Optimized preprocessing methods for marine RNA metavirome studies.

Area of Science:

• Marine virology
• Molecular biology
• Environmental science

Background:

• Marine RNA viruses are crucial to ecosystem health but difficult to study due to their small size and instability.
• Efficient concentration methods are needed to investigate marine RNA virus diversity and ecological roles.

Purpose of the Study:

• To evaluate and optimize methods for concentrating marine RNA viruses from large seawater volumes.
• To establish a reproducible protocol for capturing and analyzing marine RNA viruses.

Main Methods:

• Compared three concentration methods: membrane filtration, ultrafiltration, and ultracentrifugation.
• Tested various seawater volumes (60–300 L) and filtration parameters.
• Optimized a protocol using 0.2 μm membrane filtration, 30-kDa tangential flow filtration, and ultracentrifugation.

Main Results:

• A 0.2 μm nitrocellulose membrane filter showed a 97.3% library QC pass rate, outperforming other initial filtration methods.
• The optimized three-step method (membrane filtration, TFF, ultracentrifugation) significantly enriched RNA virus contigs to 81.5% of the virome.
• This method recovered 763 high-quality contigs from 27 families and 66 genera, demonstrating high reproducibility.

Conclusions:

• The developed concentration protocol is effective for capturing and analyzing marine RNA viruses.
• This method provides a valuable tool for advancing research into marine RNA virus diversity and ecological significance.