{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,24]],"date-time":"2025-12-24T11:39:44Z","timestamp":1766576384781,"version":"3.48.0"},"reference-count":30,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2025,12,24]],"date-time":"2025-12-24T00:00:00Z","timestamp":1766534400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"American Rescue Plan Act through USDA APHIS","award":["AP23OA000000C008"],"award-info":[{"award-number":["AP23OA000000C008"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>Affordable, flexible surveillance tools are needed to detect SARS-CoV-2 and other pathogens in wildlife. Standard nucleic acid amplification tests (NAATs) are reliable but restricted to predefined targets, limiting their ability to detect co-infections or emerging pathogens. To address this, we adapted a targeted next-generation sequencing (tNGS) panel for mesocarnivores to the Oxford Nanopore Technologies (ONT) MinION platform and combined it with a SARS-CoV-2 whole-genome sequencing assay. Merging both assays before library preparation enables simultaneous SARS-CoV-2 detection, variant identification, and broader pathogen screening. The MinION platform also improves turnaround time because sequencing can begin immediately on small numbers of samples, reducing costs in low-volume workflows. We converted our validated carnivore tNGS panel from the Ion Torrent system to MinION, optimizing amplification conditions, primer pools, and barcoding for multiplexing. Analytical sensitivity was measured using contrived wildlife samples spiked with serial dilutions of SARS-CoV-2 and tested in parallel with a commercial NAAT. Diagnostic sensitivity was assessed using contrived positives, and specificity was evaluated using NAAT-negative wildlife samples and in silico analyses. All 161 wildlife samples were NAAT-negative. MinION tNGS detected SARS-CoV-2 down to Ct 34 and produced \u2265 99% genome coverage for Ct \u2264 24 while simultaneously identifying additional pathogens. Diagnostic sensitivity and specificity were 96.7% and 100%. This workflow offers a low-cost, scalable approach for comprehensive wildlife pathogen surveillance.<\/jats:p>","DOI":"10.3390\/pathogens15010023","type":"journal-article","created":{"date-parts":[[2025,12,24]],"date-time":"2025-12-24T11:22:14Z","timestamp":1766575334000},"page":"23","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["MinION Adapted tNGS Panel for Carnivore Pathogens Including SARS-CoV-2"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4726-8584","authenticated-orcid":false,"given":"Nelly O.","family":"Elshafie","sequence":"first","affiliation":[{"name":"Willie M. Reed Animal Disease Diagnostic Laboratory, Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0391-0064","authenticated-orcid":false,"given":"Jobin J.","family":"Kattoor","sequence":"additional","affiliation":[{"name":"Department of Infectious Diseases and Microbiology, School of Public Health, University of Pittsburgh, Pittsburgh, PA 15213, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Janetta","family":"Kelly","sequence":"additional","affiliation":[{"name":"Indiana Department of Natural Resources, Division of Fish and Wildlife, Bloomington, IN 47401, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4846-0439","authenticated-orcid":false,"given":"Rebecca P.","family":"Wilkes","sequence":"additional","affiliation":[{"name":"Willie M. Reed Animal Disease Diagnostic Laboratory, Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907, USA"},{"name":"Veterinary Diagnostic Laboratory, University of Kentucky, Lexington, KY 40511, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2000045","DOI":"10.2807\/1560-7917.ES.2020.25.3.2000045","article-title":"Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR","volume":"25","author":"Corman","year":"2020","journal-title":"Eurosurveillance"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Owusu, H., and Sanad, Y.M. (2025). Comprehensive Insights into Highly Pathogenic Avian Influenza H5N1 in Dairy Cattle: Transmission Dynamics, Milk-Borne Risks, Public Health Implications, Biosecurity Recommendations, and One Health Strategies for Outbreak Control. Pathogens, 14.","DOI":"10.3390\/pathogens14030278"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"263","DOI":"10.1038\/s43856-024-00691-9","article-title":"A unified framework for diagnostic test development and evaluation during outbreaks of emerging infections","volume":"4","author":"Chaturvedi","year":"2024","journal-title":"Commun. Med."