Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Summary

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.

Abstract

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.

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 ⁶ although other methods have been developed that use chemical flocculation ⁷.
2. Enumerate viral particles using established protocols, e.g. using epifluorescence ^8-10 or flow cytometry ^11,12.
3. Aliquot viral suspension into t....

Discussion

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 react.......

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
GenomiPhi HY kit	GE Healthcare	25-6600-22
Glycoblue	Invitrogen	AM9516	15 mg/ml
LMP agarose	Invitrogen	16520100
PTFE microscope slide	Electron Microscopy Sciences	63430-04	24 well, 4 mm Diameter
SybrGreen	Invitrogen	S7585	10,000X
β-agarase	New England Biolabs	M0392S