Although shotgun metagenomic sequencing of microbiome samples enables partial reconstruction of strain-level community structure, obtaining high-quality microbial genome drafts without isolation and culture remains difficult. Here, we present an application of read clouds, short-read sequences tagged with long-range information, to microbiome samples. We present Athena, a de novo assembler that uses read clouds to improve metagenomic assemblies. We applied this approach to sequence stool samples from two healthy individuals and compared it with existing short-read and synthetic long-read metagenomic sequencing techniques. Read-cloud metagenomic sequencing and Athena assembly produced the most comprehensive individual genome drafts with high contiguity (>200-kb N50, fewer than ten contigs), even for bacteria with relatively low (20×) raw short-read-sequence coverage. We also sequenced a complex marine-sediment sample and generated 24 intermediate-quality genome drafts (>70% complete, <10% contaminated), nine of which were complete (>90% complete, <5% contaminated). Our approach allows for culture-free generation of high-quality microbial genome drafts by using a single shotgun experiment.
Marine sediments contain half of all marine microbial cells, and benthic archaea constitute an important part of those microorganisms. Studies on the activity of these archaea demonstrate these microorganisms are deeply entwined in carbon cycling within sediments and influence the availability of inorganic and organic carbon to the atmosphere and the deep subsurface. However, the metabolic capabilities of most benthic archaea are poorly characterized; therefore, their specific contributions to carbon cycling are unknown. Furthermore, the relationship between genetic and functional diversity of benthic archaeal lineages and the physicochemical and ecosystem controls on that diversity are unknown. This project aims to address these gaps in our knowledge, utilizing stable isotope probing to determine the incorporation of isotopically labelled substrates in conjunction with metagenomic and metatranscriptomic sequencing of benthic archaeal communities within sediments collected across a transect. This approach will identify the autotrophic, heterotrophic, and mixotrophic activity of known and uncharacterized benthic archaea, and assess differences in the magnitude of their activity. Furthermore, the proposed analyses will provide insight on the genetic diversity of expressed genes to help determine what genetic variations determine the archaeal community compositions in different environments and how these organisms relate to those found in the deep subsurface.
Nitrogen fixation, the microbial conversion of N2 to NH3 (i.e., diazotrophy), is the largest natural source of bioavailable nitrogen to the biosphere. Previous work has demonstrated the unexpected occurrence of N2 fixation in deep-sea sediments, particularly at sites of elevated carbon loading, including methane seeps, whale falls, and oxygen minimum zones. However, the organisms responsible for this diazotrophic activity are largely unknown. Here, we investigated diazotroph identity, diversity and activity within marine sediment (0-3 and 9-12 cmbsf) collected at 2893 m water depth within Monterey Canyon off the coast of San Francisco, CA. Through an analysis of nifH sequences, a key gene in nitrogen fixation, and a density-gradient stable-isotope-probing experiment (15N-DNA-SIP), we found evidence for a diverse assemblage of functional diazotrophs spanning multiple phylogenetic groups, including Deltaproteobacteria, Gammaproteobacteria, Planctomycetes, and Acidobacteria. Comparison to closest cultured representatives based on 16S rRNA identities suggests these putative diazotrophs are catabolically diverse. Such diversity may increase their collective resilience and ability to provide a sustained source of new nitrogen to the ecosystem. Additionally, we quantified the effect of sample preparation on isotopically-labelled archaeal cells for analysis by nanoscale secondary ion mass spectrometry (nanoSIMS), a technique to investigate activity of uncultured cells. This analysis revealed a greater effect of sample preparation than was previously reported, suggesting that previous estimates of microbial activity assessed by this method are underestimates. Together, our results reveal the identity of diverse diazotrophs in deep marine sediment, and improve our ability to quantify rates of activity in uncultured microbial cells.
Although the subsurface biosphere is now recognized as an important reservoir of life on our planet, until recently the microbial community beneath open-ocean oligotrophic gyres (making up the majority of the seafloor) has not been studied in detail (D’Hondt et al., 2004, 2009). IODP Expedition 329 has taken a first step at characterizing the microbial community beneath the South Pacific Gyre. This region has low biological surface productivity and therefore very low organic carbon burial rates (10-8 and 10-10 moles C cm-1 yr-1), deep oxygen penetration (sediments are oxidized to the basement), and low prokaryotic cell counts (106 cells cm-3 to <103 cells cm-3) (D’Hondt et al., 2009; Fischer et al., 2009, IODP Exp. 329 Preliminary Report, 2011). In these sediments, the dominant fraction of organic carbon may be aggregated or adsorbed to minerals (Arnarson & Kiel 2007). Thus the ability to colonize minerals should be an important ecological adaptation, with those microbes that are able to grow on the minerals creating potential “hotspots” of microbial activity within these oligotrophic sediments. Our project aims to determine whether there is stimulated microbial activity associated in long-term incubations with H13CO3- and 15NO3-. Specific mineral and clay fractions in the oligotrophic South Pacific Gyre sediment system were targeted using combination of magnetic and density separation and SEM-EDS. The bacterial and archaeal community were examined by CARD-FISH, CARD-FISH-nanoSIMS, and 16S rRNA tag sequencing. Overall results from this C-DEBI grant have shown the viability of magnetic separation and identification of single cells in subsurface sediments as a method for investigating mineral association in microbial communities. We have identified putatively viable cells attached to 7 Fe/Mn-rich minerals, potentially representing an unexplored strategy for low-carbon environments. We also have discovered a higher level of diversity in the paramagnetic (Fe/Mn-rich) mineral-associated bacteria and higher number of Marine Group I archaeal OTUs compared to the diamagnetic fraction in the oligotrophic subsurface sediment from the South Pacific Gyre.