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In recent years, metagenomic Next-Generation Sequencing (mNGS) has increasingly been used for an accurate assumption-free virological diagnosis. However, the systematic workflow evaluation on clinical respiratory samples and implementation of quality controls (QCs) is still lacking.

A total of 3 QCs were implemented and processed through the whole mNGS workflow: a no-template-control to evaluate contamination issues during the process; an internal and an external QC to check the integrity of the reagents, equipment, the presence of inhibitors, and to allow the validation of results for each sample. The workflow was then evaluated on 37 clinical respiratory samples from patients with acute respiratory infections previously tested for a broad panel of viruses using semi-quantitative real-time PCR assays (28 positive samples …