Benchmarking methods for genome annotation using nanopore direct RNA in a non-model crop plant.

Motivation: High-quality genome annotations are essential for transcriptomic analyses investigating plant responses to environmental stress. While nanopore long-read direct RNA sequencing offers a powerful approach for improving genome annotations, studies benchmarking optimal tools for this process have primarily focused on animal models. In this study, we benchmarked five annotation tools: StringTie3, IsoQuant, Bambu, FLAIR, and FLAMES, using direct RNA data from barley infected with Net Form Net Blotch disease. Results: We observed substantial variation across tools in isoform detection, structural completeness, splicing classification, and handling of 5' read truncation. Several tools successfully identified novel transcripts, with the two top-performing reference-guided approaches both detecting over 700 previously unannotated transcripts, including candidates with predicted roles in disease response. Our results highlight the importance of plant-specific benchmarking of bioinformatic tools and demonstrate the utility of direct RNA sequencing for improving genome annotations, supporting ongoing efforts to enhance reference resources for non-model plant species. Availability and implementation: Benchmarking code is available at https://github.com/jadedavis5/benchmarking_paper. Datasets are described in the 'Data availability' section.