• Login
    View Item 
    •   UMB Digital Archive
    • UMB Open Access Articles
    • UMB Coronavirus Publications
    • View Item
    •   UMB Digital Archive
    • UMB Open Access Articles
    • UMB Coronavirus Publications
    • View Item
    JavaScript is disabled for your browser. Some features of this site may not work without it.

    Browse

    All of UMB Digital ArchiveCommunitiesPublication DateAuthorsTitlesSubjectsThis CollectionPublication DateAuthorsTitlesSubjects

    My Account

    LoginRegister

    Statistics

    Display statistics

    A Universal Next-Generation Sequencing Protocol To Generate Noninfectious Barcoded cDNA Libraries from High-Containment RNA Viruses

    • CSV
    • RefMan
    • EndNote
    • BibTex
    • RefWorks
    Author
    Moser, L.A.
    Ramirez-Carvajal, L.
    Matthews, K.
    Date
    2016
    Journal
    mSystems
    Publisher
    American Society for Microbiology
    Type
    Article
    
    Metadata
    Show full item record
    See at
    https://doi.org/10.1128/mSystems.00039-15
    Abstract
    Several biosafety level 3 and/or 4 (BSL-3/4) pathogens are high-consequence, single-stranded RNA viruses, and their genomes, when introduced into permissive cells, are infectious. Moreover, many of these viruses are select agents (SAs), and their genomes are also considered SAs. For this reason, cDNAs and/or their derivatives must be tested to ensure the absence of infectious virus and/or viral RNA before transfer out of the BSL-3/4 and/or SA laboratory. This tremendously limits the capacity to conduct viral genomic research, particularly the application of next-generation sequencing (NGS). Here, we present a sequence-independent method to rapidly amplify viral genomic RNA while simultaneously abolishing both viral and genomic RNA infectivity across multiple single-stranded positive-sense RNA (ssRNA) virus families. The process generates barcoded DNA amplicons that range in length from 300 to 1,000 bp, which cannot be used to rescue a virus and are stable to transport at room temperature. Our barcoding approach allows for up to 288 barcoded samples to be pooled into a single library and run across various NGS platforms without potential reconstitution of the viral genome. Our data demonstrate that this approach provides full-length genomic sequence information not only from high-titer virion preparations but it can also recover specific viral sequence from samples with limited starting material in the background of cellular RNA, and it can be used to identify pathogens from unknown samples. In summary, we describe a rapid, universal standard operating procedure that generates high-quality NGS libraries free of infectious virus and infectious viral RNA. IMPORTANCE This report establishes and validates a standard operating procedure (SOP) for select agents (SAs) and other biosafety level 3 and/or 4 (BSL-3/4) RNA viruses to rapidly generate noninfectious, barcoded cDNA amenable for next-generation sequencing (NGS). This eliminates the burden of testing all processed samples derived from high-consequence pathogens prior to transfer from high-containment laboratories to lower-containment facilities for sequencing. Our established protocol can be scaled up for high-throughput sequencing of hundreds of samples simultaneously, which can dramatically reduce the cost and effort required for NGS library construction. NGS data from this SOP can provide complete genome coverage from viral stocks and can also detect virus-specific reads from limited starting material. Our data suggest that the procedure can be implemented and easily validated by institutional biosafety committees across research laboratories. Copyright 2016 Moser et al.
    Sponsors
    This project has been funded under Department of Homeland Security contract HSHQDC-13-C-B0016.
    Keyword
    Alphavirus, coronavirus
    Flavivirus, foot-and-mouth disease virus
    Genomics, picornavirus
    Next-generation sequencing
    Rhinovirus
    West Nile virus
    Identifier to cite or link to this item
    https://www.scopus.com/inward/record.uri?eid=2-s2.0-85041919744&doi=10.1128%2fmSystems.00039-15&partnerID=40&md5=0316dd9287a19670021a7fc5b701c18a; http://hdl.handle.net/10713/12401
    ae974a485f413a2113503eed53cd6c53
    10.1128/mSystems.00039-15
    Scopus Count
    Collections
    UMB Coronavirus Publications

    entitlement

     
    DSpace software (copyright © 2002 - 2021)  DuraSpace
    Quick Guide | Policies | Contact Us | UMB Health Sciences & Human Services Library
    Open Repository is a service operated by 
    Atmire NV
     

    Export search results

    The export option will allow you to export the current search results of the entered query to a file. Different formats are available for download. To export the items, click on the button corresponding with the preferred download format.

    By default, clicking on the export buttons will result in a download of the allowed maximum amount of items.

    To select a subset of the search results, click "Selective Export" button and make a selection of the items you want to export. The amount of items that can be exported at once is similarly restricted as the full export.

    After making a selection, click one of the export format buttons. The amount of items that will be exported is indicated in the bubble next to export format.