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"172","DOI":"10.1126\/science.abe5901","article-title":"Transmission of SARS-CoV-2 on mink farms between humans and mink and back to humans","volume":"371","author":"Sikkema","year":"2021","journal-title":"Science"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2488","DOI":"10.1038\/s41467-023-38202-4","article-title":"Virus diversity, wildlife-domestic animal circulation and potential zoonotic viruses of small mammals, pangolins and zoo animals","volume":"14","author":"Cui","year":"2023","journal-title":"Nat. Commun."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"2333","DOI":"10.1126\/science.286.5448.2333","article-title":"Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States","volume":"286","author":"Lanciotti","year":"1999","journal-title":"Science"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Childs, J.E., Mackenzie, J.S., and Richt, J.A. (2007). Impediments to Wildlife Disease Surveillance, Research, and Diagnostics. Wildlife and Emerging Zoonotic Diseases: The Biology, Circumstances and Consequences of Cross-Species Transmission, Springer.","DOI":"10.1007\/978-3-540-70962-6"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"e0251321","DOI":"10.1128\/spectrum.02513-21","article-title":"Emergency SARS-CoV-2 Variants of Concern: Novel Multiplex Real-Time RT-PCR Assay for Rapid Detection and Surveillance","volume":"10","author":"Chung","year":"2022","journal-title":"Microbiol. Spectr."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"6210","DOI":"10.1038\/s41467-024-49891-w","article-title":"Widespread exposure to SARS-CoV-2 in wildlife communities","volume":"15","author":"Goldberg","year":"2024","journal-title":"Nat. Commun."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1038\/nrg.2017.88","article-title":"Towards a genomics-informed, real-time, global pathogen surveillance system","volume":"19","author":"Gardy","year":"2018","journal-title":"Nat. Rev. Genet."},{"key":"ref_11","first-page":"10","article-title":"Acute Vector-Borne Viral Infection: Zika and MinION Surveillance","volume":"7","author":"Giovanetti","year":"2019","journal-title":"Microbiol. Spectr."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Goldstein, S., Beka, L., Graf, J., and Klassen, J.L. (2019). Evaluation of strategies for the assembly of diverse bacterial genomes using MinION long-read sequencing. BMC Genom., 20.","DOI":"10.1186\/s12864-018-5381-7"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"gkae1202","DOI":"10.1093\/nar\/gkae1202","article-title":"Enhanced detection and genotyping of disease-associated tandem repeats using HMMSTR and targeted long-read sequencing","volume":"53","author":"Mumm","year":"2025","journal-title":"Nucleic Acids Res."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"114180","DOI":"10.1016\/j.jviromet.2021.114180","article-title":"MinION nanopore sequencing and assembly of a complete human papillomavirus genome","volume":"294","author":"Brancaccio","year":"2021","journal-title":"J. Virol. Methods"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Park, K., Noh, J., Kim, K., Kim, J., Cho, H.-K., Kim, S.-G., Yang, E., Kim, W.-K., and Song, J.-W. (2023). A Development of Rapid Whole-Genome Sequencing of Seoul orthohantavirus Using a Portable One-Step Amplicon-Based High Accuracy Nanopore System. Viruses, 15.","DOI":"10.3390\/v15071542"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Shaw, A.E., Lebani, K., Gordon, L.G., Ihearahu, U.E., Wadsworth, J., Hicks, H.M., Polo, N., Freimanis, G., Muhanguzi, D., and Tennakoon, C. (2025). Universal amplification and sequencing of foot-and-mouth disease virus complete genomes using nanopore technology. BMC Genom., 26.","DOI":"10.1186\/s12864-025-11938-7"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Tomb\u00e1cz, D., Moldov\u00e1n, N., Bal\u00e1zs, Z., Guly\u00e1s, G., Csabai, Z., Boldogk\u0151i, M., Snyder, M., and Boldogk\u0151i, Z. (2019). Multiple Long-Read Sequencing Survey of Herpes Simplex Virus Dynamic Transcriptome. Front. Genet., 10.","DOI":"10.3389\/fgene.2019.00834"},{"key":"ref_18","first-page":"104483","article-title":"Extraction-free direct PCR from dried serum spots permits HBV genotyping and RAS identification by Sanger and minION