Analysis of Different Size Fractions Provides a More Complete Perspective of Viral Diversity in a Freshwater Embayment.

Inspired by recent discoveries of the prevalence of large viruses in the environment, we reassessed the longstanding approach of filtering water through small-pore-size filters to separate viruses from cells before metagenomic analysis. We collected samples from three sites in Hamilton Harbour, an embayment of Lake Ontario, and studied 6 data sets derived from <0.45-μm- and >0.45-μm-size fractions to compare the diversity of viruses in these fractions. At the level of virus order/family, we observed highly diverse and distinct virus communities in the >0.45-μm-size fractions, whereas the <0.45-μm-size fractions were composed primarily of The relative abundances of for which hosts could be inferred varied widely between size fractions, with higher relative abundances of cyanophages in the >0.45-μm-size fractions, potentially indicating replication within cells during ongoing infections. Many viruses of eukaryotes, such as , , , and , were detected exclusively in the often-disregarded >0.45-μm-size fractions. In addition to observing unique virus communities associated with each size fraction from every site we examined, we detected viruses common to both fractions, suggesting that these are candidates for further exploration because they could be the product of ongoing or recent lytic events. Most importantly, our observations indicate that analysis of either fraction alone provides only a partial perspective of double-stranded DNA (dsDNA) viruses in the environment, highlighting the need for more comprehensive approaches for analyzing virus communities inferred from metagenomic sequencing. Most studies of aquatic virus communities analyze DNA sequences derived from the smaller-size "free-virus" fraction. Our study demonstrates that analysis of virus communities using only the smaller-size fraction can lead to erroneously low diversity estimates for many of the larger viruses such as , , , and , whereas analyzing only the larger->0.45-μm-size fraction can lead to underestimates of diversity and relative abundance. Similarly, our data show that examining only the smaller-size fraction can lead to underestimations of virophage and cyanophage relative abundances that could, in turn, cause researchers to assume their limited ecological importance. Given the considerable differences we observed in this study, we recommend cautious interpretations of environmental virus community assemblages and dynamics when based on metagenomic data derived from different size fractions.