Abstract
ABSTRACTThe small genomes of most viruses make it difficult to fully capture viral diversity in metagenomes dominated by DNA from cellular organisms. Viral size fraction metagenomics (viromics) protocols facilitate the enrichment of viral DNA from environmental samples, and these protocols typically include DNase treatment of the post-0.2-μm-filtered viromic fraction to remove contaminating free DNA prior to virion lysis. However, DNase may also remove desirable viral genomic DNA (e.g., contained in virions compromised due to frozen storage or laboratory processing), suggesting that DNase-untreated viromes might be useful in some cases. In order to understand how virome preparation with and without DNase treatment influences the resultant data, here, we compared 15 soil viromes (7 DNase treated and 8 untreated) from 8 samples collected from agricultural fields prior to tomato planting. DNase-treated viromes yielded significantly more assembled viral contigs, contained significantly less nonviral microbial DNA, and recovered more viral populations (viral operational taxonomic units [vOTUs]) through read mapping. However, DNase-treated and untreated viromes were statistically indistinguishable in terms of ecological patterns across viral communities. Although the results suggest that DNase treatment is preferable where possible, in comparison to previously reported total metagenomes from the same samples, both DNase-treated and untreated viromes were significantly enriched in viral signatures by all metrics compared, including a 225-times-higher proportion of viral reads in untreated viromes compared to total metagenomes. Thus, even without DNase treatment, viromics was preferable to total metagenomics for capturing viral diversity in these soils, suggesting that preparation of DNase-untreated viromes can be worthwhile when DNase treatment is not possible.IMPORTANCE Viromics is becoming an increasingly popular method for characterizing soil viral communities. DNase treatment of the viral size fraction prior to DNA extraction is meant to reduce contaminating free DNA and is a common step within viromics protocols to ensure that sequences are of viral origin. However, some samples may not be amenable to DNase treatment due to viral particles being compromised either in storage (i.e., frozen) or during other sample processing steps. To date, the effect of DNase treatment on the recovery of viruses and downstream ecological interpretations of soil viral communities is not thoroughly understood. This work sheds light on these questions and indicates that while DNase treatment of soil viromes improves the recovery of viral populations, this improvement is modest in comparison to the gains made by viromics over total soil metagenomics. Furthermore, DNase treatment may not be necessary to observe the ecological patterns structuring soil viral communities.
Highlights
The small genomes of most viruses make it difficult to fully capture viral diversity in metagenomes dominated by DNA from cellular organisms
Marine viral ecology has benefited from a viromics approach, in which purified, concentrated viral particles are subjected to DNA extraction and metagenomic sequencing [12, 22, 23], most recent soil viral ecological studies have focused on recovering viral signatures from total soil metagenomes [10, 19, 20, 24]
We have shown that DNase treatment of viromes significantly reduced sequence complexity and decreased the amount of contaminating cell-derived DNA by about 2-fold
Summary
The small genomes of most viruses make it difficult to fully capture viral diversity in metagenomes dominated by DNA from cellular organisms. Bioinformatic advances in viral contig identification (e.g., through the recognition of viral hallmark genes and other viral sequence signatures) [25,26,27,28] and efforts to compile viral reference databases that include partial and putative viral genomes [1, 19, 29] have improved our ability to recognize viral genomic sequences in soil metagenomes Despite these advances, our ability to catalog soil viral diversity is still largely gated by the low prevalence of viral DNA in soil and other metagenomes, which tend to be dominated by bacterial and archaeal sequences [10, 19, 30]
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