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.

中文翻译：

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对比样品类型在水生病毒体制备中超滤和氯化铁絮凝的比较

病毒元基因组（病毒基因组）是研究病毒多样性和病毒在宿主疾病，生态学和进化中所起的核心作用的有价值的，没有针对性的工具。建立有效的方法以在测序前浓缩和纯化病毒基因组对于高质量的病毒是必不可少的。通过病毒加标和回收实验，我们逐步比较了两种不同的基质上用于浓缩病毒，超滤和氯化铁絮凝的两种常用方法：废水入水，废水二次出水，河水和海水。通过经由氯仿细胞裂解，过滤和酶促降解外病毒DNA去除细胞DNA来纯化病毒DNA。我们发现，对于所有基质，超滤浓缩的病毒基因组比氯化铁絮凝浓缩的病毒基因组高1-2个数量级，从而产生了适用于无扩增和长测序测序的更高质量的DNA。鉴于其作为病毒采样的廉价现场方法的广泛应用和实用性，我们仍然试图优化铁的絮凝效果。我们发现病毒最集中在海水中，铁浓度比所用标准高五倍；抑制DNase活性降低了净化效果；从海水中絮凝病毒所需的铁比海水多五倍。将这种广泛使用的方法应用于淡水病毒样本。全面的，我们的结果表明，在测试的基质中，超滤和纯化的性能优于氯化铁的絮凝和纯化。鉴于方法的性能取决于样品的固体含量和盐度，我们建议在浓缩和纯化与此处测试的样品类型不同的样品时，采用加标回收实验。