BACKGROUND Bacteriophages (phages) are the most numerous biological entities on Earth and play a crucial role in shaping microbial communities. Investigating the bacteriophage community from soil will shed light not only on the yet largely unknown phage diversity, but may also result in novel insights towards their functioning in the global biogeochemical nutrient cycle and their significance in earthbound ecosystems. Unfortunately, information about soil viromes is rather scarce compared to aquatic environments, due to the heterogeneous soil matrix, which rises major technical difficulties in the extraction process. Resolving these technical challenges and establishing a standardized extraction protocol is, therefore, a fundamental prerequisite for replicable results and comparative virome studies. RESULTS We here report the optimization of protocols for the extraction of phage DNA from agricultural soil preceding metagenomic analysis such that the protocol can equally be harnessed for phage isolation. As an optimization strategy, soil samples were spiked with Listeria phage A511 (Myovirus), Staphylococcus phage 2638AΔLCR (Siphovirus) and Escherichia phage T7 (Podovirus) (each 106 PFU/g soil). The efficacy of phage (i) elution, (ii) filtration, (iii) concentration and (iv) DNA extraction methods was tested. Successful extraction routes were selected based on spiked phage recovery and low bacterial 16S rRNA gene contaminants. Natural agricultural soil viromes were then extracted with the optimized methods and shotgun sequenced. Our approach yielded sufficient amounts of inhibitor-free viral DNA for shotgun sequencing devoid of amplification prior library preparation, and low 16S rRNA gene contamination levels (≤ 0.2‰). Compared to previously published protocols, the number of bacterial read contamination was decreased by 65%. In addition, 379 novel putative complete soil phage genomes (≤ 235 kb) were obtained from over 13,000 manually identified viral contigs, promising the discovery of a large, previously inaccessible viral diversity. CONCLUSION We have shown a considerably enhanced extraction of the soil phage community by protocol optimization that has proven robust in both culture-dependent as well as through viromic analyses. Our huge data set of manually curated soil viral contigs substantially increases the amount of currently available soil virome data, and provides insights into the yet largely undescribed soil viral sequence space.

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发现隐藏的多样性：从土壤中提取dsDNA噬菌体的优化方案。

背景技术噬菌体（噬菌体）是地球上数量最多的生物实体，在塑造微生物群落中起着至关重要的作用。从土壤中调查噬菌体群落不仅将揭示尚未广泛了解的噬菌体多样性，而且还将导致对其在全球生物地球化学养分循环中的功能及其在地球生态系统中的意义的新颖见解。不幸的是，由于土壤基质的不均一性，与水生环境相比，有关土壤病毒的信息相当匮乏，这在提取过程中增加了主要的技术难度。因此，解决这些技术难题并建立标准化的提取方案是可复制结果和比较病毒学研究的基本前提。结果我们在此报告了在宏基因组学分析之前从农业土壤中提取噬菌体DNA的方案的优化，因此该方案同样可以用于噬菌体分离。作为一种优化策略，土壤样品中掺有李斯特菌噬菌体A511（肌病毒），葡萄球菌噬菌体2638AΔLCR（Siphovirus）和大肠埃希氏菌噬菌体T7（Podovirus）（每个106 PFU / g土壤）。测试了噬菌体（i）洗脱，（ii）过滤，（iii）浓缩和（iv）DNA提取方法的功效。根据加标的噬菌体回收率和低细菌16S rRNA基因污染物选择成功的提取途径。然后使用优化方法提取天然农业土壤病毒并进行shot弹枪测序。我们的方法可产生足够数量的无抑制剂病毒DNA，用于散弹枪测序，无需在文库制备之前进行扩增，并且16S rRNA基因污染水平低（≤0.2‰）。与以前发布的协议相比，细菌读取污染的数量减少了65％。此外，从超过13,000个人工鉴定的病毒重叠群中获得了379个新的假定的完整土壤噬菌体基因组（≤235 kb），有望发现以前无法获得的巨大病毒多样性。结论我们已经证明，通过方案优化可以大大提高对土壤噬菌体群落的提取，这在依赖于培养物以及通过毒理学分析中均被证明是可靠的。我们庞大的人工整理土壤病毒重叠群数据集大大增加了当前可用的土壤病毒学数据量，