Single virus genomics: a new tool for virus discovery

Abstract

Whole genome amplification and sequencing of single microbial cells has significantly influenced genomics and microbial ecology by facilitating direct recovery of reference genome data. However, viral genomics continues to suffer due to difficulties related to the isolation and characterization of uncultivated viruses. We report here on a new approach called 'Single Virus Genomics', which enabled the isolation and complete genome sequencing of the first single virus particle. A mixed assemblage comprised of two known viruses; E. coli bacteriophages lambda and T4, were sorted using flow cytometric methods and subsequently immobilized in an agarose matrix. Genome amplification was then achieved in situ via multiple displacement amplification (MDA). The complete lambda phage genome was recovered with an average depth of coverage of approximately 437X. The isolation and genome sequencing of uncultivated viruses using Single Virus Genomics approaches will enable researchers to address questions about viral diversity, evolution, adaptation and ecology that were previously unattainable.

Figure 1. Flow diagram depicting SVG methodology.

Viral suspensions are sorted via flow cytometry onto PTFE slides with 24 distinct wells containing agarose beads. Viral particles are then embedded within the agarose bead by overlaying with an additional layer of agarose. Lastly, MDA is performed in situ.

Figure 2. Flow cytometric bi-plot showing SYBR-stained T4 and lambda phage mixture.

Gates were chosen to highlight T4/lambda assemblages (green), and background (blue). Particles not gated (black) were not sorted.

Figure 3. Confocal laser scanning micrograph of sorted viral particle embedded in agarose bead.

A) Three dimensional reconstruction of syber green I stained viral particle within depth of agarose bead verifying a single sorted event. Inset: higher magnification of viral particle. B) Profile plot of relative fluorescence for a stained viral particle in an agarose bead. The blue line through the viral particle (green) is the reference for the inset graph showing the relative fluorescence.

Figure 4. Phage 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 (1050 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 5. Lambda genome attributes and coverage.

A) GC plot with bars indicating %GC above or below the average of 49%, B) Genome map of lambda (adapted from http://img.jgi.doe.gov), and C) Reference mapping of SVG reads to phage lambda, x-axis is genome position, y-axis is %coverage.

Figure 6. De novo assembly of reads followed by reference mapping to evaluate assembly.

A) Filtered sequences randomly to 3400 reads, approximately 22X coverage of the lambda genome, B) All reads (99,911), C) Normalization of assembly by reducing redundancy of overrepresented sequences from (B).