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Virology

Unbiased Detection of Respiratory Viruses by Use of RNA Sequencing-Based Metagenomics: a Systematic Comparison to a Commercial PCR Panel

Erin H. Graf, Keith E. Simmon, Keith D. Tardif, Weston Hymas, Steven Flygare, Karen Eilbeck, Mark Yandell, Robert Schlaberg
A. M. Caliendo, Editor
Erin H. Graf
aUniversity of Utah School of Medicine, Department of Pathology, Salt Lake City, Utah, USA
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Keith E. Simmon
bUniversity of Utah School of Medicine, Department of Biomedical Informatics, Salt Lake City, Utah, USA
cARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah, USA
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Keith D. Tardif
cARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah, USA
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Weston Hymas
cARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah, USA
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Steven Flygare
dDepartment of Human Genetics, University of Utah, Salt Lake City, Utah, USA
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Karen Eilbeck
bUniversity of Utah School of Medicine, Department of Biomedical Informatics, Salt Lake City, Utah, USA
eUSTAR Center for Genetic Discovery, Salt Lake City, Utah, USA
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Mark Yandell
dDepartment of Human Genetics, University of Utah, Salt Lake City, Utah, USA
eUSTAR Center for Genetic Discovery, Salt Lake City, Utah, USA
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Robert Schlaberg
aUniversity of Utah School of Medicine, Department of Pathology, Salt Lake City, Utah, USA
cARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah, USA
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A. M. Caliendo
Roles: Editor
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DOI: 10.1128/JCM.03060-15
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ABSTRACT

Current infectious disease molecular tests are largely pathogen specific, requiring test selection based on the patient's symptoms. For many syndromes caused by a large number of viral, bacterial, or fungal pathogens, such as respiratory tract infections, this necessitates large panels of tests and has limited yield. In contrast, next-generation sequencing-based metagenomics can be used for unbiased detection of any expected or unexpected pathogen. However, barriers for its diagnostic implementation include incomplete understanding of analytical performance and complexity of sequence data analysis. We compared detection of known respiratory virus-positive (n = 42) and unselected (n = 67) pediatric nasopharyngeal swabs using an RNA sequencing (RNA-seq)-based metagenomics approach and Taxonomer, an ultrarapid, interactive, web-based metagenomics data analysis tool, with an FDA-cleared respiratory virus panel (RVP; GenMark eSensor). Untargeted metagenomics detected 86% of known respiratory virus infections, and additional PCR testing confirmed RVP results for only 2 (33%) of the discordant samples. In unselected samples, untargeted metagenomics had excellent agreement with the RVP (93%). In addition, untargeted metagenomics detected an additional 12 viruses that were either not targeted by the RVP or missed due to highly divergent genome sequences. Normalized viral read counts for untargeted metagenomics correlated with viral burden determined by quantitative PCR and showed high intrarun and interrun reproducibility. Partial or full-length viral genome sequences were generated in 86% of RNA-seq-positive samples, allowing assessment of antiviral resistance, strain-level typing, and phylogenetic relatedness. Overall, untargeted metagenomics had high agreement with a sensitive RVP, detected viruses not targeted by the RVP, and yielded epidemiologically and clinically valuable sequence information.

FOOTNOTES

    • Received 18 November 2015.
    • Returned for modification 14 December 2015.
    • Accepted 20 January 2016.
    • Accepted manuscript posted online 27 January 2016.
  • Supplemental material for this article may be found at http://dx.doi.org/10.1128/JCM.03060-15.

  • Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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Unbiased Detection of Respiratory Viruses by Use of RNA Sequencing-Based Metagenomics: a Systematic Comparison to a Commercial PCR Panel
Erin H. Graf, Keith E. Simmon, Keith D. Tardif, Weston Hymas, Steven Flygare, Karen Eilbeck, Mark Yandell, Robert Schlaberg
Journal of Clinical Microbiology Mar 2016, 54 (4) 1000-1007; DOI: 10.1128/JCM.03060-15

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Unbiased Detection of Respiratory Viruses by Use of RNA Sequencing-Based Metagenomics: a Systematic Comparison to a Commercial PCR Panel
Erin H. Graf, Keith E. Simmon, Keith D. Tardif, Weston Hymas, Steven Flygare, Karen Eilbeck, Mark Yandell, Robert Schlaberg
Journal of Clinical Microbiology Mar 2016, 54 (4) 1000-1007; DOI: 10.1128/JCM.03060-15
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