sequencing","volume":"129","author":"Astbury","year":"2019","journal-title":"bioRxiv"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Chan-Herur, V., Catoe, D., Duong, D., Wu, Y.C., Boehm, A.B., White, B.J., and Wolfe, M.K. (2023). SARS-CoV-2 Whole Genome Amplicon Sequencing from Wastewater Solids or Liquid Wastewater. protocols.io.","DOI":"10.17504\/protocols.io.yxmvm323bl3p\/v2"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Kattoor, J.J., Mlalazi-Oyinloye, M., Nemser, S.M., and Wilkes, R.P. (2024). Development of a Targeted NGS Assay for the Detection of Respiratory Pathogens including SARS-CoV-2 in Felines. Pathogens, 13.","DOI":"10.3390\/pathogens13040335"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Kattoor, J.J., Nikolai, E., Qurollo, B., and Wilkes, R.P. (2022). Targeted Next-Generation Sequencing for Comprehensive Testing for Selected Vector-Borne Pathogens in Canines. Pathogens, 11.","DOI":"10.3390\/pathogens11090964"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Kattoor, J.J., Anis, E., Elshafie, N.O., and Wilkes, R.P. (2025). A multiplex targeted NGS panel for identifying pathogens in canine neurological and reproductive diseases. Front. Cell. Infect. Microbiol., 15.","DOI":"10.3389\/fcimb.2025.1591145"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Elshafie, N. (2025, August 29). Oxford Nanopore Targeted Sequencing Assay for Flongle Flow Cells. Available online: https:\/\/www.protocols.io\/view\/oxford-nanopore-targeted-sequencing-assay-for-flon-g4fmbytk7.","DOI":"10.17504\/protocols.io.3byl46ewrgo5\/v1"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Reichard, M.V., Sanders, T.L., Weerarathne, P., Meinkoth, J.H., Miller, C.A., Scimeca, R.C., and Almaz\u00e1n, C. (2021). Cytauxzoonosis in North America. Pathogens, 10.","DOI":"10.3390\/pathogens10091170"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1016\/j.vetpar.2018.01.022","article-title":"Chronic Cytauxzoon felis infections in wild-caught bobcats (Lynx rufus)","volume":"252","author":"Zieman","year":"2018","journal-title":"Vet. Parasitol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1174","DOI":"10.1186\/s13071-022-05271-1","article-title":"Molecular epidemiological study on ticks and tick-borne protozoan parasites (Apicomplexa: Cytauxzoon and Hepatozoon spp.) from wild cats (Felis silvestris), Mustelidae and red squirrels (Sciurus vulgaris) in central Europe, Hungary","volume":"15","author":"Hornok","year":"2022","journal-title":"Parasites Vectors"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Meli, M.L., Meili, T., Pineroli, B., B\u00f6nzli, E., Eichenberger, R.M., Willi, B., and Hofmann-Lehmann, R. (2025). First detection of Cytauxzoon spp. DNA in questing Ixodes ricinus nymphs. bioRxiv, 2025.08.04.667914.","DOI":"10.1101\/2025.08.04.667914"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"366","DOI":"10.7589\/0090-3558-42.2.366","article-title":"Molecular Detection and Characterization of Cytauxzoon felis and a Babesia Species in Cougars from Florida","volume":"42","author":"Yabsley","year":"2006","journal-title":"J. Wildl. Dis."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"107682","DOI":"10.1016\/j.micpath.2025.107682","article-title":"First report of \u2018Candidatus mycoplasma spp.\u2019 in mustelids from two different Brazilian regions with description of three putative novel species","volume":"205","author":"Berger","year":"2025","journal-title":"Microb. Pathog."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Portik, D.M., Brown, C.T., and Pierce-Ward, N.T. (2022). Evaluation of taxonomic classification and profiling methods for long-read shotgun metagenomic sequencing datasets. BMC Bioinform., 23.","DOI":"10.1186\/s12859-022-05103-0"}],"container-title":["Pathogens"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-0817\/15\/1\/23\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,24]],"date-time":"2025-12-24T11:37:06Z","timestamp":1766576226000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-0817\/15\/1\/23"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,24]]},"references-count":30,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2026,1]]}},"alternative-id":["pathogens15010023"],"URL":"https:\/\/doi.org\/10.3390\/pathogens15010023","relation":{},"ISSN":["2076-0817"],"issn-type":[{"value":"2076-0817","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,12,24]]}}}