Recent advances in single-cell genomics provide an alternative to largely gene-centric metagenomics studies, enabling whole-genome sequencing of uncultivated bacteria. However, single-cell assembly projects are challenging due to (i) the highly nonuniform read coverage and (ii) a greatly elevated number of chimeric reads and read pairs. While recently developed single-cell assemblers have addressed the former challenge, methods for assembling highly chimeric reads remain poorly explored. We present algorithms for identifying chimeric edges and resolving complex bulges in de Bruijn graphs, which significantly improve single-cell assemblies. We further describe applications of the single-cell assembler SPAdes to a new approach for capturing and sequencing "microbial dark matter" that forms small pools of randomly selected single cells (called a mini-metagenome) and further sequences all genomes from the mini-metagenome at once. On single-cell bacterial datasets, SPAdes improves on the recently developed E+V-SC and IDBA-UD assemblers specifically designed for single-cell sequencing. For standard (cultivated monostrain) datasets, SPAdes also improves on A5, ABySS, CLC, EULER-SR, Ray, SOAPdenovo, and Velvet. Thus, recently developed single-cell assemblers not only enable single-cell sequencing, but also improve on conventional assemblers on their own turf. SPAdes is available for free online download under a GPLv2 license.

Summary Desert plants are hypothesized to survive the environmental stress inherent to these regions in part thanks to symbioses with microorganisms, and yet these microbial species, the communities they form, and the forces that influence them are poorly understood.Here we report the first comprehensive investigation of the microbial communities associated with species of Agave, which are native to semiarid and arid regions of Central and North America and are emerging as biofuel feedstocks. We examined prokaryotic and fungal communities in the rhizosphere, phyllosphere, leaf and root endosphere, as well as proximal and distal soil samples from cultivated and native agaves, through Illumina amplicon sequencing.Phylogenetic profiling revealed that the composition of prokaryotic communities was primarily determined by the plant compartment, whereas the composition of fungal communities was mainly influenced by the biogeography of the host species. Cultivated A. tequilana exhibited lower levels of prokaryotic diversity compared with native agaves, although no differences in microbial diversity were found in the endosphere.Agaves shared core prokaryotic and fungal taxa known to promote plant growth and confer tolerance to abiotic stress, which suggests common principles underpinning Agave–microbe interactions.

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Plants intimately associate with diverse bacteria. Plant-associated (PA) bacteria have ostensibly evolved genes enabling adaptation to the plant environment. However, the identities of such genes are mostly unknown and their functions are poorly characterized. We sequenced 484 genomes of bacterial isolates from roots of Brassicaceae, poplar, and maize. We then compared 3837 bacterial genomes to identify thousands of PA gene clusters. Genomes of PA bacteria encode more carbohydrate metabolism functions and fewer mobile elements than related non-plant associated genomes. We experimentally validated candidates from two sets of PA genes, one involved in plant colonization, the other serving in microbe-microbe competition between PA bacteria. We also identified 64 PA protein domains that potentially mimic plant domains; some are shared with PA fungi and oomycetes. This work expands the genome-based understanding of plant-microbe interactions and provides leads for efficient and sustainable agriculture through microbiome engineering.

Single cell genomics is a powerful and increasingly popular tool for studying the genetic make-up of uncultured microbes. A key challenge for successful single cell sequencing and analysis is the removal of exogenous DNA from whole genome amplification reagents. We found that UV irradiation of the multiple displacement amplification (MDA) reagents, including the Phi29 polymerase and random hexamer primers, effectively eliminates the amplification of contaminating DNA. The methodology is quick, simple, and highly effective, thus significantly improving whole genome amplification from single cells.

