Over the past decade, high-throughput short-read 16S rRNA gene amplicon sequencing has eclipsed clone-dependent long-read Sanger sequencing for microbial community profiling. The transition to new technologies has provided more quantitative information at the expense of taxonomic resolution with implications for inferring metabolic traits in various ecosystems. We applied single-molecule real-time sequencing for microbial community profiling, generating full-length 16S rRNA gene sequences at high throughput, which we propose to name PhyloTags. We benchmarked and validated this approach using a defined microbial community. When further applied to samples from the water column of meromictic Sakinaw Lake, we show that while community structures at the phylum level are comparable between PhyloTags and Illumina V4 16S rRNA gene sequences (iTags), variance increases with community complexity at greater water depths. PhyloTags moreover allowed less ambiguous classification. Last, a platform-independent comparison of PhyloTags and in silico generated partial 16S rRNA gene sequences demonstrated significant differences in community structure and phylogenetic resolution across multiple taxonomic levels, including a severe underestimation in the abundance of specific microbial genera involved in nitrogen and methane cycling across the Lake's water column. Thus, PhyloTags provide a reliable adjunct or alternative to cost-effective iTags, enabling more accurate phylogenetic resolution of microbial communities and predictions on their metabolic potential.

Marine oxygen minimum zones (OMZs) are widespread regions of the ocean that are currently expanding due to global warming. While inhospitable to most metazoans, OMZs are hotspots for microbial mediated biogeochemical cycling of carbon, nitrogen and sulphur, contributing disproportionately to marine nitrogen loss and climate active trace gas production. Our current understanding of microbial community responses to OMZ expansion is limited by a lack of time-resolved data sets linking multi-omic sequence information (DNA, RNA, protein) to geochemical parameters and process rates. Here, we present six years of time-resolved multi-omic observations in Saanich Inlet, a seasonally anoxic fjord on the coast of Vancouver Island, British Columbia, Canada that undergoes recurring changes in water column oxygenation status. This compendium provides a unique multi-omic framework for studying microbial community responses to ocean deoxygenation along defined geochemical gradients in OMZ waters.

Genome sequencing enhances our understanding of the biological world by providing blueprints for the evolutionary and functional diversity that shapes the biosphere. However, microbial genomes that are currently available are of limited phylogenetic breadth, owing to our historical inability to cultivate most microorganisms in the laboratory. We apply single-cell genomics to target and sequence 201 uncultivated archaeal and bacterial cells fromnine diverse habitats belonging to 29 major mostly uncharted branches of the tree of life, so-called microbial dark matter. With this additional genomic information, we are able to resolve many intra- and inter-phylum-level relationships and to propose two new superphyla. We uncover unexpected metabolic features that extend our understanding of biology and challenge established boundaries between the three domains of life. These include a novel amino acid use for the opal stop codon, an archaeal-type purine synthesis in Bacteria and complete sigma factors in Archaea similar to those in Bacteria. The single-cell genomes also served to phylogenetically anchor up to 20percent of metagenomic reads in some habitats, facilitating organism-level interpretation of ecosystem function. This study greatly expands the genomic representation of the tree of life and provides a systematic step towards a better understanding of biological evolution on our planet.

The 'Atribacteria' is a candidate phylum in the Bacteria recently proposed to include members of the OP9 and JS1 lineages. OP9 and JS1 are globally distributed, and in some cases abundant, in anaerobic marine sediments, geothermal environments, anaerobic digesters and reactors and petroleum reservoirs. However, the monophyly of OP9 and JS1 has been questioned and their physiology and ecology remain largely enigmatic due to a lack of cultivated representatives. Here cultivation-independent genomic approaches were used to provide a first comprehensive view of the phylogeny, conserved genomic features and metabolic potential of members of this ubiquitous candidate phylum. Previously available and heretofore unpublished OP9 and JS1 single-cell genomic data sets were used as recruitment platforms for the reconstruction of atribacterial metagenome bins from a terephthalate-degrading reactor biofilm and from the monimolimnion of meromictic Sakinaw Lake. The single-cell genomes and metagenome bins together comprise six species- to genus-level groups that represent most major lineages within OP9 and JS1. Phylogenomic analyses of these combined data sets confirmed the monophyly of the 'Atribacteria' inclusive of OP9 and JS1. Additional conserved features within the 'Atribacteria' were identified, including a gene cluster encoding putative bacterial microcompartments that may be involved in aldehyde and sugar metabolism, energy conservation and carbon storage. Comparative analysis of the metabolic potential inferred from these data sets revealed that members of the 'Atribacteria' are likely to be heterotrophic anaerobes that lack respiratory capacity, with some lineages predicted to specialize in either primary fermentation of carbohydrates or secondary fermentation of organic acids, such as propionate.