Respiratory viral co-infections among SARS-CoV-2 cases confirmed by virome capture sequencing

Respiratory viral co-infections among SARS-CoV-2 cases confirmed by virome capture sequencing | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Respiratory viral co-infections among SARS-CoV-2 cases confirmed by virome capture sequencing Ki Wook Kim, Ira W. Deveson, Chi Nam I. Pang, Malinna Yeang, Zin Naing, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-105996/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Feb, 2021 Read the published version in Scientific Reports → Version 1 posted You are reading this latest preprint version Abstract Accumulating evidence supports the high prevalence of co-infections among Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) patients, and their potential to worsen the clinical outcome of COVID-19. However, there are few data on Southern Hemisphere populations, and most studies to date have investigated a narrow spectrum of viruses using targeted qRT-PCR. Here we assessed respiratory viral co-infections among SARS-CoV-2 patients in Australia, through respiratory virome characterization. Nasopharyngeal swabs of 92 SARS-CoV-2-positive cases were sequenced using pan-viral hybrid-capture and the Twist Respiratory Virus Panel. In total, 8% of cases were co-infected, with rhinovirus (6%) or influenzavirus (2%). Twist capture also achieved near-complete sequencing (>90% coverage, >10-fold depth) of the SARS-CoV-2 genome in 95% of specimens with Ct<30. Our results highlight the importance of assessing all pathogens in symptomatic patients, and the dual-functionality of Twist hybrid-capture, for SARS-CoV-2 whole-genome sequencing without amplicon generation and the simultaneous identification of viral co-infections with ease. Virology Molecular Biology Bioinformatics Biochemical Research Methods SARS-CoV-2 COVID-19 co-infection hybrid-capture respiratory virome metagenomics next-generation sequencing rhinovirus influenzavirus Figures Figure 1 Figure 2 Figure 3 Figure 4 Full Text Supplementary Files SupplemenatryInformationFinalpreprint.pdf Supplementary figures and tables SupplementaryFile1.xlsx Supplementary File 1 SupplementaryTable2.xlsx Supplementary Table 2 SupplementaryTable6.xlsx Supplementary Table 6 Cite Share Download PDF Status: Published Journal Publication published 16 Feb, 2021 Read the published version in Scientific Reports → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-105996","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":4596971,"identity":"7dbe9dc0-d3d7-4fe3-a4c1-de982b09a0ed","order_by":0,"name":"Ki Wook Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYBACCSBmbDBgkIPymYnXYkyqFgaGxAaitUg28BgwzijYlr7hdvszCYYK68QG9jMGeLVIMwC1bDC4nbvhzhkzCYYz6YkNPDn4tcgx8G5gfADSciOH7QZj22GgC4nUkm5wI/3ZDcZ/QC38bwg5DKgF6LAEgxsJZjcYG4BaJAjYItnM/+HgDIPbhjNv5Jj/SDiWbtwm8awArxaJ422JD3v+3Jbnu5H+2OBDjbVsP3/yBrxaQBFxAM5JAGI2/OpHwSgYBaNgFBADADYjRjtYKxe3AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-9579-6408","institution":"School of Women’s and Children’s Health, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Ki","middleName":"Wook","lastName":"Kim","suffix":""},{"id":4596972,"identity":"96bfb3f7-5cd0-44cb-99f3-0d1bc310abf1","order_by":1,"name":"Ira W. 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VirCapSeq (n=92); and b. Twist Respiratory Virus Panel (n=83). Heatmap of viral reads in log scale and represented at the genus level. Sample IDs apply for both panels a and b, indicating overlapping samples sequenced by both approaches. Horizontal line separates respiratory viruses (above) from non-respiratory viruses (below). Bar charts indicate number of viruses detected per specimen. nCoV_neg_1 \u0026 2 are clinical control specimens from two individuals confirmed negative for SARS-CoV-2 by qRT-PCR.","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-105996/v1/31b87cad0996290cb45b7a78.png"},{"id":3663010,"identity":"04496ea8-e479-439d-b7c3-d09766249f8e","added_by":"auto","created_at":"2020-11-18 