This work was supported by the National Science Foundation through a CAREER Award [grant number DBI-1253293] and MCB-1243963; the National Institutes of Health (NIH) [grant numbers HG007233-01, R01-EB019453-01, DP2-AR068129-01]; and the Defense Advanced Research Projects Agency Living Foundries Program [contract numbers HR0011-12-C-0065, N66001-12- C-4211, HR0011-12-C-0066]. Funding for open access charge: [NIH grant number DP2-AR068129-01] and funds from Portland State University to K.M.S.
Viruses -- Evolution, Viral genomes, Viruses -- Genome mapping
Background: Viruses are incredibly diverse organisms and impact all forms of life on Earth; however, individual virions are challenging to study due to their small size and mass, precluding almost all direct imaging or molecular analysis. Moreover, like microbes, the overwhelming majority of viruses cannot be cultured, impeding isolation, replication, and study of interesting new species. Here, we introduce PCR-activated virus sorting, a method to isolate specific viruses from a heterogeneous population. Specific sorting opens new avenues in the study of uncultivable viruses, including recovering the full genomes of viruses based on genetic fragments in metagenomes, or identifying the hosts of viruses.
Methods: PAVS enables specific sorting of viruses with flow cytometry. A sample containing a virus population is processed through a microfluidic device to encapsulate it into droplets, such that the droplets contain different viruses from the sample. TaqMan PCR reagents are also included targeting specific virus species such that, upon thermal cycling, droplets containing the species become fluorescent. The target viruses are then recovered via droplet sorting. The recovered virus genomes can then be analyzed with qPCR and next generation sequencing.
Results and Conclusions: We describe the PAVS workflow and demonstrate its specificity for identifying target viruses in a heterogeneous population. In addition, we demonstrate recovery of the target viruses via droplet sorting and analysis of their nucleic acids with qPCR.
Lance, S. T., Sukovich, D. J., Stedman, K. M., & Abate, A. R. (2016). Peering below the diffraction limit: robust and specific sorting of viruses with flow cytometry. Virology Journal, 13(1), 201.