Towards optimized viral metagenomes for double-stranded and single-stranded DNA viruses from challenging soils
- Published
- Accepted
- Subject Areas
- Ecology, Microbiology, Soil Science, Virology
- Keywords
- Soil viruses, Viromes, DNA extraction, Organics, Microbiology, ssDNA viruses, dsDNA viruses, Viromics
- Copyright
- © 2019 Trubl et al.
- Licence
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ Preprints) and either DOI or URL of the article must be cited.
- Cite this article
- 2019. Towards optimized viral metagenomes for double-stranded and single-stranded DNA viruses from challenging soils. PeerJ Preprints 7:e27640v1 https://doi.org/10.7287/peerj.preprints.27640v1
Abstract
Soils impact global carbon cycling and their resident microbes are critical to their biogeochemical processing and ecosystem outputs. Based on studies in marine systems, viruses infecting soil microbes likely modulate host activities via mortality, horizontal gene transfer, and metabolic control. However, their roles remain largely unexplored due to technical challenges with separating, isolating, and extracting DNA from viruses in soils. Some of these challenges have been overcome by using whole genome amplification methods and while these have allowed insights into the identities of soil viruses and their genomes, their inherit biases have prevented meaningful ecological interpretations. Here we experimentally optimized steps for generating quantitatively-amplified viral metagenomes to better capture both ssDNA and dsDNA viruses across three distinct soil habitats along a permafrost thaw gradient. First, we assessed differing DNA extraction methods (PowerSoil, Wizard mini columns, and cetyl trimethylammonium bromide) for quantity and quality of viral DNA. This established PowerSoil as best for yield and quality of DNA from our samples, though ~1/3 of the viral populations captured by each extraction kit were unique, suggesting appreciable differential biases among DNA extraction kits. Second, we evaluated the impact of purifying viral particles after resuspension (by cesium chloride gradients; CsCl) and of viral lysis method (heat vs bead-beating) on the resultant viromes. DNA yields after CsCl particle-purification were largely non-detectable, while unpurified samples yielded 1–2-fold more DNA after lysis by heat than by bead-beating. Virome quality was assessed by the number and size of metagenome-assembled viral contigs, which showed no increase after CsCl-purification, but did from heat lysis relative to bead-beating. We also evaluated sample preparation protocols for ssDNA virus recovery. In both CsCl-purified and non-purified samples, ssDNA viruses were successfully recovered by using the Accel-NGS 1S Plus Library Kit. While ssDNA viruses were identified in all three soil types, none were identified in the samples that used bead-beating, suggesting this lysis method may impact recovery. Further, 13 ssDNA vOTUs were identified compared to 582 dsDNA vOTUs, and the ssDNA vOTUs only accounted for ~4% of the assembled reads, implying dsDNA viruses were dominant in these samples. This optimized approach was combined with the previously published viral resuspension protocol into a sample-to-virome protocol for soils now available at protocols.io, where community feedback creates ‘living’ protocols. This collective approach will be particularly valuable given the high physicochemical variability of soils, which will may require considerable soil type-specific optimization. This optimized protocol provides a starting place for developing quantitatively-amplified viromic datasets and will help enable viral ecogenomic studies on organic-rich soils.
Author Comment
This is a submission to PeerJ for review.
Supplemental Information
Experiment 1 Bioanalyzer results
Extracted DNA was run on a Bioanalyzer High Sensitivity DNA Assay for all samples and successful libraries (see methods) are shown. Each sample had 15 PCR cycles. Upper marker designated with purple and lower marker with green.
Relative abundance of vOTUs across 2 bog and 4 fen viromes with variable genome coverage cutoffs (Experiment 1)
Four heatmaps are shown comparing the relative abundances of the 516 vOTUs with different thresholds on the minimum percentage of genome covered (10%, 20%, 30%, and 75%). The relative abundance was normalized per Gbp of metagenome and log10-transformed. All mapping used a minimum nucleotide identify of 90%.
Relative abundance of vOTUs across 12 palsa viromes with variable genome coverage cutoffs (Experiment 2)
Six heatmaps are shown comparing the relative abundances of the 66 vOTUs with different thresholds on the minimum percentage of genome covered, increasing in increments of 10 (0–60%). The relative abundance was normalized per Gbp of metagenome and log10-transformed. All mapping used a minimum nucleotide identify of 90%.
Diversity metrics of vOTUs
(A) Richness (R), Pielou's evenness index (J), and Shannon’s Diversity index (H) were calculated for each virome and the viromes are plotted by habitat. Within each habitat the viromes are denoted by a circle, but displayed differently per treatment. For Experiment 1 (bog and fen), viromes are colored green for PowerSoil and blue for Wizard DNA extractions methods. Experiment 2 (palsa) viromes are outlined in red for heat treated samples or blue for bead-beating samples. The marker is filled in for samples that were CsCl purified. (B) A principal coordinate analysis of the viromes by normalized relative abundance of the 66 vOTUs from Experiment 2 based on their Bray-Curtis dissimilarity. Viromes distinguished by soil core, purification (+CsCl outlined in red), and lysis method.
ssDNA vOTUs from both Experiments
The detected ssDNA virus sequences (see methods) were clustered at 95% average nucleotide identify (ANI) across 85% of their contig length, resulting in 13 vOTUs from the 18 viromes. The ssDNA viruses from each experiment are listed along with their corresponding marker gene and habitat of origin.