14:52:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":72901,"visible":true,"origin":"","legend":"Full genome coverage of co-infecting influenzavirus. Coverage plot of sequence reads generated by Twist capture sequencing of the SARS-CoV-2 case specimen nCoV_240, aligned to the influenzavirus A reference genome across eight different segments (S1-S8). Depth represented as X fold coverage. Single nucleotide polymorphisms (SNPs) detected at positions across the genome are indicated in red, greater intensity of red indicates higher % frequency.","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-105996/v1/b81252e23f9ba45763418b1c.png"},{"id":3663012,"identity":"6ff19fcb-dd40-4992-8f85-a19699a6a9ff","added_by":"auto","created_at":"2020-11-18 14:52:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":305174,"visible":true,"origin":"","legend":"Complete SARS-CoV-2 genome coverage by sequences generated using the Twist Respiratory Virus Panel. a. The number of sequence reads generated by VirCapSeq (blue) and Twist (red) hybrid-capture sequencing, aligned to SARS-CoV-2 in case samples (n=83) with varying viral load determined by the qRT-PCR cycle threshold (Ct) value. b. The fraction of SARS-CoV-2 genome covered at \u003e10X depth. Violin plot (left) shows the distribution of genome coverage in samples sequenced by VirCapSeq (blue) and Twist (red) capture with horizontal line indicating the median fraction of genome covered. Vertical dotted line indicates the Ct 30 border (right). c. Distribution of aligned sequence reads across the SARS-CoV-2 reference genome (MN908947.3) and depth of coverage at each position in the genome (50-bp windows) normalized to the average coverage across the whole genome for a given sample.","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-105996/v1/534257066455d68b1a3acf59.png"},{"id":3663014,"identity":"786be1ab-d4ff-450c-8064-d2e56e4534aa","added_by":"auto","created_at":"2020-11-18 14:52:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":174386,"visible":true,"origin":"","legend":"Confirmation of a 328 nt ORF8 deletion in the SARS-CoV-2. Genome browser view of Twist enriched Illumina (upper) and amplicon-based WGS ONT (lower) sequencing reads aligned across the SARS-CoV-2 genome of nCoV_200 case specimen, zoomed in at the site of 328 nt ORF8 deletion. ONT sequence alignment shows loss of coverage in the region targeted by the A7 amplicon primers, due to the deletion of a primer-binding site within ORF8. In contrast, Twist sequence reads align continuously across this region (Genes orf3a to orf7), providing greater breadth of coverage of the reference genome (MN908947.3).","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-105996/v1/26473e019bae08f2356093b2.png"},{"id":13556795,"identity":"8ad64360-02ba-4601-a5c9-60edaf235276","added_by":"auto","created_at":"2021-09-17 02:50:08","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1045396,"visible":true,"origin":"","legend":"","description":"","filename":"ManuscriptFinalpreprint.pdf","url":"https://assets-eu.researchsquare.com/files/rs-105996/v1_covered.pdf"},{"id":3663005,"identity":"1c0f73b8-cb4c-421a-a007-38cad0d1f785","added_by":"auto","created_at":"2020-11-18 14:52:45","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1387363,"visible":true,"origin":"","legend":"","description":"","filename":"ManuscriptFinalpreprint.pdf","url":"https://assets-eu.researchsquare.com/files/rs-105996/v1_stamped.pdf"},{"id":3663009,"identity":"60bf5350-48a8-44a6-8d79-fe2491a67cc8","added_by":"auto","created_at":"2020-11-18 14:52:47","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1460898,"visible":true,"origin":"","legend":"Supplementary figures and tables","description":"","filename":"SupplemenatryInformationFinalpreprint.pdf","url":"https://assets-eu.researchsquare.com/files/rs-105996/v1/c491e4824e774c19074fb8b4.pdf"},{"id":3663011,"identity":"0436376c-a4bd-4ccd-8678-fbe8e1155cb5","added_by":"auto","created_at":"2020-11-18 14:52:47","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":39552,"visible":true,"origin":"","legend":"Supplementary