As the vast majority of microorganisms have yet to be cultivated in a laboratory setting, access to their genetic makeup has largely been limited to cultivation-independent methods. These methods, namely metagenomics and more recently single-cell genomics, have become cornerstones for microbial ecology and environmental microbiology. One ultimate goal is the recovery of genome sequences from each cell within an environment to move toward a better understanding of community metabolic potential and to provide substrate for experimental work. As single-cell sequencing has the ability to decipher all sequence information contained in an individual cell, this method holds great promise in tackling such challenge. Methodological limitations and inherent biases however do exist, which will be discussed here based on environmental and benchmark data, to assess how far we are from reaching this goal.

Asgardarchaeota have been proposed as the closest living relatives to eukaryotes, and a total of 72 metagenome-assembled genomes (MAGs) representing six primary lineages in this archaeal phylum have thus far been described. These organisms are predicted to be fermentative heterotrophs contributing to carbon cycling in sediment ecosystems. Here, we double the genomic catalogue of Asgardarchaeota by obtaining 71 MAGs from a range of habitats around the globe, including the deep subsurface, brackish shallow lakes, and geothermal spring sediments. Phylogenomic inferences followed by taxonomic rank normalisation confirmed previously established Asgardarchaeota classes and revealed four additional lineages, two of which were consistently recovered as monophyletic classes. We therefore propose the names Candidatus Sifarchaeia class nov. and Ca. Jordarchaeia class nov., derived from the gods Sif and Jord in Norse mythology. Metabolic inference suggests that both classes represent hetero-organotrophic acetogens, which also have the ability to utilise methyl groups such as methylated amines, with acetate as the probable end product in remnants of a methanogen-derived core metabolism. This inferred mode of energy conservation is predicted to be enhanced by genetic code expansions, i.e., stop codon recoding, allowing the incorporation of the rare 21st and 22nd amino acids selenocysteine (Sec) and pyrrolysine (Pyl). We found Sec recoding in Jordarchaeia and all other Asgardarchaeota classes, which likely benefit from increased catalytic activities of Sec-containing enzymes. Pyl recoding, on the other hand, is restricted to Sifarchaeia in the Asgardarchaeota, making it the first reported non-methanogenic archaeal lineage with an inferred complete Pyl machinery, likely providing members of this class with an efficient mechanism for methylamine utilisation. Furthermore, we identified enzymes for the biosynthesis of ester-type lipids, characteristic of bacteria and eukaryotes, in both newly described classes, supporting the hypothesis that mixed ether-ester lipids are a shared feature among Asgardarchaeota.

Background

A major obstacle in single-cell sequencing is sample contamination with foreign DNA. To guarantee clean genome assemblies and to prevent the introduction of contamination into public databases, considerable quality control efforts are put into post-sequencing analysis. Contamination screening generally relies on reference-based methods such as database alignment or marker gene search, which limits the set of detectable contaminants to organisms with closely related reference species. As genomic coverage in the tree of life is highly fragmented, there is an urgent need for a reference-free methodology for contaminant identification in sequence data.

Results

We present acdc, a tool specifically developed to aid the quality control process of genomic sequence data. By combining supervised and unsupervised methods, it reliably detects both known and de novo contaminants. First, 16S rRNA gene prediction and the inclusion of ultrafast exact alignment techniques allow sequence classification using existing knowledge from databases. Second, reference-free inspection is enabled by the use of state-of-the-art machine learning techniques that include fast, non-linear dimensionality reduction of oligonucleotide signatures and subsequent clustering algorithms that automatically estimate the number of clusters. The latter also enables the removal of any contaminant, yielding a clean sample. Furthermore, given the data complexity and the ill-posedness of clustering, acdc employs bootstrapping techniques to provide statistically profound confidence values. Tested on a large number of samples from diverse sequencing projects, our software is able to quickly and accurately identify contamination. Results are displayed in an interactive user interface. Acdc can be run from the web as well as a dedicated command line application, which allows easy integration into large sequencing project analysis workflows.

Conclusions

Acdc can reliably detect contamination in single-cell genome data. In addition to database-driven detection, it complements existing tools by its unsupervised techniques, which allow for the detection of de novo contaminants. Our contribution has the potential to drastically reduce the amount of resources put into these processes, particularly in the context of limited availability of reference species. As single-cell genome data continues to grow rapidly, acdc adds to the toolkit of crucial quality assurance tools.

