Short- and long-read hybrid-capture viral metagenomics reach high sensitivity and specificity with data-driven cut-offs compared with routine diagnostics in CSF and BAL specimens

Hybrid-capture viral metagenomics detected 22 of 23 expected viral targets in CSF EQA samples (95.7% sensitivity) with 100% specificity on both AVITI and ONT platforms.

• Why it matters: Routine diagnostics can miss low-level or unexpected viruses, and cut-off rules for metagenomic pathogen calling have not been rigorously validated on real clinical specimens across sequencing platforms.
• What they did: Using the Twist Comprehensive Viral Research Panel, the workflow was benchmarked on 12 CSF EQA mock samples and 14 CSF plus 21 BAL clinical specimens with AVITI short-read and ONT long-read sequencing. Data-driven pathogen-calling cut-offs were set via Latin hypercube sampling across genome coverage, abundance, and bleed-ratio parameters, then validated on independent EQA datasets.
• The result: In clinical samples, AVITI confirmed 26 of 30 (86.7%) and ONT 25 of 30 (83.3%) routine viral detections while keeping 100% diagnostic specificity; sensitivity was 100% in CSF but lower in BAL, and all detections below Cq 32 were found by both platforms. Metagenomics also flagged a potentially clinically relevant pathogen not included in routine testing in two CSF samples.

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