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Article
Peering Below the Diffraction Limit: Robust and Specific Sorting of Viruses with Flow Cytometry
Virology Journal
  • Shea T. Lance, University of California, San Francisco
  • David J. Sukovich, University of California, San Francisco
  • Kenneth M. Stedman, Portland State University
  • Adam R. Abate, University of California, San Francisco
Document Type
Article
Publication Date
12-1-2016
Subjects
  • Viruses -- Evolution,
  • Viral genomes,
  • Viruses -- Genome mapping
Disciplines
Abstract
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.
Description

© The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Supplementary material is located below in Additional Files.

DOI
10.1186/s12985-016-0655-7
Persistent Identifier
http://archives.pdx.edu/ds/psu/18933
Citation Information
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.