Virus Hunting: Discovery of New Episomal Circular Viruses by Rolling Circle Techniques

Bert Vanmechelen1, Annabel Rector1, Piet Maes1

1 KU Leuven—University of Leuven, Department of Microbiology and Immunology, Laboratory of Clinical Virology, Rega Institute, Leuven
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 1E.12
DOI:  10.1002/cpmc.23
Online Posting Date:  February, 2017
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Abstract

Many methods for the discovery of novel viruses are based on amplification of the virus using consensus or degenerate PCR primers. A downside of this approach is that it requires prior knowledge of the viral nucleotide sequence to be applicable. Presented in this unit is a method for the sequence‐independent amplification of circular viral genomes that is based on the rolling‐circle mechanism used by certain viruses in their natural replication cycle. The amplification of the virus of interest is coupled to the isolation of the viral genome by gel extraction following a restriction digestion. Once isolated, the sequence of the viral genome can be determined by nanopore sequencing, a rapid and inexpensive next‐generation sequencing technology that generates long reads in real time. The method described in this unit was originally developed for the discovery of papillomaviruses, but can be used for the identification of all types of circular DNA viruses. © 2017 by John Wiley & Sons, Inc.

Keywords: virus identification; papillomavirus; rolling‐circle amplification; nanopore sequencing

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Enrichment of Circular Virus DNA by Multiply Primed Rolling‐Circle Amplification (RCA)
  • Basic Protocol 2: DNA Isolation and Amplification of Virus of Interest
  • Basic Protocol 3: Sequencing of Amplified Viral DNA Using the Oxford Nanopore MinION
  • Alternate Protocol 1: Rapid Sequencing of Amplified Viral DNA using the Oxford Nanopore MinION
  • Basic Protocol 4: Isolation of Papillomavirus Reads from the MinION Sequencing Data and Assembly of the Viral Genome
  • Alternate Protocol 2: Identification of Unknown DNA Viruses by Viral Cloning and Sanger Sequencing
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Enrichment of Circular Virus DNA by Multiply Primed Rolling‐Circle Amplification (RCA)

  Materials
  • Purified DNA template containing a circular DNA virus (obtained using a QIAamp DNA Blood mini kit)
  • Illustra TempliPhi 100/500 Amplification Kit (GE Healthcare Life Sciences)
    • Sample buffer
    • Reaction buffer
    • Enzyme mix
  • dNTP stock solution (25 mM each dNTP; available from several molecular biology suppliers)
  • 30°C, 65°C, and 95°C heating blocks

Basic Protocol 2: DNA Isolation and Amplification of Virus of Interest

  Materials
  • RCA product ( protocol 1)
  • Nuclease‐free water
  • Set of restriction enzymes (20 U/µl) and appropriate buffers:
    • EcoRI (New England Biolabs, cat. no. R0101S)
    • BamHI (New England Biolabs, cat. no. R0136S)
    • HindIII (New England Biolabs, cat. no. R0104S)
    • SalI (New England Biolabs, cat. no. R0138S)
    • PureLink Quick Gel Extraction Kit (Invitrogen)
  • Qiagen REPLI‐g Mini Kit (Qiagen)
  • Qubit dsDNA HS Assay Kit (Invitrogen)
  • 37°C heating block
  • UV transilluminator
  • Qubit fluorometer (Invitrogen)
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, 2000)

Basic Protocol 3: Sequencing of Amplified Viral DNA Using the Oxford Nanopore MinION

  Materials
  • Amplified viral DNA ( protocol 2)
  • NEBNext Ultra II End Repair/dA‐Tailing Module (New England Biolabs, cat. no. E7546):
    • Ultra II End‐prep reaction buffer
    • Ultra II End‐prep enzyme mix
  • Agencourt AMPure XP beads (Beckman Coulter, cat. no. A63880)
  • 70% molecular‐grade ethanol in nuclease‐free water
  • Nuclease‐free water
  • Nanopore Sequencing Kit ((Oxford Nanopore Technologies, cat. no. SQK‐NSK007):
    • Adapter mix (AMX)
    • Hairpin adapter (HPA)
    • Hairpin tether (HPT)
    • Bead binding buffer (BBB)
    • Elution buffer (ELB)
    • Running buffer with fuel mix (RBF1)
  • Blunt/TA Ligase Master Mix (New England Biolabs, cat. no. M0367)
  • MyOne C1 beads (Invitrogen, cat. no. 65001)
  • Qubit dsDNA HS Assay Kit (Invitrogen)
  • 1.5‐ml DNA LoBind tubes (Eppendorf)
  • 20°C, 37°C, and 65°C heating blocks
  • Magnetic rack
  • Qubit fluorometer (Invitrogen)
  • Oxford Nanopore MinION Mk 1B coupled to a computer
  • SpotON Flow Cell Mk I

Alternate Protocol 1: Rapid Sequencing of Amplified Viral DNA using the Oxford Nanopore MinION

  Materials
  • Amplified viral DNA ( protocol 2)
  • Nuclease‐free water
  • Nanopore Rapid Sequencing Kit (Oxford Nanopore Technologies, cat. no. SQK‐RAD001)
    • Fragmentation mix (FRM)
    • Rapid adapter (RAD)
    • Running buffer with fuel mix (RBF1)
  • Blunt/TA Ligase Master Mix (New England Biolabs, cat. no. M0367)
  • 1.5‐ml DNA LoBind tubes (Eppendorf)
  • 30°C and 75°C heating blocks (or a thermal cycler)
  • Oxford Nanopore MinION Mk 1B coupled to a computer
  • SpotON Flow Cell Mk I