The archaeal phylum 'Diapherotrites' was recently proposed based on phylogenomic analysis of genomes recovered from an underground water seep in an abandoned gold mine (Homestake mine in Lead, SD, USA). Here we present a detailed analysis of the metabolic capabilities and genomic features of three single amplified genomes (SAGs) belonging to the 'Diapherotrites'. The most complete of the SAGs, Candidatus 'Iainarchaeum andersonii' (Cand. IA), had a small genome (∼1.24 Mb), short average gene length (822 bp), one ribosomal RNA operon, high coding density (∼90.4%), high percentage of overlapping genes (27.6%) and low incidence of gene duplication (2.16%). Cand. IA genome possesses limited catabolic capacities that, nevertheless, could theoretically support a free-living lifestyle by channeling a narrow range of substrates such as ribose, polyhydroxybutyrate and several amino acids to acetyl-coenzyme A. On the other hand, Cand. IA possesses relatively well-developed anabolic capabilities, although it remains auxotrophic for several amino acids and cofactors. Phylogenetic analysis suggests that the majority of Cand. IA anabolic genes were acquired from bacterial donors via horizontal gene transfer. We thus propose that members of the 'Diapherotrites' have evolved from an obligate symbiotic ancestor by acquiring anabolic genes from bacteria that enabled independent biosynthesis of biological molecules previously acquired from symbiotic hosts. 'Diapherotrites' 16S rRNA genes exhibit multiple mismatches with the majority of archaeal 16S rRNA primers, a fact that could be responsible for their observed rarity in amplicon-generated data sets. The limited substrate range, complex growth requirements and slow growth rate predicted could be responsible for its refraction to isolation.

Ecogenomic investigation of a methanogenic bioreactor degrading terephthalate (TA) allowed elucidation of complex synergistic networks of uncultivated microorganisms, including those from candidate phyla with no cultivated representatives. Our previous metagenomic investigation proposed that Pelotomaculum and methanogens may interact with uncultivated organisms to degrade TA; however, many members of the community remained unaddressed because of past technological limitations. In further pursuit, this study employed state-of-the-art omics tools to generate draft genomes and transcriptomes for uncultivated organisms spanning 15 phyla and reports the first genomic insight into candidate phyla Atribacteria, Hydrogenedentes and Marinimicrobia in methanogenic environments. Metabolic reconstruction revealed that these organisms perform fermentative, syntrophic and acetogenic catabolism facilitated by energy conservation revolving around H2 metabolism. Several of these organisms could degrade TA catabolism by-products (acetate, butyrate and H2) and syntrophically support Pelotomaculum. Other taxa could scavenge anabolic products (protein and lipids) presumably derived from detrital biomass produced by the TA-degrading community. The protein scavengers expressed complementary metabolic pathways indicating syntrophic and fermentative step-wise protein degradation through amino acids, branched-chain fatty acids and propionate. Thus, the uncultivated organisms may interact to form an intricate syntrophy-supported food web with Pelotomaculum and methanogens to metabolize catabolic by-products and detritus, whereby facilitating holistic TA mineralization to CO2 and CH4.

The candidate phylum Poribacteria is one of the most dominant and widespread members of the microbial communities residing within marine sponges. Cell compartmentalization had been postulated along with their discovery about a decade ago and their phylogenetic association to the Planctomycetes, Verrucomicrobia, Chlamydiae superphylum was proposed soon thereafter. In the present study we revised these features based on genomic data obtained from six poribacterial single cells. We propose that Poribacteria form a distinct monophyletic phylum contiguous to the PVC superphylum together with other candidate phyla. Our genomic analyses supported the possibility of cell compartmentalization in form of bacterial microcompartments. Further analyses of eukaryote-like protein domains stressed the importance of such proteins with features including tetratricopeptide repeats, leucin rich repeats as well as low density lipoproteins receptor repeats, the latter of which are reported here for the first time from a sponge symbiont. Finally, examining the most abundant protein domain family on poribacterial genomes revealed diverse phyH family proteins, some of which may be related to dissolved organic posphorus uptake.

