SS-VIME: a single-source virome-microbiome extraction protocol toward comprehensive soil community analysis

Integrated analysis of soil microbiomes and their associated viromes is critical for understanding ecosystem function, yet is hampered by the profound spatial heterogeneity of soil, which introduces significant bias when using separate extraction workflows and/or subsampling strategies to capture fungal, bacterial, and viral communities. Here, we present single-source extraction for unified soil virome-microbiome profiling (SS-VIME), a protocol that overcomes this limitation. Based on extended cellulose column chromatography, this method sequentially elutes distinct DNA and double-stranded RNA (dsRNA) fractions from a single soil lysate. We validated the protocol using sterilized soil co-spiked with a ZymoBIOMICS microbial community standard and a synthetic viral dsRNA fragment. Sequencing confirmed that the DNA fraction accurately recovered the theoretical bacterial (16S rRNA gene) and fungal (ITS) community profiles, while the dsRNA fraction demonstrated highly specific recovery of the target viral signature. The protocol was then successfully applied to characterize the complex native communities in environmental soil samples. The SS-VIME protocol provides a streamlined approach for isolating high-quality nucleic acids suitable for downstream applications. By using dsRNA as a proxy for viral activity and eliminating subsample bias, this method provides a robust, accessible, and unified platform to investigate virus-host dynamics <i>in situ</i>, paving the way for a more holistic understanding of the soil microbiome.IMPORTANCEThe study of soil microbes and their viruses, which are central to ecosystem health, is fundamentally limited by technical barriers. Separate extraction workflows for each group introduce sampling bias, obscuring the true ecological relationships within soil's spatially complex micro-environments. Our single-source virome-microbiome extraction (SS-VIME) protocol directly overcomes this by efficiently recovering both microbial DNA and viral double-stranded RNA (dsRNA) from one sample. This unified approach is not only cost-effective but, by using dsRNA as a signature of viral activity, captures a more accurate and representative profile of the soil active virome. SS-VIME provides the foundation for robustly investigating how viruses modulate soil health, carbon cycling, and agricultural productivity, moving the field from correlational studies toward a direct, integrated view of the soil ecosystem.