Comparison of ultrafiltration and iron chloride flocculation in the preparation of aquatic viromes from contrasting sample types.

Viral metagenomes (viromes) are a valuable untargeted tool for studying viral diversity and the central roles viruses play in host disease, ecology, and evolution. Establishing effective methods to concentrate and purify viral genomes prior to sequencing is essential for high quality viromes. Using virus spike-and-recovery experiments, we stepwise compared two common approaches for virus concentration, ultrafiltration and iron chloride flocculation, across diverse matrices: wastewater influent, wastewater secondary effluent, river water, and seawater. Viral DNA was purified by removing cellular DNA via chloroform cell lysis, filtration, and enzymatic degradation of extra-viral DNA. We found that viral genomes were concentrated 1-2 orders of magnitude more with ultrafiltration than iron chloride flocculation for all matrices and resulted in higher quality DNA suitable for amplification-free and long-read sequencing. Given its widespread use and utility as an inexpensive field method for virome sampling, we nonetheless sought to optimize iron flocculation. We found viruses were best concentrated in seawater with five-fold higher iron concentrations than the standard used, inhibition of DNase activity reduced purification effectiveness, and five-fold more iron was needed to flocculate viruses from freshwater than seawater-critical knowledge for those seeking to apply this broadly used method to freshwater virome samples. Overall, our results demonstrated that ultrafiltration and purification performed better than iron chloride flocculation and purification in the tested matrices. Given that the method performance depended on the solids content and salinity of the samples, we suggest spike-and-recovery experiments be applied when concentrating and purifying sample types that diverge from those tested here.

Iron Chloride Flocculation of Bacteriophages from Seawater.

Viruses influence ecosystem dynamics by modulating microbial host population dynamics, evolutionary trajectories and metabolic outputs. While they are ecologically important across diverse ecosystems, viruses are challenging to study due to minimal biomass often obtained when sampling natural communities. Here we describe a technique using chemical flocculation, filtration and resuspension to recover bacteriophages from seawater and other natural waters. The method uses iron to precipitate viruses which are recovered by filtration onto large-pore size membranes and then resuspended using a buffer containing magnesium and a reductant (ascorbic acid or oxalic acid) at slightly acid pH (6-6.5). The recovery of bacteriophages using iron flocculation is efficient (>90%), inexpensive and reliable, resulting in preparations that are amenable to downstream analysis by next generation DNA sequencing, proteomics and, in some cases, can be used to study virus-host interactions.