The "Latescibacteria" (formerly WS3), member of the Fibrobacteres-Chlorobi-Bacteroidetes (FCB) superphylum, represents a ubiquitous candidate phylum found in terrestrial, aquatic, and marine ecosystems. Recently, single-cell amplified genomes (SAGs) representing the "Latescibacteria" were obtained from the anoxic monimolimnion layers of Sakinaw Lake (British Columbia, Canada), and anoxic sediments of a coastal lagoon (Etoliko lagoon, Western Greece). Here, we present a detailed in-silico analysis of the four SAGs to gain some insights on their metabolic potential and apparent ecological roles. Metabolic reconstruction suggests an anaerobic fermentative mode of metabolism, as well as the capability to degrade multiple polysaccharides and glycoproteins that represent integral components of green (Charophyta and Chlorophyta) and brown (Phaeophycaea) algae cell walls (pectin, alginate, ulvan, fucan, hydroxyproline-rich glycoproteins), storage molecules (starch and trehalose), and extracellular polymeric substances (EPSs). The analyzed SAGs also encode dedicated transporters for the uptake of produced sugars and amino acids/oligopeptides, as well as an extensive machinery for the catabolism of all transported sugars, including the production of a bacterial microcompartment (BMC) to sequester propionaldehyde, a toxic intermediate produced during fucose and rhamnose metabolism. Finally, genes for the formation of gas vesicles, flagella, type IV pili, and oxidative stress response were found, features that could aid in cellular association with algal detritus. Collectively, these results indicate that the analyzed "Latescibacteria" mediate the turnover of multiple complex organic polymers of algal origin that reach deeper anoxic/microoxic habitats in lakes and lagoons. The implications of such process on our understanding of niche specialization in microbial communities mediating organic carbon turnover in stratified water bodies are discussed.

Since the discovery of the genetic code and protein translation mechanisms (1), a limited number of variations of the standard assignment between unique base triplets (codons) and their encoded amino acids and translational stop signals have been found in bacteria and phages (2-3). Given the apparent ubiquity of the canonical genetic code, the design of genomically recoded organisms with non-canonical codes has been suggested as a means to prevent horizontal gene transfer between laboratory and environmental organisms (4). It is also predicted that genomically recoded organisms are immune to infection by viruses, under the assumption that phages and their hosts must share a common genetic code (5). This paradigm is supported by the observation of increased resistance of genomically recoded bacteria to phages with a canonical code (4). Despite these assumptions and accompanying lines of evidence, it remains unclear whether differential and non-canonical codon usage represents an absolute barrier to phage infection and genetic exchange between organisms.Our knowledge of the diversity of genetic codes and their use by viruses and their hosts is primarily derived from the analysis of cultivated organisms. Advances in single-cell sequencing and metagenome assembly technologies have enabled the reconstruction of genomes of uncultivated bacterial and archaeal lineages (6). These initial findings suggest that large scale systematic studies of uncultivated microorganisms and viruses may reveal the extent and modes of divergence from the canonical genetic code operating in nature.To explore alternative genetic codes, we carried out a systematic analysis of stop codon reassignments from the canonical TAG amber, TGA opal, and TAA ochre codons in assembled metagenomes from environmental and host-associated samples, single-cell genomes of uncultivated bacteria and archaea, and a collection of phage sequences

Single amplified genomes and genomes assembled from metagenomes have enabled the exploration of uncultured microorganisms at an unprecedented scale. However, both these types of products are plagued by contamination. Since these genomes are now being generated in a high-throughput manner and sequences from them are propagating into public databases to drive novel scientific discoveries, rigorous quality controls and decontamination protocols are urgently needed. Here, we present ProDeGe (Protocol for fully automated Decontamination of Genomes), the first computational protocol for fully automated decontamination of draft genomes. ProDeGe classifies sequences into two classes--clean and contaminant--using a combination of homology and feature-based methodologies. On average, 84% of sequence from the non-target organism is removed from the data set (specificity) and 84% of the sequence from the target organism is retained (sensitivity). The procedure operates successfully at a rate of ~0.30 CPU core hours per megabase of sequence and can be applied to any type of genome sequence.