Library preparation strategy critically impacts RNA virus sensitivity in clinical metagenomics

Clinical metagenomics uses sequencing for culture-independent identification of pathogens directly from clinical specimens. While a number of protocols claim to be pathogen agnostic, sensitivity for RNA viruses is likely lower than for bacteria or fungi, as it requires additional processing steps including conversion to cDNA. Sequence-independent, single-primer amplification (SISPA) was first described in 1991, yet how it preferentially enriches viral molecules has never been described. Here we propose that single-primer amplification exploits the PCR suppression effect, which selectively amplifies longer viral molecules over shorter host-derived cDNA fragments on the basis of size. This model predicts that any upstream processing step that disrupts fragment length will prevent this enrichment occurring. To test this, we systematically compared two adapter introduction strategies - during cDNA synthesis and via tagmentation - followed by single primer amplification, using the ZeptoMetrix Respiratory Panel 2.1 containing 16 RNA and 3 DNA virus strains. SISPA-based approaches recovered all of the viral genomes in the control, whereas using tagmentation to amplify cDNA recovered none. We then spiked the controls into extracted clinical samples and found that SISPA-based methods performed best in all background settings, however in high-background settings no viral genomes were recovered by any approach. Finally, using a modified SMART-9N protocol, we demonstrated that single-primer PCR is critical to overall performance, indicating that direct tagmentation of cDNA and dual-primer PCR should be avoided in protocols for clinical metagenomics where high sensitivity for RNA viruses is critical. These findings demonstrate that library preparation strategy fundamentally determines RNA virus sensitivity and offer mechanistic insights for protocol optimisation with direct relevance to clinical metagenomics.