Optimizing and evaluating the reconstruction of Metagenome-assembled microbial genomes.

BACKGROUND: Microbiome/host interactions describe characteristics that affect the host's health. Shotgun metagenomics includes sequencing a random subset of the microbiome to analyze its taxonomic and metabolic potential. Reconstruction of DNA fragments into genomes from metagenomes (called metagenome-assembled genomes) assigns unknown fragments to taxa/function and facilitates discovery of novel organisms. Genome reconstruction incorporates sequence assembly and sorting of assembled sequences into bins, characteristic of a genome. However, the microbial community composition, including taxonomic and phylogenetic diversity may influence genome reconstruction. We determine the optimal reconstruction method for four microbiome projects that had variable sequencing platforms (IonTorrent and Illumina), diversity (high or low), and environment (coral reefs and kelp forests), using a set of parameters to select for optimal assembly and binning tools. METHODS: We tested the effects of the assembly and binning processes on population genome reconstruction using 105 marine metagenomes from 4 projects. Reconstructed genomes were obtained from each project using 3 assemblers (IDBA, MetaVelvet, and SPAdes) and 2 binning tools (GroopM and MetaBat). We assessed the efficiency of assemblers using statistics that including contig continuity and contig chimerism and the effectiveness of binning tools using genome completeness and taxonomic identification. RESULTS: We concluded that SPAdes, assembled more contigs (143,718 ± 124 contigs) of longer length (N50 = 1632 ± 108 bp), and incorporated the most sequences (sequences-assembled = 19.65%). The microbial richness and evenness were maintained across the assembly, suggesting low contig chimeras. SPAdes assembly was responsive to the biological and technological variations within the project, compared with other assemblers. Among binning tools, we conclude that MetaBat produced bins with less variation in GC content (average standard deviation: 1.49), low species richness (4.91 ± 0.66), and higher genome completeness (40.92 ± 1.75) across all projects. MetaBat extracted 115 bins from the 4 projects of which 66 bins were identified as reconstructed metagenome-assembled genomes with sequences belonging to a specific genus. We identified 13 novel genomes, some of which were 100% complete, but show low similarity to genomes within databases. CONCLUSIONS: In conclusion, we present a set of biologically relevant parameters for evaluation to select for optimal assembly and binning tools. For the tools we tested, SPAdes assembler and MetaBat binning tools reconstructed quality metagenome-assembled genomes for the four projects. We also conclude that metagenomes from microbial communities that have high coverage of phylogenetically distinct, and low taxonomic diversity results in highest quality metagenome-assembled genomes.

Aura-biomes are present in the water layer above coral reef benthic macro-organisms.

As coral reef habitats decline worldwide, some reefs are transitioning from coral- to algal-dominated benthos with the exact cause for this shift remaining elusive. Increases in the abundance of microbes in the water column has been correlated with an increase in coral disease and reduction in coral cover. Here we investigated how multiple reef organisms influence microbial communities in the surrounding water column. Our study consisted of a field assessment of microbial communities above replicate patches dominated by a single macro-organism. Metagenomes were constructed from 20 L of water above distinct macro-organisms, including (1) the coral Mussismilia braziliensis, (2) fleshy macroalgae (Stypopodium, Dictota and Canistrocarpus), (3) turf algae, and (4) the zoanthid Palythoa caribaeorum and were compared to the water microbes collected 3 m above the reef. Microbial genera and functional potential were annotated using MG-RAST and showed that the dominant benthic macro-organisms influence the taxa and functions of microbes in the water column surrounding them, developing a specific "aura-biome". The coral aura-biome reflected the open water column, and was associated with Synechococcus and functions suggesting oligotrophic growth, while the fleshy macroalgae aura-biome was associated with Ruegeria, Pseudomonas, and microbial functions suggesting low oxygen conditions. The turf algae aura-biome was associated with Vibrio, Flavobacterium, and functions suggesting pathogenic activity, while zoanthids were associated with Alteromonas and functions suggesting a stressful environment. Because each benthic organism has a distinct aura-biome, a change in benthic cover will change the microbial community of the water, which may lead to either the stimulation or suppression of the recruitment of benthic organisms.

Oxygen minimum zones harbour novel viral communities with low diversity.

Oxygen minimum zones (OMZs) are oceanographic features that affect ocean productivity and biodiversity, and contribute to ocean nitrogen loss and greenhouse gas emissions. Here we describe the viral communities associated with the Eastern Tropical South Pacific (ETSP) OMZ off Iquique, Chile for the first time through abundance estimates and viral metagenomic analysis. The viral-to-microbial ratio (VMR) in the ETSP OMZ fluctuated in the oxycline and declined in the anoxic core to below one on several occasions. The number of viral genotypes (unique genomes as defined by sequence assembly) ranged from 2040 at the surface to 98 in the oxycline, which is the lowest viral diversity recorded to date in the ocean. Within the ETSP OMZ viromes, only 4.95% of genotypes were shared between surface and anoxic core viromes using reciprocal BLASTn sequence comparison. ETSP virome comparison with surface marine viromes (Sargasso Sea, Gulf of Mexico, Kingman Reef, Chesapeake Bay) revealed a dissimilarity of ETSP OMZ viruses to those from other oceanic regions. From the 1.4 million non-redundant DNA sequences sampled within the altered oxygen conditions of the ETSP OMZ, more than 97.8% were novel. Of the average 3.2% of sequences that showed similarity to the SEED non-redundant database, phage sequences dominated the surface viromes, eukaryotic virus sequences dominated the oxycline viromes, and phage sequences dominated the anoxic core viromes. The viral community of the ETSP OMZ was characterized by fluctuations in abundance, taxa and diversity across the oxygen gradient. The ecological significance of these changes was difficult to predict; however, it appears that the reduction in oxygen coincides with an increased shedding of eukaryotic viruses in the oxycline, and a shift to unique viral genotypes in the anoxic core.