title: Peering below the diffraction limit: robust and specific sorting of viruses with flow cytometry.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, California, USA.|California Institute for Quantitative Biosciences, University of California San Francisco, San Francisco, California, USA.|UC Berkeley-UCSF Graduate Program in Bioengineering, University of California San Francisco, San Francisco, CA, USA.
authors.forename: Shea T
authors.initials: ST
authors.lastname: Lance
authors.affiliation: Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, California, USA.|California Institute for Quantitative Biosciences, University of California San Francisco, San Francisco, California, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Sukovich
authors.affiliation: Center for Life in Extreme Environments, Biology Department, Portland State University, Portland, Oregon, USA.
authors.forename: Kenneth M
authors.initials: KM
authors.lastname: Stedman
authors.affiliation: Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, California, USA. adam@abatelab.org.|California Institute for Quantitative Biosciences, University of California San Francisco, San Francisco, California, USA. adam@abatelab.org.|UC Berkeley-UCSF Graduate Program in Bioengineering, University of California San Francisco, San Francisco, CA, USA. adam@abatelab.org.
authors.forename: Adam R
authors.initials: AR
authors.lastname: Abate
mesh_terms: D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D056656:Lab-On-A-Chip Devices; D056186:Metagenomics / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D014773:Virology / Q000379:methods*; D014780:Viruses / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*
keywords: Droplet microfluidics; Next generation sequencing; Single virus genomics