Title: Towards optimized viral metagenomes for double-stranded and single-stranded DNA viruses from challenging soils

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 viralmore » 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 atprotocols.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.« less

Authors:

Trubl, Gareth ^[1]; Roux, Simon ^[2]; Solonenko, Natalie ^[3]; Li, Yueh-Fen ^[3]; Bolduc, Benjamin ^[3]; Rodríguez-Ramos, Josué ^[4]; Eloe-Fadrosh, Emiley A. ^[2]; Rich, Virginia I. ^[3]; Sullivan, Matthew B. ^[5]

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+ Show Author Affiliations

1. Department of Microbiology, The Ohio State University, Columbus, OH, United States of America, Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, CA, United States of America
2. United States Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Walnut Creek, CA, United States of America
3. Department of Microbiology, The Ohio State University, Columbus, OH, United States of America
4. Department of Microbiology, The Ohio State University, Columbus, OH, United States of America, Department of Soil and Crop Sciences, Colorado State University, Fort Collins, CO, United States of America
5. Department of Microbiology, The Ohio State University, Columbus, OH, United States of America, Department of Civil, Environmental and Geodetic Engineering, The Ohio State University, Columbus, OH, United States of America

Publication Date:

Thu Jul 04 00:00:00 EDT 2019

Research Org.:

Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); Lawrence Livermore National Laboratory (LLNL), Livermore, CA (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Biological and Environmental Research (BER); USDOE National Nuclear Security Administration (NNSA)

OSTI Identifier:

1630370

Alternate Identifier(s):

OSTI ID: 1559173; OSTI ID: 1569170

Report Number(s):

LLNL-JRNL-789611
Journal ID: ISSN 2167-8359; e7265

Grant/Contract Number:  

SC0014664; AC02-05CH11231; DBI-073519; DBI-1265383; AC52-07NA27344

Resource Type:

Published Article

Journal Name:

PeerJ

Additional Journal Information:

Journal Name: PeerJ Journal Volume: 7; Journal ID: ISSN 2167-8359

Publisher:

PeerJ

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; Soil viruses; Viromes; DNA extraction; Organics; Microbiology; ssDNA viruses; dsDNA viruses; Viromics; Environmental sciences