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

Abstract 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 5-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. Importance Viruses in diverse habitats numerically dominate microbial community members and are consequential for host fate and responsible for disease outbreaks. Metagenomics has provided novel insights into their presence, diversity, and ecosystem impacts. Yet the approach is hindered by the challenge that, despite their numbers, viral genomes comprise a minor portion of total community DNA in a sample. To comprehensively describe a viral community with metagenomics, viral genomes must be effectively concentrated and purified to capture low abundance and rare viruses amidst the overwhelming presence of cellular genomic DNA. We evaluated two commonly applied methods for concentrating and purifying viral DNA, ultrafiltration and iron chloride flocculation, across four aquatic sample types that differ in their solids content and salinity. Our findings demonstrated the importance of sample characteristics for method effectivity and provided performance measures for a variety of matrices. These findings contribute a more comprehensive understanding of the impacts of virome sample preparation across diverse matrices and are thus important for guiding future virome studies, given the wide range of habitats in which viruses are studied.