Metagenome-assembled Genomes Research Articles

A single amplified genome catalog reveals the dynamics of mobilome and resistome in the human microbiome

BackgroundThe increase in metagenome-assembled genomes (MAGs) has advanced our understanding of the functional characterization and taxonomic assignment within the human microbiome. However, MAGs, as population consensus genomes, often aggregate heterogeneity among species and strains, thereby obfuscating the precise relationships between microbial hosts and mobile genetic elements (MGEs). In contrast, single amplified genomes (SAGs) derived via single-cell genome sequencing can capture individual genomic content, including MGEs.ResultsWe introduce the first substantial SAG dataset (bbsag20) from the human oral and gut microbiome, comprising 17,202 SAGs above medium-quality without co-assembly. This collection unveils a diversity of bacterial lineages across 312 oral and 647 gut species, demonstrating different taxonomic compositions from MAGs. Moreover, the SAGs showed cellular-level evidence of the translocation of oral bacteria to the gut. We also identified broad-host-range MGEs harboring antibiotic resistance genes (ARGs), which were not detected in the MAGs.ConclusionsThe difference in taxonomic composition between SAGs and MAGs indicates that combining both methods would be effective in expanding the genome catalog. By connecting mobilomes and resistomes in individual samples, SAGs could meticulously chart a dynamic network of ARGs on MGEs, pinpointing potential ARG reservoirs and their spreading patterns in the microbial community.DX-3H-wDoVG35Spx9tFU_UVideo

Metagenomic analysis reveals abundance of mixotrophic, heterotrophic, and homoacetogenic bacteria in a hydrogen-based membrane biofilm reactor

Heterotrophic microorganisms are frequently observed in hydrogenotrophic denitrification systems and are presumed to contribute to their improved performance. However, their roles and metabolic pathways in the hydrogen-based membrane biofilm reactor (H2−MBfR) system remain unclear. The objective of this study was to elucidate the underlying mechanisms driving heterotrophic denitrification. For this purpose, metagenomic analysis was conducted on an H2−MBfR showing higher denitrification performance, focusing on the metabolic function of the microbial community. Functional genes related to H2 oxidation, organic carbon metabolism, and denitrification were the major targets of interest. This analysis revealed a substantial number of genes associated with the oxidation of organic carbon compounds in the biofilm, suggesting its potential for heterotrophic denitrification. Investigation of the genes of interest in metagenome-assembled genomes (MAGs) has demonstrated a predominance of mixotrophs or heterotrophs rather than obligate autotrophs. Notably, MAGs exhibiting the highest abundance of genes of interest were affiliated with Hydrogenophaga and Thauera, implying their significant role in denitrifying the H2−MBfR as mixotrophs utilizing both H2 and organic substrates. The identification of 11 MAGs, presumed to originate from homoacetogens suggested that acetate might contribute to the proliferation of heterotrophs. Based on these metagenomic findings, possible metabolic pathways were identified to explain heterotrophic denitrification within the H2−MBfR biofilms.

Metagenome sequencing and 982 microbial genomes from Kermadec and Diamantina Trenches sediments

Deep-sea trenches representing an intriguing ecosystem for exploring the survival and evolutionary strategies of microbial communities in the highly specialized deep-sea environments. Here, 29 metagenomes were obtained from sediment samples collected from Kermadec and Diamantina trenches. Notably, those samples covered a varying sampling depths (from 5321 m to 9415 m) and distinct layers within the sediment itself (from 0~40 cm in Kermadec trench and 0~24 cm in Diamantina trench). Through metagenomic binning process, we reconstructed 982 metagenome assembled genomes (MAGs) with completeness >60% and contamination <5%. Within them, completeness of 351 MAGs were >90%, while an additional 331 were >80%. Phylogenomic analysis for the MAGs revealed nearly all of them were distantly related to known cultivated isolates. The abundant bacterial MAGs affiliated to phyla of Proteobacteria, Planctomycetota, Nitrospirota, Acidobacteriota, Actinobacteriota, and Chlorofexota, while the abundant archaeal phyla affiliated with Nanoarchaeota and Thermoproteota. These results provide a dataset available for further interrogation of diversity, distribution and ecological function of deep-sea microbes existed in the trenches.

