induction of temperate viruses (by means of, for instance, mitomycin C treatment) prior to applying this method, can provide accession to the dormant phage fraction. One possible limitation of the technique presented here that could mislead the assignment of the virus-host pair is the co-sorting of a cell with a free virus. However, the single-cell sorting mode used here will sort a drop when it contains only one cell in its center and no other detectable free particles in the drop27, which makes the co-sorting of a cell with a virus very unlikely. Other potential limitations of the technique are the co-sorting of the host cell with unspecific viruses attached to it or with free viruses placed in its shade, known in flow cytometry as detection28. However, our microarray hybridization ‘swarm’ data does not indicate contamination with free viruses, suggesting an insignificant contribution of detection during cell sorting. Nevertheless, a simple ‘swarm’ step to remove most free viruses could be routinely implemented before sorting, if needed. In the case of unspecific attached viruses, our data does not support that hypothesis but rather confirms previous assignment data5. Here, we have provided a tool that can be used to analyze VMINs over any range of spatiotemporal scales and to draw valuable information on the evolution of virus genomes and the co-evolution with their hosts. This approach can be used to assign viruses to hosts even without previous information about either of them, which makes it suitable for the exploration of microbial dark matter. Methods Sample collection. A 50 µl water sample from CR30, a crystallizer pond of Bras del Port salterns (Santa Pola, Spain, 38º12’N, 0º36’W) taken in June 2011 was used for single cell sorting. The salinity of the sample was 37.2% and harbored 1.74x107 cells/ml. Cell
induction of temperate viruses (by means of, for instance, mitomycin C treatment) prior to applying this method, can provide accession to the dormant phage fraction. One possible limitation of the technique presented here that could mislead the assignment of the virus-host pair is the co-sorting of a cell with a free virus. However, the single-cell sorting mode used here will sort a drop when it contains only one cell in its center and no other detectable free particles in the drop27, which makes the co-sorting of a cell with a virus very unlikely. Other potential limitations of the technique are the co-sorting of the host cell with unspecific viruses attached to it or with free viruses placed in its shade, known in flow cytometry as ‘swarm’ detection28. However, our microarray hybridization data does not indicate contamination with free viruses, suggesting an insignificant contribution of ‘swarm’ detection during cell sorting. Nevertheless, a simple step to remove most free viruses could be routinely implemented before sorting, if needed. In the case of unspecific attached viruses, our data does not support that hypothesis but rather confirms previous assignment data5. Here, we have provided a tool that can be used to analyze VMINs over any range of spatiotemporal scales and to draw valuable information on the evolution of virus genomes and the co-evolution with their hosts. This approach can be used to assign viruses to hosts even without previous information about either of them, which makes it suitable for the exploration of microbial dark matter. Methods Sample collection. A 50 µl water sample from CR30, a crystallizer pond of Bras del Port salterns (Santa Pola, Spain, 38º12’N, 0º36’W) taken in June 2011 was used for single cell sorting. The salinity of the sample was 37.2% and harbored 1.74x107 cells/ml. Cell
2014-Nat-Com-Assigning-viruses-to-hosts_revised.docx