A Universal Next-Generation Sequencing Protocol To Generate Noninfectious Barcoded cDNA Libraries from High-Containment RNA Viruses.
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.IMPORTANCEThis 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.
Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Lindsey A
LA
Moser
Plum Island Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, Greenport, New York, USA; Plum Island Animal Disease Center-Oak Ridge Institute for Science and Education (ORISE) Research Participation Program, Oak Ridge, Tennessee, USA.
Lisbeth
L
Ramirez-Carvajal
Virology Group, J. Craig Venter Institute, Rockville, Maryland, USA.
Vinita
V
Puri
Plum Island Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, Greenport, New York, USA.
Steven J
SJ
Pauszek
Department of Microbiology and Immunology, University of Maryland at Baltimore, Baltimore, Maryland, USA.
Krystal
K
Matthews
Virology Group, J. Craig Venter Institute, Rockville, Maryland, USA.
Kari A
KA
Dilley
Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Clancy
C
Mullan
Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Jennifer
J
McGraw
Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Michael
M
Khayat
Sequencing Group, J. Craig Venter Institute, La Jolla, California, USA.
Karen
K
Beeri
Virology Group, J. Craig Venter Institute, Rockville, Maryland, USA.
Anthony
A
Yee
Virology Group, J. Craig Venter Institute, Rockville, Maryland, USA.
Vivien
V
Dugan
Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Mark T
MT
Heise
Department of Microbiology and Immunology, University of Maryland at Baltimore, Baltimore, Maryland, USA.
Matthew B
MB
Frieman
Plum Island Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, Greenport, New York, USA.
Luis L
LL
Rodriguez
Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Kristen A
KA
Bernard
Virology Group, J. Craig Venter Institute, Rockville, Maryland, USA.
David E
DE
Wentworth
Virology Group, J. Craig Venter Institute, Rockville, Maryland, USA.
Timothy B
TB
Stockwell
Virology Group, J. Craig Venter Institute, Rockville, Maryland, USA.
Reed S
RS
Shabman
West Nile virus; alphavirus; coronavirus; flavivirus; foot-and-mouth disease virus; genomics; next-generation sequencing; picornavirus; rhinovirus