Integrated metatranscriptomic and metagenomic analyses of stratified microbial assemblages in the open ocean

As part of an ongoing survey of microbial community gene expression in the ocean, we sequenced and compared ~38 Mbp of community transcriptomes and ~157 Mbp of community genomes from four bacterioplankton samples, along a defined depth profile at Station ALOHA in North Pacific subtropical gyre (NPSG). Taxonomic analysis suggested that the samples were dominated by three taxa: Prochlorales, Consistiales and Cenarchaeales, which comprised 36–69% and 29–63% of the annotated sequences in the four DNA and four cDNA libraries, respectively. The relative abundance of these taxonomic groups was sometimes very different in the DNA and cDNA libraries, suggesting differential relative transcriptional activities per cell. For example, the 125 m sample genomic library was dominated by Pelagibacter (~36% of sequence reads), which contributed fewer sequences to the community transcriptome (~11%). Functional characterization of highly expressed genes suggested taxon-specific contributions to specific biogeochemical processes. Examples included Roseobacter relatives involved in aerobic anoxygenic phototrophy at 75 m, and an unexpected contribution of low abundance Crenarchaea to ammonia oxidation at 125 m. Read recruitment using reference microbial genomes indicated depth-specific partitioning of coexisting microbial populations, highlighted by a transcriptionally active high-light-like Prochlorococcus population in the bottom of the photic zone. Additionally, nutrient-uptake genes dominated Pelagibacter transcripts, with apparent enrichment for certain transporter types (for example, the C4-dicarboxylate transport system) over others (for example, phosphate transporters). In total, the data support the utility of coupled DNA and cDNA analyses for describing taxonomic and functional attributes of microbial communities in their natural habitats.Gordon and Betty Moore FoundationUnited States. Dept. of EnergyNational Science Foundation (U.S.) (Science and Technology Center Award EF0424599

Practical application of self-organizing maps to interrelate biodiversity and functional data in NGS-based metagenomics

Next-generation sequencing (NGS) technologies have enabled the application of broad-scale sequencing in microbial biodiversity and metagenome studies. Biodiversity is usually targeted by classifying 16S ribosomal RNA genes, while metagenomic approaches target metabolic genes. However, both approaches remain isolated, as long as the taxonomic and functional information cannot be interrelated. Techniques like self-organizing maps (SOMs) have been applied to cluster metagenomes into taxon-specific bins in order to link biodiversity with functions, but have not been applied to broad-scale NGS-based metagenomics yet. Here, we provide a novel implementation, demonstrate its potential and practicability, and provide a web-based service for public usage. Evaluation with published data sets mimicking varyingly complex habitats resulted into classification specificities and sensitivities of close to 100% to above 90% from phylum to genus level for assemblies exceeding 8 kb for low and medium complexity data. When applied to five real-world metagenomes of medium complexity from direct pyrosequencing of marine subsurface waters, classifications of assemblies above 2.5 kb were in good agreement with fluorescence in situ hybridizations, indicating that biodiversity was mostly retained within the metagenomes, and confirming high classification specificities. This was validated by two protein-based classifications (PBCs) methods. SOMs were able to retrieve the relevant taxa down to the genus level, while surpassing PBCs in resolution. In order to make the approach accessible to a broad audience, we implemented a feature-rich web-based SOM application named TaxSOM, which is freely available at http://www.megx.net/toolbox/taxsom. TaxSOM can classify reads or assemblies exceeding 2.5 kb with high accuracy and thus assists in linking biodiversity and functions in metagenome studies, which is a precondition to study microbial ecology in a holistic fashion

High-resolution SAR11 ecotype dynamics at the Bermuda Atlantic Time-series Study site by phylogenetic placement of pyrosequences

Advances in next-generation sequencing technologies are providing longer nucleotide sequence reads that contain more information about phylogenetic relationships. We sought to use this information to understand the evolution and ecology of bacterioplankton at our long-term study site in the Western Sargasso Sea. A bioinformatics pipeline called PhyloAssigner was developed to align pyrosequencing reads to a reference multiple sequence alignment of 16S ribosomal RNA (rRNA) genes and assign them phylogenetic positions in a reference tree using a maximum likelihood algorithm. Here, we used this pipeline to investigate the ecologically important SAR11 clade of Alphaproteobacteria. A combined set of 2.7 million pyrosequencing reads from the 16S rRNA V1–V2 regions, representing 9 years at the Bermuda Atlantic Time-series Study (BATS) site, was quality checked and parsed into a comprehensive bacterial tree, yielding 929 036 Alphaproteobacteria reads. Phylogenetic structure within the SAR11 clade was linked to seasonally recurring spatiotemporal patterns. This analysis resolved four new SAR11 ecotypes in addition to five others that had been described previously at BATS. The data support a conclusion reached previously that the SAR11 clade diversified by subdivision of niche space in the ocean water column, but the new data reveal a more complex pattern in which deep branches of the clade diversified repeatedly Q2 across depth strata and seasonal regimes. The new data also revealed the presence of an unrecognized clade of Alphaproteobacteria, here named SMA-1 (Sargasso Mesopelagic Alphaproteobacteria, group 1), in the upper mesopelagic zone. The high-resolution phylogenetic analyses performed herein highlight significant, previously unknown, patterns of evolutionary diversification, within perhaps the most widely distributed heterotrophic marine bacterial clade, and strongly links to ecosystem regimes

Single-cell enabled comparative genomics of a deep ocean SAR11 bathytype

Bacterioplankton of the SAR11 clade are the most abundant microorganisms in marine systems, usually representing 25% or more of the total bacterial cells in seawater worldwide. SAR11 is divided into subclades with distinct spatiotemporal distributions (ecotypes), some of which appear to be specific to deep water. Here we examine the genomic basis for deep ocean distribution of one SAR11 bathytype (depth-specific ecotype), subclade Ic. Four single-cell Ic genomes, with estimated completeness of 55%-86%, were isolated from 770 m at station ALOHA and compared with eight SAR11 surface genomes and metagenomic datasets. Subclade Ic genomes dominated metagenomic fragment recruitment below the euphotic zone. They had similar COG distributions, high local synteny and shared a large number (69%) of orthologous clusters with SAR11 surface genomes, yet were distinct at the 16S rRNA gene and amino-acid level, and formed a separate, monophyletic group in phylogenetic trees. Subclade Ic genomes were enriched in genes associated with membrane/cell wall/envelope biosynthesis and showed evidence of unique phage defenses. The majority of subclade Ic-specfic genes were hypothetical, and some were highly abundant in deep ocean metagenomic data, potentially masking mechanisms for niche differentiation. However, the evidence suggests these organisms have a similar metabolism to their surface counterparts, and that subclade Ic adaptations to the deep ocean do not involve large variations in gene content, but rather more subtle differences previously observed deep ocean genomic data, like preferential amino-acid substitutions, larger coding regions among SAR11 clade orthologs, larger intergenic regions and larger estimated average genome size