Evaluation of a Probe-Based Enrichment Protocol for Nanopore Sequencing of Zoonotic Viruses

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Hawes, Kailin
Greene, Benjamin
Weishampel, Zachary A
Beare, Paul A
van Tol, Sarah
Schaughency, Paul
Kuhn, Skyler
Peel, Alison J
Munster, Vincent J
Yinda, Claude Kwe
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2025
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The detection of high-consequence viral pathogens is essential for spillover prevention and reduction in transmission but is limited by the low sensitivity of next-generation sequencing technology. Low-titer field samples from a variety of hosts are primarily composed of non-viral genomic material, reducing the probability of obtaining usable sequence data. Targeted enrichment, such as VirCapSeq-VERT, removes background genomic material to improve virus detection but is mainly used for sequencing clinical samples. We customized the VirCapSeq-VERT probe system to aid in the detection of zoonotic viruses of interest and adapted it for use on the Oxford Nanopore sequencing platform. We validated the method on a variety of samples, including a mock virome consisting of seven RNA viruses, samples from an animal laboratory study, and a set of animal field samples. We also developed Nanite, a lightweight bioinformatics pipeline, to perform bioinformatic analyses. Results indicated that the optimized enrichment protocol improved sequencing by enhancing the detection of viruses, increasing read lengths, and, in some cases, improving genomic coverage. Most importantly, the sequencing of zoonotic viruses was improved in field samples with low titers, suggesting that this protocol is a useful tool for increasing the efficacy of Oxford Nanopore sequencing for field-oriented applications.

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Viruses

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17

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11

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© 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

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Virology

Microbiology

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Hawes, K; Greene, B; Weishampel, ZA; Beare, PA; van Tol, S; Schaughency, P; Kuhn, S; Peel, AJ; Munster, VJ; Yinda, CK, Evaluation of a Probe-Based Enrichment Protocol for Nanopore Sequencing of Zoonotic Viruses, Viruses, 17 (11), pp. 1465

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