Spatial Total RNA Sequencing of Formalin‐Fixed Paraffin‐Embedded Tissue by spRandom‐seq

The molecular pathogenesis of infectious diseases and cancer is orchestrated by nanoscale of host and microbial RNA transcripts within the tissue microenvironment. Nevertheless, spatially resolving the comprehensive transcriptional landscape within complex clinical tissues, like formalin-fixed paraffin-embedded (FFPE) specimens, still poses a formidable challenge. Here, we present spRandom-seq, a random primer-based spatial total RNA sequencing technology designed to spatially resolve complete transcriptomes from host, bacteria, and even nanoscale viruses in FFPE tissues. Capitalizing on the random primer design, our technology not only facilitated the discovery of specific lncRNAs and alternative splicing events in mouse brain and olfactory bulb, but also delineated pronounced spatial heterogeneity in clinical FFPE sections-across distinct tumor regions in breast cancer and microbial infection sites in Klebsiella pneumoniae-infected tissues. Importantly, integrated analysis of host and viral RNAs in FFPE samples from hepatitis B virus (HBV)‑positive hepatocellular carcinoma (HCC) demonstrated that complement and coagulation pathways were specifically activated across expansive HBV‑infected tumor areas, which also exhibited an increased burden of copy number variations (CNVs). Owing to its compatibility with existing spatial transcriptomics platforms and minimal operational complexity, spRandom-seq represents a practical and scalable approach for clinical pathology applications and infection diagnostics.