File 1","description":"","filename":"SupplementaryFile1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-105996/v1/b9db4317e535a2f17b74231e.xlsx"},{"id":3663013,"identity":"1657474b-b927-4fd0-bd01-263b85b93d05","added_by":"auto","created_at":"2020-11-18 14:52:48","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":15669,"visible":true,"origin":"","legend":"Supplementary Table 2","description":"","filename":"SupplementaryTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-105996/v1/8844e8263f3bde9d0b13d0a6.xlsx"},{"id":3663015,"identity":"6f3f8a11-90eb-4d2f-82f8-2a89a7afdf49","added_by":"auto","created_at":"2020-11-18 14:52:48","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":13476,"visible":true,"origin":"","legend":"Supplementary Table 6","description":"","filename":"SupplementaryTable6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-105996/v1/510486748392a0212d0c3814.xlsx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eRespiratory viral co-infections among SARS-CoV-2 cases confirmed by virome capture sequencing\u003c/p\u003e","fulltext":[{"header":"Full Text","content":"\u003cp\u003eThis preprint is available for \u003ca href='/article/rs-105996/latest.pdf' target='_blank'\u003edownload as a PDF\u003c/a\u003e.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"SARS-CoV-2, COVID-19, co-infection, hybrid-capture, respiratory virome, metagenomics, next-generation sequencing, rhinovirus, influenzavirus","lastPublishedDoi":"10.21203/rs.3.rs-105996/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-105996/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAccumulating evidence supports the high prevalence of co-infections among Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) patients, and their potential to worsen the clinical outcome of COVID-19. However, there are few data on Southern Hemisphere populations, and most studies to date have investigated a narrow spectrum of viruses using targeted qRT-PCR. Here we assessed respiratory viral co-infections among SARS-CoV-2 patients in Australia, through respiratory virome characterization. Nasopharyngeal swabs of 92 SARS-CoV-2-positive cases were sequenced using pan-viral hybrid-capture and the Twist Respiratory Virus Panel. In total, 8% of cases were co-infected, with rhinovirus (6%) or influenzavirus (2%). Twist capture also achieved near-complete sequencing (\u0026gt;90% coverage, \u0026gt;10-fold depth) of the SARS-CoV-2 genome in 95% of specimens with Ct\u0026lt;30. Our results highlight the importance of assessing all pathogens in symptomatic patients, and the dual-functionality of Twist hybrid-capture, for SARS-CoV-2 whole-genome sequencing without amplicon generation and the simultaneous identification of viral co-infections with ease.\u003c/p\u003e","manuscriptTitle":"Respiratory viral co-infections among SARS-CoV-2 cases confirmed by virome capture sequencing","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-11-18 14:44:11","doi":"10.21203/rs.3.rs-105996/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"99d23103-eb12-4b79-9f96-17b3b0d40d3b","owner":[],"postedDate":"November 18th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":1112782,"name":"Virology"},{"id":1112783,"name":"Molecular Biology"},{"id":1112784,"name":"Bioinformatics"},{"id":1112785,"name":"Biochemical Research Methods"}],"tags":[{"value":"featured","date":"2020-11-23 21:19:41"}],"updatedAt":"2021-02-19T13:37:31+00:00","versionOfRecord":{"articleIdentity":"rs-105996","link":"https://doi.org/10.1038/s41598-021-83642-x","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2021-02-16 13:00:00","publishedOnDateReadable":"February 16th, 2021"},"versionCreatedAt":"2020-11-18 14:44:11","video":"","vorDoi":"10.1038/s41598-021-83642-x","vorDoiUrl":"https://doi.org/10.1038/s41598-021-83642-x","workflowStages":[]},"version":"v1","identity":"rs-105996","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-105996","identity":"rs-105996","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}