Fluorescence-Based Sorting of Individual Virus Particles for Genomic Analysis

Abstract

Source: Zeigler Allen, L., et al. Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'. J. Vis. Exp. (2013).

This video demonstrates how flow cytometry enables the isolation of individual, genome-containing viruses from mixed populations through fluorescence-based detection and precise particle sorting, followed by agarose embedding for subsequent genomic characterization.

Protocol

1. Preparation of Viral Suspensions

Before isolation of single virions via flow cytometry, prepare unfixed viral suspensions.

1. Isolate viral particles using previously established protocols making sure final viral suspension is contained in 0.1 μm-filtered TE (Tris-EDTA, pH 8). We recommend using tangential flow filtration (TFF) approaches although other methods have been developed that use chemical flocculation.
2. Enumerate viral particles using established protocols, e.g. using epifluorescence 8-10 or flow cytometry.
3. Aliquot viral suspension into two Eppendorf tubes (final volume is recommended to be 1,000 μl).

2. Flow Cytometry

1. To sort viruses using the BD FACSAria II Flow Cytometer equipped with a custom Forward Scatter PMT (FSC PMT), dilute viral particles in 0.1 μm-filtered TE, pH 8.0 to an appropriate titer for an event rate of 200 events sec^-1.
2. Set thresholds to maximize signal-to-noise rations, e.g. for a mixed assemblage containing T4 and lambda phage particles the following are recommended: FSC PMT at 1,000 and SSC at 200.
3. Run 100 μl of "blank" samples containing only 1) 0.1 μm-filtered TE and 2) 0.1 μm-filtered TE and SybrGreen1 at 1e-4 dilution of stock (10,000X).
4. Run a small aliquot (100 μl is recommended) of unstained viral suspension to assess background, followed by the stained viral suspension (SybrGreen1 at 1e-4 dilution of stock), viral particles to a total of 5,000 events each. This is to check concentration and adjust sorting gates, if necessary, prior to sort. We recommend a tight gate in the center of viral particles. Also, for generating gates we use the SYBR Green and FSC PMT plot.
5. Add 5 μl of 1% low melting point (LMP) agarose (in TBE buffer) cooled to 37 °C to each well on a polytetrafluoroethylene (PTFE) microscope slide (Electron Microscopy Sciences).
6. Load the PTFE microscope slide into the flow cytometer to capture sorted viral particles.
7. Begin sort of SYBR green stained viral particles directly onto PTFE slide with LMP agarose. We recommend sorting 10 or more events (viral particles) in some wells to aid in the detection of the depth of viruses during confocal laser scanning microscopy (CSLM).
8. When sorting is complete, remove the slide from the flow cytometer and add 5 μl more LMP agarose cooled to 37 °C to each well, thus embedding the viral particle(s).

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1X TE buffer	Invitrogen	12090-015
Buffer-saturated Phenol	Invitrogen	15513-039
LMP agarose	Invitrogen	16520100
PTFE microscope slide	Electron Microscopy Sciences	63430-04	24 well, 4 mm Diameter
β-agarase	New England Biolabs	M0392S