Abstract Rationale Accurate characterization of the respiratory virome is essential for understanding the contribution of viruses to respiratory infections. However, sputum remains a technically challenging specimen due to its complex matrix, low viral biomass, and the predominance of host and bacterial nucleic acids. To overcome these challenges, we aimed to optimize sputum preprocessing and viral enrichment workflows to improve viral recovery and minimize background contamination in metagenomic virome profiling. Methods Sputum samples collected from patients with Influenza A infection were subjected to a factorial experimental design combining nuclease digestion (N), 0.45 μm membrane filtration (F), and physical enrichment by ultrafiltration (UF) or ultracentrifugation (UC). Metagenomic sequencing was used to quantify viral read proportion, viral richness, and the relative abundance of bacterial and host nucleic acids under each condition. Results In untreated sputum, only 4.3% of sequencing reads were nonhost, among which eukaryotic reads represented 82.6%, bacterial 12.7%, and viral 0.82%. Nuclease digestion was the primary determinant of viral signal enhancement, leading to a 36fold increase in viral reads and a 1.77fold increase in viral richness compared with untreated controls. Combining nuclease treatment with filtration and ultrafiltration or ultracentrifugation further boosted viral read proportions (10 to 417fold) and richness (1.53 to 1.83fold). Physical enrichment alone yielded limited gains and often coconcentrated host and bacterial materials, with UC showing greater dilution of viral signals than UF. Incorporating 0.45 μm filtration prior to UF markedly reduced bacterial sequences (0.21fold) while maintaining the highest viral recovery (17.7%) and diversity (1.83-fold). Notably, nuclease treatment alone emerged as an efficient, lowcost, and scalable strategy for routine virome analyses. Conclusions Nuclease digestion is the key driver of viral signal recovery, while filtration substantially improves bacterial depletion. The F + UF +N workflow achieves optimal enrichment performance, whereas nuclease treatment alone provides a practical, resourceefficient solution for largescale respiratory virome investigations. This abstract is funded by: Natural Science Foundation of China (82341113)
Yao et al. (Fri,) studied this question.