Uncovering a hidden diversity: optimized protocols for the extraction of dsDNA bacteriophages from soil

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 10(6) 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.