{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,15]],"date-time":"2026-07-15T07:04:28Z","timestamp":1784099068505,"version":"3.55.0"},"reference-count":35,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,7,15]],"date-time":"2026-07-15T00:00:00Z","timestamp":1784073600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>In the post-pandemic era, co-circulation of multiple respiratory RNA viruses has increased the need for timely diagnosis and reliable recognition of mixed infections. Although reverse transcription quantitative polymerase chain reaction (RT-qPCR) remains the clinical standard for respiratory virus detection, its target-restricted design limits the detection of unexpected or coinfecting pathogens. Conventional metagenomic next-generation sequencing (mNGS) provides hypothesis-free pathogen detection, but routine clinical use is still limited by long turnaround times and complex library preparation. Therefore, a sequencing-based strategy that preserves broad, unbiased detection while offering a simplified workflow and clinically acceptable turnaround time is needed.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>We optimized and clinically validated CATCH, a rapid RNA\/DNA hybrid tagmentation-based mNGS workflow, for respiratory RNA virus detection. Analytical performance was assessed using standardized reference materials, including SARS-CoV-2 and influenza A virus, with evaluations of sensitivity, reproducibility, short-term stability, and host-background interference. Clinical validation was performed in retrospective and prospective respiratory infection cohorts, and assay performance was benchmarked against RT-qPCR and multiplex PCR. The same sequencing data were further examined for semiquantitative viral assessment, coinfection detection, and exploratory respiratory microbial profiling.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>The optimized CATCH workflow shortened library preparation to approximately 3\u202fh, with about 35\u202fmin of hands-on time, enabling same-day sequencing-based diagnostics. Broad detection was achieved across seven clinically relevant respiratory RNA viruses. Sequencing-derived viral abundance showed a significant overall correlation with viral input concentration, supporting semiquantitative interpretation, although virus- and subtype-specific variability highlighted biological constraints on absolute quantification. Using SARS-CoV-2 and influenza A virus as representative targets, CATCH achieved clinically actionable limits of detection with high reproducibility and stability. In clinical cohorts, CATCH showed high concordance with routine molecular assays and identified mixed respiratory infections missed by targeted testing. Exploratory analyses also demonstrated the feasibility of respiratory microbial community profiling from the same sequencing dataset.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>CATCH is a rapid and clinically deployable RNA virus mNGS workflow that helps bridge targeted molecular diagnostics and conventional metagenomic sequencing. By combining broad pathogen detection, coinfection identification, and semiquantitative assessment within a streamlined workflow, CATCH provides a practical framework for comprehensive respiratory RNA virus diagnosis and syndromic surveillance.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.3389\/fmicb.2026.1849991","type":"journal-article","created":{"date-parts":[[2026,7,15]],"date-time":"2026-07-15T06:05:35Z","timestamp":1784095535000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Rapid clinical validation of an RNA\/DNA hybrid tagmentation-based metagenomic workflow for respiratory RNA virus detection"],"prefix":"10.3389","volume":"17","author":[{"given":"Xia","family":"Ma","sequence":"first","affiliation":[{"name":"First Hospital of Shanxi Medical University","place":["Taiyuan, Shanxi, China"]},{"name":"Beijing Shijingshan Hospital","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shan","family":"Guo","sequence":"additional","affiliation":[{"name":"Beijing Institute of Hepatology, Beijing YouAn Hospital, Capital Medical University","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yangyang","family":"Feng","sequence":"additional","affiliation":[{"name":"Beijing Institute of Hepatology, Beijing YouAn Hospital, Capital Medical University","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Murong","family":"Su","sequence":"additional","affiliation":[{"name":"Department of Public Health Sciences, University of California, Irvine","place":["Irvine, CA, United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Feili","family":"Wei","sequence":"additional","affiliation":[{"name":"Beijing