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

Abstract

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.

Mimicking prophage induction in the body: induction in the lab with pH gradients

“…It is possible that the phage protein coats were not denatured during this heat treatment. While amending the protocol to include phage lysate DNA extraction may circumvent this issue, the efficacy of DNA extraction protocols varies based on sample type ( Kleiner, Hooper & Duerkop, 2015 ; Castro-Mejía et al, 2015 , p.; Verbanic et al, 2019 ; Burgener et al, 2020 ; Göller et al, 2020 , p.). Second, the induced phage may have a low burst size, below the detection threshold of standard PCR, recently shown to be 32 copies of the genome per 1 µL input DNA ( Purcell et al, 2016 ).…”

Viromes outperform total metagenomes in revealing the spatiotemporal patterns of agricultural soil viral communities

“…Moreover, taxonomic classification of the recovered 16S rRNA gene reads revealed clear differences in the microbial profiles associated with each approach: total metagenomes were significantly enriched in Acidobacteria, Actinobacteria, Firmicutes, and Thaumarchaeota, whereas viromes were significantly enriched in Armatimonadetes, Saccharibacteria, and Parcubacteria (SFig 2A-B). These last two taxa belong to the Candidate Phyla Radiation (CPR) clade and are typified by small cells (56)(57)(58), which would be more likely to pass through the 0.22 um filter that we used for viral particle purification (36).…”

“…Until recently, large amounts of soil input were required to avoid these amplification biases, but recent improvements in library construction from nanograms of DNA now make it practical to work with manageable amounts of soil (~50 g or less) per sample (30). Continuous development of optimized extraction workflows has enabled virome preparation from a variety of soils (15,36), greatly expanding our ability to examine viral diversity in these environments.…”

Minnesota peat viromes reveal terrestrial and aquatic niche partitioning for local and global viral populations

“…Although we now have an array of laboratory and bioinformatics methods for soil viral ecology [7,15,23,31,34,[46][47][48][49][50][51], we lack a thorough comparative understanding of these approaches and best practices. As one specific example, viral size-fraction metagenomes (viromes) from grassland and agricultural soils have been shown to be substantially enriched in viral and ultrasmall cellular organismal DNA, compared to total metagenomes that tend to be more enriched in DNA from cellular organisms too large to easily pass through the 0.2 µm filters used for viral enrichment [35,52].…”