Bacteriophages, which are viruses that prey on bacteria, play an outsized role in environmental and human health. Metagenomics are restricted by an inability to isolate and identify specific components in a microbiota, in particular rare and closely related species. This and the fact that abundant species dominate analyses are some of the major obstacles to progress in the field. Considering the astronomical number of assumed viral species (~10<sup>31</sup>), it is not surprising that unknown species and sequences monopolize virome data. Rapid isolation, characterization, and host-speciation of bacteriophage are essential for the logarithmic expansion of effort necessary to detail the most abundant organisms on Earth. We developed a capillary-electrophoretic (CE) fractionation that separates intact phages based on the chemical properties of the intact particle resulting in non-destructive purification of populations from a diverse environmental mixture. Nanoliter volumes of bacteriophage suspensions are pneumatically injected into a fused silica capillary for separation. Bacteriophages migrate to the distal end of the capillary where they are mixed with deposition buffer and deposited into a collection plate. In CE, the conditions of the separation can be modified to influence the mobility of a particular analyte, but the molecular and chemical basis of the separation is unknown. We optimized conditions for reconstructed mixtures of diverse phage, <i>E. coli</i> specific bacteriophage and mixed microorganisms isolated from primary sewage effluent. The data established three principal findings: 1.) In mixed microbiome populations, we achieve complete isolation of viral populations from bacterial species. 2.) Modification of electrophoretic conditions produces bacteriophage enriched to near-homogeneity, reducing traditional manual plating/isolation techniques. 3.) Phage fractions collected directly onto electron microscopy grids facilitate morphological classification, and help define the molecular basis for migration. These data illustrate that intact phage populations can be fractionated for subsequent detection, or offline use. The combination of separation and TEM imaging of intact viruses will be highly beneficial for characterization of novel organisms and cataloging them for therapeutic use.

Electrophoretic separation of bacteriophage from complex mixed microbiomes

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