Institute of Hepatology, Beijing YouAn Hospital, Capital Medical University","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xuejun","family":"Liu","sequence":"additional","affiliation":[{"name":"First Hospital of Shanxi Medical University","place":["Taiyuan, Shanxi, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,7,15]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"101174","DOI":"10.1016\/j.lanmic.2025.101174","article-title":"Rapid pan-microbial metagenomics for pathogen detection and personalised therapy in the intensive care unit: a single-Centre prospective observational study","volume":"6","author":"Alcolea-Medina","year":"2025","journal-title":"Lancet Microbe"},{"key":"ref2","doi-asserted-by":"publisher","first-page":"596","DOI":"10.1128\/JVI.02005-07","article-title":"The influenza virus resource at the National Center for biotechnology information","volume":"82","author":"Bao","year":"2008","journal-title":"J. Virol."},{"key":"ref3","doi-asserted-by":"publisher","first-page":"421","DOI":"10.1186\/1471-2105-10-421","article-title":"BLAST+: architecture and applications","volume":"10","author":"Camacho","year":"2009","journal-title":"BMC Bioinformatics"},{"key":"ref4","doi-asserted-by":"publisher","first-page":"239","DOI":"10.2471\/BLT.22.288220","article-title":"Global genomic surveillance strategy for pathogens with pandemic and epidemic potential 2022\u20132032","volume":"100","author":"Carter","year":"2022","journal-title":"Bull. World Health Organ."},{"key":"ref5","doi-asserted-by":"publisher","first-page":"213","DOI":"10.1186\/s40001-024-01806-7","article-title":"Assessment and clinical utility of metagenomic next-generation sequencing for suspected lower respiratory tract infections","volume":"29","author":"Chen","year":"2024","journal-title":"Eur. J. Med. Res."},{"key":"ref6","doi-asserted-by":"publisher","first-page":"i884","DOI":"10.1093\/bioinformatics\/bty560","article-title":"Fastp: an ultra-fast all-in-one FASTQ preprocessor","volume":"34","author":"Chen","year":"2018","journal-title":"Bioinformatics"},{"key":"ref7","doi-asserted-by":"publisher","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":"ref8","doi-asserted-by":"publisher","first-page":"46","DOI":"10.1186\/s40168-019-0658-x","article-title":"Sunbeam: an extensible pipeline for analyzing metagenomic sequencing experiments","volume":"7","author":"Clarke","year":"2019","journal-title":"Microbiome"},{"key":"ref9","volume-title":"Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures","year":"2012"},{"key":"ref10","doi-asserted-by":"publisher","first-page":"106501","DOI":"10.1016\/j.jinf.2025.106501","article-title":"Impact of co-infection with SARS-CoV-2 and other respiratory viruses on illness: pooled analyses of 11 COVID-19 cohorts","volume":"90","author":"de Hoog","year":"2025","journal-title":"J. Infect."},{"key":"ref11","doi-asserted-by":"publisher","first-page":"2886","DOI":"10.1073\/pnas.1919800117","article-title":"RNA sequencing by direct tagmentation of RNA\/DNA hybrids","volume":"117","author":"Di","year":"2020","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref12","doi-asserted-by":"publisher","first-page":"1570","DOI":"10.1038\/s41564-025-02035-2","article-title":"Guidelines for preventing and reporting contamination in low-biomass microbiome studies","volume":"10","author":"Fierer","year":"2025","journal-title":"Nat. Microbiol."},{"key":"ref13","doi-asserted-by":"publisher","first-page":"giaf004","DOI":"10.1093\/gigascience\/giaf004","article-title":"Benchmarking short-read metagenomics tools for removing host contamination","volume":"14","author":"Gao","year":"2025","journal-title":"Gigascience"},{"key":"ref14","doi-asserted-by":"publisher","first-page":"e01267-22","DOI":"10.1128\/jcm.01267-22","article-title":"Clinical metagenomics for infectious diseases: progress toward operational value","volume":"61","author":"Gaston","year":"2023","journal-title":"J. Clin. Microbiol."},{"key":"ref15","doi-asserted-by":"publisher","first-page":"e1245","DOI":"10.1093\/infdis\/jiae226","article-title":"Validation of an automated, end-to-end metagenomic sequencing assay for agnostic detection of respiratory viruses","volume":"230","author":"Gauthier","year":"2024","journal-title":"J. Infect. Dis."},{"key":"ref16","doi-asserted-by":"publisher","first-page":"108317","DOI":"10.1016\/j.ijid.2025.108317","article-title":"Associations between virus single infection or coinfection and respiratory symptoms in young children: a community-based cohort study","volume":"164","author":"Hauser-van Westrhenen","year":"2026","journal-title":"Int. J. Infect. Dis."