A universal next generation sequencing protocol to generate non-infectious barcoded cDNA libraries from high containment RNA viruses

Authors: MOSER, LINDSEY - University Of Wisconsin; RAMIREZ-CARVAJAL, LISBETH - Oak Ridge Institute For Science And Education (ORISE); PURI, VINITA - J Craig Venter Institute; Pauszek, Steven; MATTHEWS, KRYSTAL - University Of Maryland; DILLEY, KARI - J Craig Venter Institute; MULLAN, CLANCY - University Of North Carolina; MCGRAW, JENNIFER - University Of North Carolina; YEE, ANTHONY - J Craig Venter Institute; DUGAN, VIVIEN - J Craig Venter Institute; HEISE, MARK - University Of North Carolina; FRIEMAN, MATTHEW - University Of Maryland; Rodriguez, Luis; BERNARD, KRISTEN - University Of Wisconsin; WENTWORTH, DAVID - J Craig Venter Institute; STOCKWELL, TIMOTHY - J Craig Venter Institute; SHABMAN, REED - J Craig Venter Institute

Submitted to: mSystems
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/5/2016
Publication Date: 6/7/2016
Citation: Moser, L.A., Ramirez-Carvajal, L., Puri, V., Pauszek, S.J., Matthews, K., Dilley, K., Mullan, C., Mcgraw, J., Yee, A., Dugan, V., Heise, M.T., Frieman, M.B., Rodriguez, L.L., Bernard, K.A., Wentworth, D.E., Stockwell, T.B., Shabman, R.S. 2016. A universal next generation sequencing protocol to generate non-infectious barcoded cDNA libraries from high containment RNA viruses. mSystems. doi: 10.1128/mSystems.00039-15.

Interpretive Summary: Highly infectious pathogens of humans and animals such as foot-and-mouth disease can only be studied in high containment laboratories termed biosafety levels (BSL) 3 or 4. The genetic code (RNA) of some of these viruses is infectious, meaning that procedures that will inactivate other virus pathogens will not completely inactivate these viruses. Moreover many of these viruses are Select Agents (SAs), and their genetic codes are also considered SAs. Special procedures are necessary to safely bring the genetic material of these viruses out of the BSL3/4 laboratories for genetic sequencing. Here we describe methodology that assures inactivation of the pathogens while preserving their genetic sequence. Furthermore, we present a sequence-independent method to rapidly amplify viral genomic material while simultaneously abolishing both viral and genomic RNA infectivity across multiple virus families. The process generates barcoded genetic material which cannot be used to rescue a virus and is stable to transport at room temperature. Our barcoding approach allows for up to 288 barcoded-samples to be pooled into a single library to derive their complete genetic sequence using next generation sequence (NGS) methods. Our data demonstrate this approach provides full-length genomic sequence information not only from high titer virus preparations, but can recover specific viral sequence from virus-infected tissue samples, and can be used to identify pathogens from unknown samples. In summary we describe a rapid, universal standard operating procedure that generates high quality genetic sequence while removing viral infectivity. This methodology is useful to safely carry out research and diagnostic procedures of high consequence virus pathogens.

Technical Abstract: Several biosafety level (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 positive-sense single stranded RNA (ssRNA+) virus families. The process generates barcoded DNA amplicons in the range of 300-1000 base pairs in length, 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 this approach provides full-length genomic sequence information not only from high titer virion preparations, but can recover specific viral sequence from samples with limited starting material in the background of cellular RNA, and 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.