Performance Comparison of Probe-Capture Enrichment Sequencing of Viruses in Wastewater Influent and Settled Solids from California’s Central Valley
Publication: Lavon et al., bioRxiv (2026), preprint posted October 9, 2026
Overview
This study evaluated the performance of Nanotrap® Magnetic Virus Particles for concentrating viruses from wastewater influent prior to probe-capture enrichment sequencing, compared with conventional settled-solids processing. Researchers analyzed nine wastewater samples collected from three treatment facilities in California's Central Valley, processing matched samples using either Nanotrap particle concentration of whole influent or settling and dewatering of solids. Extracted DNA and RNA were sequenced using Illumina's Viral Surveillance Panels (VSP v1 and v2), targeting 66 and 242 viral genomes, respectively. The objective was to determine how sample preparation methods influence viral diversity, pathogen detection, and sequencing performance in wastewater-based epidemiology.
Key Findings
Nanotrap-enriched influent enabled substantially broader viral detection
The Nanotrap workflow identified a median of 358 viral species per sample versus 184 in settled solids using the VSP v2 sequencing configuration.
With VSP v1, Nanotrap-processed influent yielded 90 viral species versus 25 in settled solids, representing a 3.6-fold difference.
Rarefaction analysis indicated that influent generally supported greater viral species diversity, particularly at higher sequencing depths.
The influent libraries also received more sequencing reads, contributing to the observed differences in species detection.
Nanotrap concentration supported detection of public health-relevant viruses
Using VSP v2, 46 panel-associated viral species were detected in Nanotrap-processed influent compared with 36 in settled solids across the study.
With VSP v1, the corresponding totals were 26 and 23 species.
Detected viruses included human astrovirus, norovirus, adenoviruses, polyomaviruses, and sapoviruses, demonstrating compatibility with broad pathogen surveillance.
Probe-capture enrichment reduced differences in targeted viral detection between sample types compared with overall viral species detection.
Influent processing favored RNA virus representation
Nanotrap-processed influent exhibited a higher relative abundance of RNA viruses, whereas settled solids contained proportionally more DNA viruses.
Among 27 RNA virus targets detected using VSP v2, 22 were identified in influent compared with 13 in solids.
For DNA viruses, 19 were detected in influent compared with 15 in solids.
These results suggest that Nanotrap-based influent processing may be particularly valuable for surveillance programs targeting RNA viruses, although additional validation is required.
Nanotrap workflow supported strong viral genome coverage
For most endogenous viruses examined, influent samples demonstrated greater average genome coverage depth than settled solids, with human mastadenovirus F as an exception.
The bovine coronavirus process control also exhibited substantially greater sequencing coverage in influent than solids, although different spike concentrations and processing conditions prevent direct recovery comparisons.
The workflow used 75 µL Nanotrap Magnetic Virus Particles and 50 µL Enhancement Reagent 1 per approximately 5 mL wastewater sample, followed by automated KingFisher™ Apex processing and MagMAX nucleic acid extraction.
Implications for Nanotrap Technology
This study provides independent evidence supporting Nanotrap Magnetic Virus Particles as an effective sample preparation technology for wastewater viral metagenomics and targeted sequencing. The Nanotrap-based influent workflow enabled broader viral species detection, greater representation of RNA viruses, and generally higher genome coverage than the settled-solids workflow under the tested conditions.
Importantly, the study expands the demonstrated applications of Nanotrap technology beyond targeted PCR-based wastewater surveillance to broad viral discovery and hybridization probe-capture sequencing. Compatibility with Illumina's Viral Surveillance Panels and automated KingFisher processing supports potential integration into scalable, multi-pathogen wastewater surveillance programs.
Conclusion
The authors conclude that both wastewater influent and settled solids are compatible with probe-capture enrichment sequencing, but sample preparation significantly influences viral diversity and relative abundance. Nanotrap-processed influent yielded nearly twice as many viral species as settled solids with VSP v2 (358 versus 184) and detected more public health-relevant viral targets.
The findings support Nanotrap Magnetic Virus Particles as a promising technology for broad-spectrum wastewater virome analysis, particularly for surveillance applications requiring simultaneous detection of diverse RNA and DNA viruses. Further controlled comparisons using matched sequencing depths, standardized inputs, and quantitative recovery measurements would help establish the specific contribution of Nanotrap enrichment to improved viral detection.
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