Arinobu Tojo

Probe-based capture represents a highly sensitive and cost-effective approach for overcoming host background and enriching viruses in metagenomic NGS (mNGS) libraries. Using clinical specimens collected globally from patients with fever or respiratory illness, we generated mNGS libraries by random priming and Nextera XT tagmentation, followed by target enrichment (teNGS) with Comprehensive Viral Research Panel (CVRP) probes. Capture pool sizes and total reads were optimized, and libraries were initially sequenced separately. Using only 3–4% of reads required for standard mNGS, teNGS achieved increased sensitivity, 100–10,000× increases in depth, and >50% genome coverage for pathogens with titers ≥ 1000 cp/mL. Application to >2000 clinical specimens from various matrices and to contrived samples containing viruses absent from the CVRP probe set enabled detection of diverse viral families and established a minimum 65% nucleotide identity for hybridization, respectively. To save time and resources, teNGS and mNGS libraries were then combined into one sequencing run: teamNGS. In addition to streamlining the workflow, teamNGS also improved genome recovery. Coupling methods maintain the sensitivity and coverage for viruses achieved by enrichment alone while also ensuring comprehensive recovery of non-viral microbes. teamNGS has the potential to improve patient management and lower the rates of unnecessary testing and antibiotic use.