Hybrid assembly approach enhances aquatic RNA virome recovery through nanopore direct RNA sequencing

Aquatic viromics provides a powerful framework for profiling viral diversity in environmental water samples. Nanopore direct RNA sequencing (NDRS) is a promising protocol for detecting RNA viruses in water systems. We developed an NDRS-based hybrid assembly approach by screening RNA virus assemblers to select the appropriate annotation tools for accurately reading virus genomes. Flye was chosen for individual assemblies of RNA virus sequences, Unicycler was best for hybrid assemblies, and geNomad and Kraken2 were used for virus annotation. The applicability and advantages of this NDRS protocol were comprehensively evaluated. Compared to traditional direct cDNA sequencing, the NDRS -based hybrid assembly approach obtained higher data quality, achieved 99 % sequencing depth and coverage, assembled more contigs, and increased virus annotation accuracy. Using the NDRS-based hybrid assembly method, pathogenic RNA viruses were successfully identified in the Yangtze River. Overall, this study presents a high-resolution, low-bias workflow for RNA virus detection in aquatic environments and provides a theoretical foundation for the high-throughput monitoring of low-abundance RNA viruses in complex ecosystems.