Library strategies differentially shape microbial, functional, and host signals in clinical metagenomic sequencing.

RNA-based library preparation provides the most comprehensive recovery of the clinical infectome, especially for RNA viruses, cellular pathogens, and host immune signals.

• Why it matters: Understanding how library strategies influence the signals recovered from clinical samples is crucial for accurate infectious disease diagnostics and research, yet this remains poorly defined.
• What they did: The study performed a within-sample comparison of three mNGS library strategies—DNA-based, RNA-based, and total nucleic acid-based—across diverse clinical specimens and sample types.
• The result: Findings indicate that library strategies are not interchangeable; RNA-based sequencing excels in detecting RNA viruses and immune signatures, DNA-based favors DNA viruses and host genomes, and total nucleic acid approaches serve as intermediates, guiding strategy selection for specific diagnostic goals.