pangenomes are increasingly accessible and achievable due to advances in third-generation sequencing and assembly algorithms, but genome annotation remains a critical bottleneck. Existing gene annotation pipelines often require complex installations, multiple steps, long runtimes, and produce variable results, which impede quality and the downstream usage of the annotations. ResultsWe developed RAGNAROK (RApid GeNe Annotation ROcKs), a modular annotation pipeline built on Nextflow and Apptainer that integrates ab initio prediction, transcriptome and protein evidence, repeat annotation, functional assignment, and quality assessment into a single reproducible workflow. Moreover, RAGNAROK utilizes GPU acceleration and parallelization to produce high-quality gene annotations. We benchmarked against BRAKER3 across five diverse, reference-quality plant genomes and demonstrated that RAGNAROK achieved higher sensitivity, precision, and F1 scores at the exon, transcript, and gene levels. Furthermore, we demonstrated RAGNAROKs improvement in re-annotating a suite of five Rosaceae genomes that were previously annotated using MAKER. Overall, RAGNAROK produced more ideal mono:multi-exonic gene ratios, improved BUSCO completeness scores, and reduced missing gene content compared to other pipelines. Additionally, RAGNAROK consistently outperformed BRAKER3 in runtime, scaling efficiently from small to gigabase-scale genomes. RAGNAROK provides a flexible, rapid, scalable, and accurate solution for de novo and re-annotation of plant genomes. Its modular design and workflow scalability lay the foundation for future extensions to animal, fungal, and other eukaryotic genomes. Availability and ImplementationRAGNAROK is available as a GitHub repository at https://github.com/ryandkuster/ragnarok.