{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,26]],"date-time":"2026-05-26T05:04:26Z","timestamp":1779771866834,"version":"3.53.1"},"reference-count":15,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,5,20]],"date-time":"2026-05-20T00:00:00Z","timestamp":1779235200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Swine Health information Center","award":["24-007"],"award-info":[{"award-number":["24-007"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>Respiratory disease remains one of the mostly costly challenges in the U.S. swine industry and is frequently associated with polymicrobial infections. Routine qPCR assays are highly sensitive but are limited in multiplexing capacity and generally do not provide sequencing information for pathogen characterization. We hypothesized that a target next-generation sequencing (tNGS) panel could provide the broad, simultaneous detection of swine respiratory pathogens while preserving clinically relevant sensitivity. A multiplex Ion Torrent tNGS panel was developed and analytically validated using 20 serially diluted qPCR-positive clinical samples and synthetic gBlock controls, followed by diagnostic validation with 25 qPCR positive and 25 qPCR negative respiratory samples. Most targets were detected across clinically relevant pathogens concentrations. Actinobacillus suis primers showed nonspecific amplification, streptococcus suis serotyping was not consistently achievable in clinical samples, and porcine reproductive and respiratory syndrome virus typing was limited to distinguishing North American and European genotypes. Diagnostic agreement with routine qPCR was high (Cohen\u2019s \u03ba = 0.84), although sensitivity decreased for low-abundance targets. The assay detected mixed infections and additional organisms outside routine qPCR panels. These findings support tNGS as a complementary diagnostic and surveillance tool for swine respiratory disease.<\/jats:p>","DOI":"10.3390\/microorganisms14051159","type":"journal-article","created":{"date-parts":[[2026,5,20]],"date-time":"2026-05-20T14:12:54Z","timestamp":1779286374000},"page":"1159","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Analytic and Diagnostic Validation of a Targeted Next-Generation Sequencing Panel for Common and Emerging Swine Respiratory Pathogens"],"prefix":"10.3390","volume":"14","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":[{"vocabulary":"crossref","role":"author"}]},{"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":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"80","DOI":"10.1186\/s13567-020-00807-8","article-title":"Coinfections and their molecular consequences in the porcine respiratory tract","volume":"51","author":"Saade","year":"2020","journal-title":"Vet. Res."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Sarli, G., D\u2019Annunzio, G., Gobbo, F., Benazzi, C., and Ostanello, F. (2021). The Role of Pathology in the Diagnosis of Swine Respiratory Disease. Vet. Sci., 8.","DOI":"10.3390\/vetsci8110256"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"102936","DOI":"10.1016\/j.jiph.2025.102936","article-title":"Targeted next-generation sequencing (tNGS): An upcoming application for pathogen identification in clinical diagnosis","volume":"18","author":"Liang","year":"2025","journal-title":"J. Infect. Public Health"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Yang, F., Jiang, L., Cao, Q., Yi, M., and Zhao, Q. (2025). Evaluation of targeted next-generation sequencing for microbiological diagnosis of acute lower respiratory infection. Front. Microbiol., 16.","DOI":"10.3389\/fmicb.2025.1615965"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1186\/s12967-025-06342-4","article-title":"Integrating DNA and RNA sequencing for enhanced pathogen detection in respiratory infections","volume":"23","author":"Gu","year":"2025","journal-title":"J. Transl. Med."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"319","DOI":"10.1146\/annurev-pathmechdis-012418-012751","article-title":"Clinical Metagenomic Next-Generation Sequencing for Pathogen Detection","volume":"14","author":"Gu","year":"2019","journal-title":"Annu. Rev. Pathol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"341","DOI":"10.1038\/s41576-019-0113-7","article-title":"Clinical metagenomics","volume":"20","author":"Chiu","year":"2019","journal-title":"Nat. Rev. Genet."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"e00399-18","DOI":"10.1128\/JCM.00399-18","article-title":"Evaluation of Targeted Next-Generation Sequencing for Detection of Bovine Pathogens in Clinical Samples","volume":"56","author":"Anis","year":"2018","journal-title":"J. Clin. Microbiol."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Neujahr, A.C., Loy, D.S., Loy, J.D., Brodersen, B.W., and Fernando, S.C. (2024). 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Sci., 12.","DOI":"10.3389\/fvets.2025.1726749"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"3788","DOI":"10.14202\/vetworld.2025.3788-3805","article-title":"Advances and emerging technologies in the diagnosis of viral infections in pigs: Progress, challenges, and One Health perspectives","volume":"18","author":"Nazerke","year":"2025","journal-title":"Vet. World"}],"container-title":["Microorganisms"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2607\/14\/5\/1159\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,26]],"date-time":"2026-05-26T04:30:06Z","timestamp":1779769806000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2607\/14\/5\/1159"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,20]]},"references-count":15,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2026,5]]}},"alternative-id":["microorganisms14051159"],"URL":"https:\/\/doi.org\/10.3390\/microorganisms14051159","relation":{},"ISSN":["2076-2607"],"issn-type":[{"value":"2076-2607","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,5,20]]}}}