Basic Protocol 4: Isolation of Papillomavirus Reads from the MinION Sequencing Data and Assembly of the Viral Genome

  Materials
  • EPI2ME v2.42 (Metrichor)
  • Poretools v0.6.0 (Loman and Quinlan, ); run in Linux
  • BLAST (The National Center for Biotechnology Information; NCBI); run in Linux
  • Canu v1.3 (Koren et al., ); run in Linux
  • Nanopolish v0.5.0 (Simpson, 2016); run in Linux
  • Open Reading Frame Finder (The National Center for Biotechnology Information; NCBI)
  • MEGA7 (Kumar et al., )
  • SeaView (Gouy et al., )

Alternate Protocol 2: Identification of Unknown DNA Viruses by Viral Cloning and Sanger Sequencing

  Materials
  • Set of restriction enzymes (100 U/µl)
    • EcoRI (New England Biolabs, cat. no. R0101T)
    • BamHI (New England Biolabs, cat. no. R0136T)
    • HindIII (New England Biolabs, cat. no. R0104T)
    • SalI (New England Biolabs, cat. no. R0138T)
  • Appropriate restriction enzyme buffers
  • Cloning vector pUC18
  • Viral genome DNA, amplified using RCA (see protocol 1)
  • Nuclease‐free water
  • 1U/μl Calf intestine alkaline phosphatase (CIAP; Thermo Fisher Scientific, cat. no. 18009027)
  • Geneclean kit (MP Biomedicals)
  • Qubit dsDNA HS Assay Kit (Invitrogen)
  • Rapid DNA Ligation Kit (Roche):
    • 2× T4 DNA ligation buffer
    • 5× DNA dilution buffer
    • 5 U/µl T4 DNA ligase
  • One Shot MAX Efficiency DH5α‐T1 Escherichia coli (Invitrogen, cat. no. 12297016)
  • SOC medium (Invitrogen)
  • 2% X‐gal (see recipe)
  • LB agar plates, supplemented with 100 μg/ml ampicillin ( appendix 4A; Stevenson, )
  • LB/ampicillin: LB broth, supplemented 100 μg/ml ampicillin ( appendix 4A; Stevenson, )
  • QIAprep Spin Miniprep Kit (Qiagen)
  • Big Dye reaction mixture (Applied Biosystems)
  • M13 forward primer (Thermo Fisher Scientific, cat. no. N52002)
  • M13 reverse primer (Thermo Fisher Scientific, cat. no. N53002)
  • 70% molecular‐grade ethanol in nuclease‐free Water
  • 3 M sodium acetate (NaOAc), pH 5 ( appendix 2A)
  • Formamide
  • 37°C, 42°C, 50°C, 85°C, and 95°C heating blocks
  • UV light source
  • Qubit fluorometer (Invitrogen)
  • 37°C shaking incubator
  • Thermal cycler
  • ABI 3130xl genetic analyzer (Applied Biosystems)
  • MEGA7 software (Kumar et al., )
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, 2000) and restriction digestion ( protocol 2, steps 2 to 3)
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Figures

Videos

Literature Cited

  Burnett, L.C., Lunn, G., and Coico, R. 2009. Biosafety: Guidelines for working with pathogenic and infectious microorganisms. Curr. Protoc. Microbiol. 13:1A.1.1‐1A.1.14.
  Gouy, M., Guindon, S., and Gascuel, O. 2010. SeaView version 4: A multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol. Biol. Evol. 27:221‐224. doi: 10.1093/molbev/msp259.
  Koren, S., Walenz, B.P., Berlin, K., Miller, J.R., and Phillippy, A.M. 2016. Canu: Scalable and accurate long‐read assembly via adaptive k‐mer weighting and repeat separation. bioRxiv. doi:https://doi.org/10.1101/071282.
  Kumar, S., Stecher, G., and Tamura, K. 2016. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33:1870‐1874. doi: 10.1093/molbev/msw054.
  Loman, N.J. and Quinlan, A.R. 2014. Poretools: A toolkit for analyzing nanopore sequence data. Bioinformatics 30:3399‐3401. doi: 10.1093/bioinformatics/btu555.
  Rector, A., Tachezy, R., and Van Ranst, M. 2004. A sequence‐independent strategy for detection and cloning of circular DNA virus genomes by using multiply primed rolling‐circle amplification. J. Virol. 78:4993‐4998. doi: 10.1128/JVI.78.10.4993‐4998.2004.
  Stevens, H., Rector, A., and Van Ranst, M. 2010. Multiply primed rolling‐circle amplification method for the amplification of circular DNA viruses. Cold Spring Harb. Protoc. 2010:pdb prot5415. doi: 10.1101/pdb.prot5415.
  Stevenson, B. 2006. Common bacterial culture techniques and media. Curr. Protoc. Microbiol. 00:4A.1‐A.4A.8. doi:10.1002/9780471729259.mca04as00.
  Voytas, D. 2001. Agarose gel electrophoresis. Curr. Protoc. Mol. Biol. 51:2.5A.1‐2.5A.9.]
Internet Resources
  https://nanoporetech.com/
  The Web site of Oxford Nanopore Technologies, the developers of the MinION.
  https://www.ncbi.nlm.nih.gov/orffinder
  Orffinder: The National Center for Biotechnology Information (NCBI).
  https://blast.ncbi.nlm.nih.gov/Blast.cgi
  The National Center for Biotechnology Information (NCBI). Blast.
  http://www.metrichor.com
  Metrichor, supplier of EPI2ME.
  https://github.com/jts/nanopolish
  Simpson, J., 2016. Nanopolish.
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