Limited effect of short- to mid-term storage conditions on an Australian farmland soil RNA virome.

Soils represent one of the largest and most diverse reservoirs of microbial life on Earth, yet their associated RNA viruses remain underexplored compared to animal and aquatic systems. Viral discovery in soils has been further limited by technical hurdles, particularly difficulties in obtaining sufficient yields of high-quality RNA for sequencing. To address this, we evaluated a range of storage and preservation strategies, including the use of commercial preservative solutions and ultra-cold snap-freezing, followed by standardized RNA extraction, sequencing, and virus discovery pipelines. This work aimed to establish minimum sample storage requirements that maintain RNA integrity, generate sufficient RNA sequencing data, and subsequently enable reliable soil virome characterization. While no preservative solution proved effective, "neat" soil samples were stable at 2°C-8°C and -30°C for at least 2 weeks, and at -80°C for at least 3 months, with no measurable reduction in RNA quality, sequencing data, or viral abundance and diversity. From 32 resulting libraries, we identified 1,475 putative novel RNA viruses, with the majority belonging to the microbe-associated phylum