Preparation method shapes the recovery and ecological interpretation of DNA and RNA soil viral communities.

Deciphering viral ecology in soils is challenging due to soil's high physicochemical and microbial community complexity. To enhance detection of DNA and RNA viruses, we applied different preparation methods to soils collected from a grassland field experiment. Analyses included metagenomics and metatranscriptomics of size-fractionated extracellular viruses, total soil metagenomics and metatranscriptomics, total soil metatranscriptomics with polyadenylation enrichment, and metagenomics of bacteria/archaea as well as eukaryote-enriched samples. DNA viromes outperformed total soil metagenomes in viral detection and quality. Contrastingly, RNA viromes and total soil metatranscriptomes performed similarly for viral recovery, though RNA viromes yielded higher-quality genomes. Together, our results highlight how different preparation methods can influence the recovery and quality of DNA and RNA vOTUs. Further, we demonstrate the power of different methods in identifying distinct viral communities with unique host predictions, which in turn can have significant implications for ecological investigations related to interkingdom interactions.