Biology subjects

Steen, A. D.

Publications and source records attributed to Steen, A. D..

3 recordsLinked to original sources

bioRxiv2018

Simulation-based approaches to characterize the effect of sequencing depth on the quantity and quality of metagenome-assembled genomes

We applied simulation-based approaches to characterize how microbial community structure influences the amount of sequencing effort to reconstruct metagenomes that are assembled from short read sequences. An initial analysis evaluated the quantity, completion, and contamination of complete-metagenome-assembled genome (complete-MAG) equivalents, a bioinformatic-pipeline normalized metric for MAG quantity, as a function of sequencing effort, on four preexisting sequence read datasets taken from a maize soil, an estuarine sediment, the surface ocean, and the human gut. These datasets were subsampled to varying degrees of completeness in order to simulate the effect of sequencing effort on MAG retrieval. Modeling suggested that sequencing efforts beyond what is typical in published experiments (1 to 10 Gbp) would generate diminishing returns in terms of MAG binning. A second analysis explored the theoretical relationship between sequencing effort and the proportion of available metagenomic DNA sequenced during a sequencing experiment as a function of community richness, evenness, and genome size. Simulations from this analysis demonstrated that while community richness and evenness influenced the amount of sequencing required to sequence a community metagenome to exhaustion, the effort necessary to sequence an individual genome to a target fraction of exhaustion was only dependent on the relative abundance of the corresponding organism and its genome size. A software tool, GRASE, was created to assist investigators further explore this relationship. Re-evaluation of the relationship between sequencing effort and binning success in the context of the relative abundance of genomes, as opposed to base pairs, provides a framework to design sequencing experiments based on the relative abundance of microbes in an environment rather than arbitrary levels of sequencing effort.

bioinformatics↗

bioRxiv2018

Phylogenetically novel uncultured microbial cells dominate Earth microbiomes

To unequivocally determine a microbes physiology, including its metabolism, environmental roles, and growth characteristics, it must be grown in a laboratory culture. Unfortunately, many phylogenetically-novel groups have never been cultured, so their physiologies have only been inferred from genomics and environmental characteristics. Although the diversity, or number of different taxonomic groups, of uncultured clades has been well-studied, their global abundances, or number of cells in any given environment, have not been assessed. We quantified the degree of similarity of 16S rRNA gene sequences from diverse environments in publicly-available metagenome and metatranscriptome databases, which we show are largely free of the culture-bias present in primer-amplified 16S rRNA gene surveys, to their nearest cultured relatives. Whether normalized to scaffold read depths or not, the highest abundance of metagenomic 16S rRNA gene sequences belong to phylogenetically novel uncultured groups in seawater, freshwater, terrestrial subsurface, soil, hypersaline environments, marine sediment, hot springs, hydrothermal vents, non-human hosts, snow and bioreactors (22-87% uncultured genera to classes and 0-64% uncultured phyla). The exceptions were human and human-associated environments which were dominated by cultured genera (45-97%). We estimate that uncultured genera and phyla could comprise 7.3 x 1029 (81%) and 2.2 x 1029 (25%) microbial cells, respectively. Uncultured phyla were over-represented in meta transcript omes relative to metagenomes (46-84% of sequences in a given environment), suggesting that they are viable, and possibly more active than cultured clades. Therefore, uncultured microbes, often from deeply phylogenetically divergent groups, dominate non-human environments on Earth, and their undiscovered physiologies may matter for Earth systems.

microbiology↗

bioRxiv2016

Extracellular peptidases in subsurface sediments of the White Oak River estuary, NC, suggest microbial community adaptation to oxidize degraded organic matter

Anoxic subsurface sediments contain communities of heterotrophic microorganisms that metabolize organic carbon at extraordinarily slow rates. In order to assess the mechanisms by which subsurface microorganisms access detrital sedimentary organic matter, we measured kinetics of a range of extracellular peptidases in anoxic sediments of the White Oak River estuary, NC. Nine distinct peptidase substrates were enzymatically hydrolyzed at all depths. Potential peptidase activities (Vmax) decreased with increasing sediment depth, although Vmax expressed on a per cell basis was approximately the same at all depths. Half-saturation constants (Km) decreased with depth, indicating peptidases that functioned more efficiently at low substrate concentrations. Potential activities of extracellular peptidases acting on molecules that are enriched in degraded organic matter (D-phenylalanine and L-ornithine) increased relative to enzymes that act on L-phenylalanine, further suggesting microbial community adaptation to access degraded organic matter. Nineteen classes of predicted, exported peptidases were identified in genomic data from the same site, of which genes for class C25 (gingipain-like) peptidases represented more than 40% at each depth. Methionine aminopeptidases, zinc carboxypeptidases, and class S24-like peptidases, which are involved in single-stranded DNA repair, were also abundant. These results suggest a subsurface heterotrophic microbial community that primarily accesses low-quality detrital organic matter via a diverse suite of well-adapted extracellular enzymes.\n\nIO_SCPLOWMPORTANCEC_SCPLOWBurial of organic carbon in marine and estuarine sediments represents a long-term sink for atmospheric carbon dioxide. Globally, [~]40% of organic carbon burial occurs in anoxic estuaries and deltaic systems. However, the ultimate controls on the amount of organic matter that is buried in sediments, versus oxidized into CO2, are poorly constrained. Here we used a combination of enzyme assays and metagenomic analysis to identify how subsurface microbial communities catalyze the first step of proteinaceous organic carbon degradation. Our results show that microbial communities in deeper sediments are adapted to access molecules characteristic of degraded organic matter, suggesting that those heterotrophs are adapted to life in the subsurface.

ecology↗