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1Department of Microbial and Environmental Genomics, The J. Craig Venter Institute
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Single Virus Genomics (SVG) is a method to isolate and amplify the genomes of single virons. Viral suspensions of a mixed assemblage are sorted using flow cytometry onto a microscope slide with discrete wells containing agarose, thereby capturing the virion and reducing genome shearing during downstream processing. Whole genome amplification is achieved using multiple displacement amplification (MDA) resulting in genomic material that is suitable for sequencing.
Keywords: Genetics, Issue 75, Microbiology, Immunology, Virology, Molecular Biology, Environmental Sciences, Genomics, environmental genomics, Single virus, single virus genomics, SVG, whole genome amplification, flow cytometry, viral ecology, virion, genome analysis, DNA, PCR, sequencing
Zeigler Allen, L., Ishoey, T., Novotny, M. A., McLean, J. S., Lasken, R. S., Williamson, S. J. Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'. J. Vis. Exp. (75), e3899, doi:10.3791/3899 (2013).
Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which are ubiquitous and the most numerous entities on our planet 4 and important in all environments 5, have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.
1. Preparation of Viral Suspensions
Before isolation of single virions via flow cytometry, prepare unfixed viral suspensions.
2. Flow Cytometry
3. Visualizing Single Virions using Confocal Microscopy
If a single virion is desired, then determining if each well contains an individual virus is imperative and CLSM is necessary to visualize the embedded virion(s) in 3D to verify that only a single particle is present and that multiple viruses are not stacked on top of each other.
4. Whole Genome Amplification
5. Representative Results
Figure 1 summarizes the SVG process. These methods use flow cytometry to sort viral particles onto PTFE microscope slides containing agarose to isolate single virions from a mixed assemblage followed by MDA to obtain quantities of gDNA that are sufficient for sequencing.
As a proof-of-concept, bacteriophage lambda and T4 were mixed and subjected to the SVG process 11. Once virions were captured and embedded in agarose, CLSM was used to visualize and confirm that a single virion was obtained; results are shown in Figure 2. Once wells with single virions were identified, they were selected for MDA of gDNA. We then used multiplex PCR specific for T4 and lambda to identify the virion isolated, Figure 3. An isolated phage lambda was selected for genome sequencing.
Genome sequencing was performed using 454-Titanium technology and sequencing reads were subjected to reference mapping to identify the level of coverage obtained using SVG. Figure 4 shows that almost the entire lambda genome was recovered (with the exception of the first 5 bp).
Figure 1. SVG methodology. Viral suspensions containing a mixed assemblage are reduced to single virus particles via flow cytometry that are then sorted onto individual agarose "beads". The virus is embedded within the agarose bead by overlaying with an additional layer of agarose post flow cytometric sorting. Lastly, whole genome amplification is performed in situ.
Figure 2. Confocal laser scanning micrograph of a sorted viral particle embedded in an agarose bead. A) Three dimensional reconstruction of a Sybr Green I-stained viral particle within an agarose bead. Inset: higher magnification of the isolated viral particle. B) Profile plot of relative fluorescence of the stained single viral particle within an agarose bead.
Figure 3. Bacteriophage identification using PCR. A) Multiplex PCR using T4 and lambda-specific primers to genotype, Lanes: 1. TrackIt 1 kb plus ladder (Invitrogen), 2. lambda integrase (750 bp), 3. T4 major capsid protein (1,050 bp), 4. Mix of lambda integrase and T4 major capsid protein. B) Subsequent lambda specific PCR with additional loci to further confirm phage genome isolation, Lanes: 1. Lambda integrase (750 bp), 2. Lambda repressor (356 bp) 3. Lambda sie (superinfection exclusion) (456 bp) 4. TrackIt 1 kb plus ladder (Invitrogen).
Figure 4. Lambda genome attributes and coverage. A) GC plot, B) Genome map of lambda (adapted from http://img.jgi.doe.gov), and C) Mapping of SVG reads to the reference bacteriophage lambda, x-axis is genome position, y-axis is % coverage.
A number of important factors must be taken into consideration when applying SVG approaches. Genotyping, as was performed during the proof-of-concept experiment 13, is not a valid option for environmental or unknown isolates as conserved primers are not available across all viral groups. Also, background DNA synthesis or nonspecific amplification is commonly reported during amplification using the MDA reaction 14. Nonspecific amplification has been attributed to contaminating DNA emerging from reaction reagents and/or through a mechanism that enables amplification from the random hexamers within the reaction mixture. When working with viral assemblages, there is perhaps a higher likelihood of nonspecific DNAs preferentially amplified due to the lower quantity of template viral DNA as opposed to single bacterial cells as a result of the significant difference in particle (cell) size and genomic DNA content (25-100 nm; ~1.5 fg for viruses, as opposed to 0.2-1.5 um; ~14 fg for bacteria). The following methods are recommended to reduce the level of nonspecific amplification: treatment of viral assemblages with DNase I, examination of virus containing agarose beads using CLSM, reduction of MDA incubation time and increased sequencing depth (i.e. as is capable with next-generation sequencing technologies like 454-Titanium and Illumina).
When performing SVG on environmental samples, optimized de novo assembly is imperative to obtain complete or near-complete genome sequences. We have found that reducing the redundant reads prior to assembly is necessary to obtain greater coverage 13.
No conflicts of interest declared.
We would like to thank Ken Nealson for his insight and advice throughout the manuscript preparation process.
|1X TE buffer||Invitrogen||12090-015|
|GenomiPhi HY kit||GE Healthcare||25-6600-22|
|PTFE microscope slide||Electron Microscopy Sciences||63430-04||24 well, 4 mm Diameter|
|β-agarase||New England Biolabs||M0392S|
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