Optimized preprocessing methods for marine RNA metavirome studies

The pathogenicity of marine RNA viruses has been extensively studied, as they infect a wide range of hosts. Recently, interest in the diversity and ecological roles of marine RNA viruses has been increasing. Marine RNA viruses are generally smaller than DNA viruses, making them more challenging to concentrate, and their inherent instability leads to rapid degradation. Therefore, optimizing an efficient RNA virus concentration system is crucial. In this study, we conducted 95 experiments with seawater volumes ranging from 60 L to 300 L to evaluate the capture efficiency of RNA viruses using three concentration methods: membrane filtration, ultrafiltration, and ultracentrifugation. Filtering 100 L of seawater through a 0.2 μm nitrocellulose disc yielded a 97.3 % library QC pass rate, whereas an equivalent 0.2 μm TFF cassette and Fe³⁺ flocculation each yielded 0 % QC success. The optimal method to concentrate marine RNA viruses involved filtering 100 L of seawater through a 0.2 μm membrane, followed by a primary concentration using a 30-kDa tangential flow filtration system and finally ultracentrifugation. Ultracentrifugation enriched RNA-virus contigs to 81.5 % of the virome, which was three-fold higher than 30 kDa centrifugal ultrafiltration (25.7 %; p < 0.05), and recovered 763 high-quality contigs spanning 27 families and 66 genera. This approach demonstrated high reproducibility. Our results present an effective method for capturing and analyzing RNA viruses in marine environments, providing a valuable tool for further investigating their diversity and ecological roles.