},{"key":"ref17","doi-asserted-by":"publisher","first-page":"e02473-21","DOI":"10.1128\/spectrum.02473-21","article-title":"Improving suspected pulmonary infection diagnosis by bronchoalveolar lavage fluid metagenomic next-generation sequencing: a multicenter retrospective study","volume":"10","author":"Jin","year":"2022","journal-title":"Microbiol. Spectrum"},{"key":"ref18","doi-asserted-by":"publisher","first-page":"3211","DOI":"10.1093\/bioinformatics\/bts611","article-title":"SortMeRNA: fast and accurate filtering of ribosomal RNAs in metatranscriptomic data","volume":"28","author":"Kopylova","year":"2012","journal-title":"Bioinformatics"},{"key":"ref19","doi-asserted-by":"publisher","first-page":"648","DOI":"10.1111\/resp.12741","article-title":"Systematic review and meta-analysis of respiratory viral coinfections in children","volume":"21","author":"Lim","year":"2016","journal-title":"Respirology"},{"key":"ref20","doi-asserted-by":"publisher","first-page":"e54919","DOI":"10.7554\/eLife.54919","article-title":"Transposase-assisted tagmentation of RNA\/DNA hybrid duplexes","volume":"9","author":"Lu","year":"2020","journal-title":"eLife"},{"key":"ref21","doi-asserted-by":"publisher","first-page":"93","DOI":"10.1186\/s12915-024-01887-0","article-title":"Meta-analysis of the human upper respiratory tract microbiome reveals robust taxonomic associations with health and disease","volume":"22","author":"Quinn-Bohmann","year":"2024","journal-title":"BMC Biol."},{"key":"ref22","doi-asserted-by":"publisher","first-page":"87","DOI":"10.1186\/s12915-014-0087-z","article-title":"Reagent and laboratory contamination can critically impact sequence-based microbiome analyses","volume":"12","author":"Salter","year":"2014","journal-title":"BMC Biol."},{"key":"ref23","doi-asserted-by":"publisher","first-page":"776","DOI":"10.5858\/arpa.2016-0539-RA","article-title":"Validation of metagenomic next-generation sequencing tests for universal pathogen detection","volume":"141","author":"Schlaberg","year":"2017","journal-title":"Arch. Pathol. Lab Med."},{"key":"ref24","doi-asserted-by":"publisher","first-page":"9339","DOI":"10.1038\/s41467-024-53566-x","article-title":"Microbial dynamics and pulmonary immune responses in COVID-19 secondary bacterial pneumonia","volume":"15","author":"Spottiswoode","year":"2024","journal-title":"Nat. Commun."},{"key":"ref25","doi-asserted-by":"publisher","first-page":"9016","DOI":"10.1038\/s41467-024-51470-y","article-title":"Laboratory validation of a clinical metagenomic next-generation sequencing assay for respiratory virus detection and discovery","volume":"15","author":"Tan","year":"2024","journal-title":"Nat. Commun."},{"key":"ref26","doi-asserted-by":"publisher","first-page":"1531084","DOI":"10.3389\/fcimb.2025.1531084","article-title":"Unveiling the role of the upper respiratory tract microbiome in susceptibility and severity to COVID-19","volume":"15","author":"von Ameln Lovison","year":"2025","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"ref27","doi-asserted-by":"publisher","DOI":"10.1101\/2020.05.12.20099754","article-title":"Laboratory validation of an RNA\/DNA hybrid tagmentation based mNGS workflow on SARS-CoV-2 and other respiratory RNA viruses detection","author":"Wei","year":"2020","journal-title":"medRxiv"},{"key":"ref28","doi-asserted-by":"publisher","first-page":"257","DOI":"10.1186\/s13059-019-1891-0","article-title":"Improved metagenomic analysis with kraken 2","volume":"20","author":"Wood","year":"2019","journal-title":"Genome Biol."},{"key":"ref29","volume-title":"Implementing the Integrated Sentinel Surveillance of Influenza and Other Respiratory Viruses of Epidemic and Pandemic Potential by the GISRS","year":"2024"},{"key":"ref30","volume-title":"Expanding the Global Influenza Surveillance and Response System to Include Other Respiratory Viruses with Epidemic or Pandemic Potential","year":"2025"},{"key":"ref31","doi-asserted-by":"publisher","first-page":"e01488-22","DOI":"10.1128\/spectrum.01488-22","article-title":"Application of metagenomic next-generation sequencing (mNGS) using bronchoalveolar lavage fluid (BALF) in diagnosing pneumonia of children","volume":"10","author":"Yang","year":"2022","journal-title":"Microbiol. 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Med."},{"key":"ref35","doi-asserted-by":"publisher","first-page":"106659","DOI":"10.1016\/j.jinf.2025.106659","article-title":"A comparative study of DNA- and RNA-metagenomic next-generation sequencing for pathogen detection in lower respiratory tract infections","volume":"91","author":"Zhu","year":"2025","journal-title":"J. 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