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Technical variation in metagenomic analysis must be minimized to confidently assess the contributions of microbiota to human health. Here we tested 21 representative DNA extraction protocols on the same fecal samples and quantified differences in observed microbial community composition. We compared them with differences due to library preparation and sample storage, which we contrasted with observed biological variation within the same specimen or within an individual over time. We found that DNA extraction had the largest effect on the outcome of metagenomic analysis. To rank DNA extraction protocols, we considered resulting DNA quantity and quality, and we ascertained biases in estimates of community diversity and the ratio between Gram-positive and Gram-negative bacteria. We recommend a standardized DNA extraction method for human fecal samples, for which transferability across labs was established and which was further benchmarked using a mock community of known composition. Its adoption will improve comparability of human gut microbiome studies and facilitate meta-analyses.

Microbes are dominant drivers of biogeochemical processes, yet drawing a global picture of functional diversity, microbial community structure, and their ecological determinants remains a grand challenge. We analyzed 7.2 terabases of metagenomic data from 243 Tara Oceans samples from 68 locations in epipelagic and mesopelagic waters across the globe to generate an ocean microbial reference gene catalog with >40 million nonredundant, mostly novel sequences from viruses, prokaryotes, and picoeukaryotes. Using 139 prokaryote-enriched samples, containing >35, 000 species, we show vertical stratification with epipelagic community composition mostly driven by temperature rather than other environmental factors or geography. We identify ocean microbial core functionality and reveal that >73% of its abundance is shared with the human gut microbiome despite the physicochemical differences between these two ecosystems.

Most current approaches for analyzing metagenomic data rely on comparisons to reference genomes, but the microbial diversity of many environments extends far beyond what is covered by reference databases. De novo segregation of complex metagenomic data into specific biological entities, such as particular bacterial strains or viruses, remains a largely unsolved problem. Here we present a method, based on binning co-abundant genes across a series of metagenomic samples, that enables comprehensive discovery of new microbial organisms, viruses and co-inherited genetic entities and aids assembly of microbial genomes without the need for reference sequences. We demonstrate the method on data from 396 human gut microbiome samples and identify 7,381 co-abundance gene groups (CAGs), including 741 metagenomic species (MGS). We use these to assemble 238 high-quality microbial genomes and identify affiliations between MGS and hundreds of viruses or genetic entities. Our method provides the means for comprehensive profiling of the diversity within complex metagenomic samples.

The cnidom of the sea anemone Sagartiogeton viduatus (Muller, 1776) is described from interference-contrast light micrographs (LMs) and scanning electron micrographs (SEMs). Special attention is given to nematocyst maturation, including the differentiation of the shaft into proximal and main regions as helical folding of the shaft wall proceeds. Comparisons are made with Metridium senile (Linnaeus, 1761), whose cnidom, with a few exceptions, is closely similar to that of S.viduatus. The two anemones possess b- and p-mastigophores, p-amastigophores, isorhizas and spirocysts. Although the majority of cnidae in S.viduatus is smaller than corresponding ones in M.senile, they are grouped into the same size classes as those of M.senile, namely small, medium and large. The main differences from M.senile cnidae are the followings: (1) Large acontia p-amastigophores are the largest nematocysts in S.viduatus. (2) They are noticeably larger than the large acontia b-mastigophores, and (3) they are separated from the p-amastigophores of M.senile by the sinusoid pattern of their U-shaped capsular matrix. (4) The large acontia b-mastigophores are microbasic and not mesobasic as in M.Senile, and (5) they do not produce darts. (6) Another difference from M.senile is the absence of catch-tentacle isorhizas.

To quantify known and unknown microorganisms at species-level resolution using shotgun sequencing data, we developed a method that establishes metagenomic operational taxonomic units (mOTUs) based on single-copy phylogenetic marker genes. Applied to 252 human fecal samples, the method revealed that on average 43% of the species abundance and 58% of the richness cannot be captured by current reference genome-based methods. An implementation of the method is available at http://www.bork.embl.de/software/mOTU/.

Despite increasing concerns over inappropriate use of antibiotics in medicine and food production, population-level resistance transfer into the human gut microbiota has not been demonstrated beyond individual case studies. To determine the "antibiotic resistance potential" for entire microbial communities, we employ metagenomic data and quantify the totality of known resistance genes in each community (its resistome) for 68 classes and subclasses of antibiotics. In 252 fecal metagenomes from three countries, we show that the most abundant resistance determinants are those for antibiotics also used in animals and for antibiotics that have been available longer. Resistance genes are also more abundant in samples from Spain, Italy, and France than from Denmark, the United States, or Japan. Where comparable country-level data on antibiotic use in both humans and animals are available, differences in these statistics match the observed resistance potential differences. The results are robust over time as the antibiotic resistance determinants of individuals persist in the human gut flora for at least a year.

In examining large acontia b-mastigophore and p-amastigophore nematocysts of the sea anemone Metridium senile (Linnaeus, 1761) darts were observed by interference-contrast light microscopy (LM) and scanning electron microscopy (SEM). The darts consist of closely packed spines detached from the shafts, still in three helical rows. Their spines form a hollow cylinder with a sharp tip and indented base, its width similar to that of an undischarged shaft but varying in length. b-mastigophore darts were more common than those of p-mastigophores and many were longer.

Acontia and mesentery nematocysts of Metridium senile (Linnaeus, 1761) are described from interference-contrast light micrographs (LMs) and scanning electron micrographs (SEMs). The acontia have 2 nematocyst categories grouped into small, medium and large size-classes, including 5 types: of these, large b-mastigophores and large p-amastigophores are the largest and most abundant. Mesenterial tissues, characterised by small p-mastigophores and medium p-amastigophores, have 3 nematocyst categories grouped as small and medium, including 6 types. Attention is given to nematocyst maturation, especially to the differentiation of the shaft into proximal and main regions as helical folding of the shaft wall proceeds. Groups of differentiating nematoblasts occur along acontia, and near the junction between acontia and mesenterial filaments. Nematoblasts are sparsely found throughout mesenterial tissues.

Tentacle cnidae of Metridium senile (Linnaeus, 1761) were examined by light microscopy. In addition to spirocysts, feeding-tentacles had 3 nematocyst categories grouped into medium and small size-classes, including 5 types. Spirocysts dominated, especially distally, followed by medium b-mastigophores. The density of cnidae decreased towards the tentacle base. Early cnidoblasts were numerous on the tentacle tip. Late cnicloblasts appeared in a moderate number on the mid-tentacle. Catch-tentacles, found in two Metridium specimens, had a maturity gradient of isorhizas and gland cells along their length. Their tip had two distinct types of mature isorhizas in great numbers and large gland cells, but lacked spirocysts. Mature isorhizas and gland cells decreased in number towards the tentacle base. On the mid-tentacle differentiating ages of isorhizas were numerous. Ordinary feeding-tentacle cnidae, abundant at the tentacle base, decreased in number distally along the tentacle.