Ammonia-oxidizing archaea (AOA) of the phylum Thaumarchaeota are widespread in marine and terrestrial habitats, playing a major role in the global nitrogen cycle. However, their evolutionary history remains unexplored, which limits our understanding of their adaptation mechanisms. Here, our comprehensive phylogenomic tree of Thaumarchaeota supports three sequential events: origin of AOA from terrestrial non-AOA ancestors, colonization of the shallow ocean, and expansion to the deep ocean. Careful molecular dating suggests that these events coincided with the Great Oxygenation Event around 2300 million years ago (Mya), and oxygenation of the shallow and deep ocean around 800 and 635-560 Mya, respectively. The first transition was likely enabled by the gain of an aerobic pathway for energy production by ammonia oxidation and biosynthetic pathways for cobalamin and biotin that act as cofactors in aerobic metabolism. The first transition was also accompanied by the loss of dissimilatory nitrate and sulfate reduction, loss of oxygen-sensitive pyruvate oxidoreductase, which reduces pyruvate to acetyl-CoA, and loss of the Wood-Ljungdahl pathway for anaerobic carbon fixation. The second transition involved gain of a K + transporter and of the biosynthetic pathway for ectoine, which may function as an osmoprotectant. The third transition was accompanied by the loss of the uvr system for repairing ultraviolet light-induced DNA lesions. We conclude that oxygen availability drove the terrestrial origin of AOA and their expansion to the photic and dark oceans, and that the stressors encountered during these events were partially overcome by gene acquisitions from Euryarchaeota and Bacteria, among other sources.

Yellowstone Lake is central to the balanced functioning of the Yellowstone ecosystem, yet little is known about the microbial component of its food chain. A remotely operated vehicle provided video documentation (http://www.tbi.montana.edu/media/videos/) and allowed sampling of dilute surface zone waters and enriched lake floor hydrothermal vent fluids. Vent emissions contained substantial H(2)S, CH(4), CO(2) and H(2), although CH(4) and H(2) levels were also significant throughout the lake. Pyrosequencing and near full-length sequencing of Bacteria 16S rRNA gene diversity associated with two vents and two surface water environments demonstrated that this lake contains significant bacterial diversity. Biomass was size-fractionated by sequentially filtering through 20-µm-, 3.0-µm-, 0.8-µm- and 0.1-µm-pore-size filters, with the >0.1 to <0.8 µm size class being the focus of this study. Major phyla included Acidobacteria, Actinobacteria, Bacteroidetes, α- and β-Proteobacteria and Cyanobacteria, with 21 other phyla represented at varying levels. Surface waters were dominated by two phylotypes: the Actinobacteria freshwater acI group and an α-Proteobacteria clade tightly linked with freshwater SAR11-like organisms. We also obtained evidence of novel thermophiles and recovered Prochlorococcus phylotypes (97-100% identity) in one near surface photic zone region of the lake. The combined geochemical and microbial analyses suggest that the foundation of this lake's food chain is not simple. Phototrophy presumably is an important driver of primary productivity in photic zone waters; however, chemosynthetic hydrogenotrophy and methanotrophy are likely important components of the lake's food chain.

The Yellowstone geothermal complex has yielded foundational discoveries that have significantly enhanced our understanding of the Archaea. This study continues on this theme, examining Yellowstone Lake and its lake floor hydrothermal vents. Significant Archaea novelty and diversity were found associated with two near-surface photic zone environments and two vents that varied in their depth, temperature and geochemical profile. Phylogenetic diversity was assessed using 454-FLX sequencing (B51 000 pyrosequencing reads; V1 and V2 regions) and Sanger sequencing of 200 near-full-length polymerase chain reaction (PCR) clones. Automated classifiers (Ribosomal Database Project (RDP) and Greengenes) were problematic for the 454-FLX reads (wrong domain or phylum), although BLAST analysis of the 454-FLX reads against the phylogenetically placed fulllength Sanger sequenced PCR clones proved reliable. Most of the archaeal diversity was associated with vents, and as expected there were differences between the vents and the near-surface photic zone samples. Thaumarchaeota dominated all samples: vent-associated organisms corresponded to the largely uncharacterized Marine Group I, and in surface waters, B69-84% of the 454-FLX reads matched archaeal clones representing organisms that are Nitrosopumilus maritimus-like (96-97% identity). Importance of the lake nitrogen cycling was also suggested by 45% of the alkaline vent phylotypes being closely related to the nitrifier Candidatus Nitrosocaldus yellowstonii. The Euryarchaeota were primarily related to the uncharacterized environmental clones that make up the Deep Sea Euryarchaeal Group or Deep Sea Hydrothermal Vent Group-6. The phylogenetic parallels of Yellowstone Lake archaea to marine microorganisms provide opportunities to examine interesting evolutionary tracks between freshwater and marine lineages.