ABSTRACT
Microbes play fundamental roles in shaping natural ecosystem properties and functions, but do so under constraints imposed by their viral predators. However, studying viruses in nature can be challenging due to low biomass and the lack of universal gene markers. Though metagenomic short-read sequencing has greatly improved our virus ecology toolkit-and revealed many critical ecosystem roles for viruses-microdiverse populations and fine-scale genomic traits are missed. Some of these microdiverse populations are abundant and the missed regions may be of interest for identifying selection pressures that underpin evolutionary constraints associated with hosts and environments. Though long-read sequencing promises complete virus genomes on single reads, it currently suffers from high DNA requirements and sequencing errors that limit accurate gene prediction. Here we introduce VirION2, an integrated short- and long-read metagenomic wet-lab and informatics pipeline that updates our previous method (VirION) to further enhance the utility of long-read viral metagenomics. Using a viral mock community, we first optimized laboratory protocols (polymerase choice, DNA shearing size, PCR cycling) to enable 76% longer reads (now median length of 6,965 bp) from 100-fold less input DNA (now 1 nanogram). Using a virome from a natural seawater sample, we compared viromes generated with VirION2 against other library preparation options (unamplified, original VirION, and short-read), and optimized downstream informatics for improved long-read error correction and assembly. VirION2 assemblies combined with short-read based data ('enhanced' viromes), provided significant improvements over VirION libraries in the recovery of longer and more complete viral genomes, and our optimized error-correction strategy using long- and short-read data achieved 99.97% accuracy. In the seawater virome, VirION2 assemblies captured 5,161 viral populations (including all of the virus populations observed in the other assemblies), 30% of which were uniquely assembled through inclusion of long-reads, and 22% of the top 10% most abundant virus populations derived from assembly of long-reads. Viral populations unique to VirION2 assemblies had significantly higher microdiversity means, which may explain why short-read virome approaches failed to capture them. These findings suggest the VirION2 sample prep and workflow can help researchers better investigate the virosphere, even from challenging low-biomass samples. Our new protocols are available to the research community on protocols.io as a 'living document' to facilitate dissemination of updates to keep pace with the rapid evolution of long-read sequencing technology.
ABSTRACT
BACKGROUND: Microorganisms drive high rates of methanogenesis and carbon mineralization in wetland ecosystems. These signals are especially pronounced in the Prairie Pothole Region of North America, the tenth largest wetland ecosystem in the world. Sulfate reduction rates up to 22 µmol cm-3 day-1 have been measured in these wetland sediments, as well as methane fluxes up to 160 mg m-2 h-1-some of the highest emissions ever measured in North American wetlands. While pore waters from PPR wetlands are characterized by high concentrations of sulfur species and dissolved organic carbon, the constraints on microbial activity are poorly understood. Here, we utilized metagenomics to investigate candidate sulfate reducers and methanogens in this ecosystem and identify metabolic and viral controls on microbial activity. RESULTS: We recovered 162 dsrA and 206 dsrD sequences from 18 sediment metagenomes and reconstructed 24 candidate sulfate reducer genomes assigned to seven phyla. These genomes encoded the potential for utilizing a wide variety of electron donors, such as methanol and other alcohols, methylamines, and glycine betaine. We also identified 37 mcrA sequences spanning five orders and recovered two putative methanogen genomes representing the most abundant taxa-Methanosaeta and Methanoregulaceae. However, given the abundance of Methanofollis-affiliated mcrA sequences, the detection of F420-dependent alcohol dehydrogenases, and millimolar concentrations of ethanol and 2-propanol in sediment pore fluids, we hypothesize that these alcohols may drive a significant fraction of methanogenesis in this ecosystem. Finally, extensive viral novelty was detected, with approximately 80% of viral populations being unclassified at any known taxonomic levels and absent from publicly available databases. Many of these viral populations were predicted to target dominant sulfate reducers and methanogens. CONCLUSIONS: Our results indicate that diversity is likely key to extremely high rates of methanogenesis and sulfate reduction observed in these wetlands. The inferred genomic diversity and metabolic versatility could result from dynamic environmental conditions, viral infections, and niche differentiation in the heterogeneous sediment matrix. These processes likely play an important role in modulating carbon and sulfur cycling in this ecosystem.
