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

High-quality genome annotations are essential for transcriptomic analyses investigating plant responses to environmental stress. While Nanopore long-read direct RNA (dRNA) 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: StringTie2, IsoQuant, Bambu, FLAIR, and FLAMES, using dRNA data from barley infected with Net Form Net Blotch disease. 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 top-performing reference-guided approach detecting 994 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 dRNA sequencing for improving genome annotations, supporting ongoing efforts to enhance reference resources for non-model plant species.