Peering below the diffraction limit: robust and specific sorting of viruses with flow cytometry
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RESEARCH
Open Access
Peering below the diffraction limit: robust and specific sorting of viruses with flow cytometry Shea T. Lance1,2,3, David J. Sukovich1,2, Kenneth M. Stedman4 and Adam R. Abate1,2,3*
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. Keywords: Droplet microfluidics, Next generation sequencing, Single virus genomics
Introduction Viruses impact every form of life on earth, from applying evolutionary stresses to enhancing the transfer of genes between organisms [1–4]. Many human diseases are caused by viruses, including acute diseases like Ebola [5] and influenza [6], and chronic diseases caused by Epstein-Barr Virus (EBV) [7], Human Immunodeficiency Virus (HIV) [8], and Zika virus [9]. Studying viruses is thus important to human health, but also for elucidating the incredible mechanisms they’ve evolved to survive, replicate, and spread; these discoveries may lead to new molecular techniques and methods for treating disease. * Correspondence: [email protected] 1 Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, California, USA 2 California Institute for Quantitative Biosciences, University of California San Francisco, San Francisco, California, USA Full list of author information is available at the end of the article
Studying viruses, however, can be challenging. They are usually much smaller than the diffraction limit of light and thus not directly visible with optical microscopy. They contain miniscule amounts of nucleic acid and protein, making direct sequencing or prote
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