Insights into the carbon and nitrogen metabolism pathways in mixed-autotrophy/heterotrophy anammox consortia in response to temperature reduction

While the multi-coupled anammox system boasts a substantial research foundation, the specific characteristics of its synergistic metabolic response to decreased temperatures, particularly within the range of 13-15°C, remained elusive. In this study, we delve into the intricate carbon and nitrogen metabolism pathways of mixed-autotrophy/heterotrophy anammox consortia under conditions of temperature reduction. Our macrogenomic analyses reveal a compelling phenomenon: the stimulation of functional genes responsible for complete denitrification, suggesting an enhancement of this process during temperature reduction. This adaptation likely contributes to maintaining system performance amidst environmental challenges. Further metabolic functional recombination analyses highlight a dramatic shift in microbial community composition, with denitrifying MAGs (metagenome-assembled genomes) experiencing a substantial increase in abundance (up to 200 times) compared to autotrophic MAGs. This proliferation underscores the strong stimulatory effect of temperature reduction on denitrifying species. Notably, autotrophic MAGs play a pivotal role in supporting the glycolytic processes of denitrifying MAGs, underscoring the intricate interdependencies within the consortia. Moreover, metabolic variations in amino acid composition among core MAGs emerge as a crucial adaptation mechanism. These differences facilitate the preservation of enzyme activity and enhance the consortia's resilience to low temperatures. Together, these findings offer a comprehensive understanding of the microbial synergistic metabolism within mixed-autotrophy/heterotrophy anammox consortia under temperature reduction, shedding light on their metabolic flexibility and resilience in dynamic environments.

Genome-resolved metagenomics revealed novel microbial taxa with ancient metabolism from macroscopic microbial mat structures inhabiting anoxic deep reefs of a Maldivian Blue Hole.

Blue holes are vertical water-filled openings in carbonate rock that exhibit complex morphology, ecology, and water chemistry. In this study, macroscopic microbial mat structures found in complete anoxic conditions in the Faanu Mudugau Blue Hole (Maldives) were studied by metagenomic methods. Such communities have likely been evolutionary isolated from the surrounding marine environment for more than 10,000 years since the Blue Hole formation during the last Ice Age. A total of 48 high-quality metagenome-assembled genomes (MAGs) were recovered, predominantly composed of the phyla Chloroflexota, Proteobacteria and Desulfobacterota. None of these MAGs have been classified to species level (<95% ANI), suggesting the discovery of several new microbial taxa. In particular, MAGs belonging to novel bacterial genera within the order Dehalococcoidales accounted for 20% of the macroscopic mat community. Genome-resolved metabolic analysis of this dominant microbial fraction revealed a mixotrophic lifestyle based on energy conservation via fermentation, hydrogen metabolism and anaerobic CO2 fixation through the Wood-Ljungdahl pathway. Interestingly, these bacteria showed a high proportion of ancestral genes in their genomes providing intriguing perspectives on mechanisms driving microbial evolution in this peculiar environment. Overall, our results provide new knowledge for understanding microbial life under extreme conditions in blue hole environments.

"Metagenomics reveal the potential for geosmin and 2-methylisoborneol production across multiple bacterial phyla in recirculating aquaculture systems".

Geosmin and 2-methylisoborneol (MIB) are known to cause taste-and-odour problems in recirculating aquaculture systems (RAS). Both geosmin and MIB are microbial metabolites belonging to terpenoids. Precursors for terpenoids are biosynthesized via the methylerythritol phosphate (MEP) and the mevalonate (MVA) pathways. We carried out a metagenomic analysis of 50 samples from five RAS to investigate terpenoid biosynthesis and metabolic potential for geosmin and MIB production in RAS microbiomes. A total of 1008 metagenome-assembled genomes (MAGs) representing 26 bacterial and three archaeal phyla were recovered. Although most archaea are thought to use the MVA pathway for terpenoid precursor biosynthesis, an Iainarchaeota archaeal MAG is shown to harbour a complete set of genes encoding the MEP pathway but lacking genes associated with the MVA pathway. In this study, a total of 16 MAGs affiliated with five bacterial phyla (Acidobacteriota, Actinobacteriota, Bacteroidota, Chloroflexota, and Myxococcota) were identified as possessing potential geosmin or MIB synthases. These putative taste and odour producers were diverse, many were taxonomically unidentified at the genus or species level, and their relative abundance differed between the investigated RAS farms. The metagenomic study of the RAS microbiomes revealed a previously unknown phylogenetic diversity of the potential to produce geosmin and MIB.

Genome-centric metagenomic analysis unveils the influence of temperature on the microbiome in anaerobic digestion

Temperature plays a crucial role in shaping microbial ecosystems during anaerobic digestion. However, the specific microbial communities and their functions across a wide temperature range still remain elusive. This study employed a genome-centric metagenomic approach to explore microbial metabolic pathways and synergistic networks at temperatures of 35℃, 44℃, 53℃, 55℃, and 65℃. A total of 84 metagenome assembled genomes (MAGs) were assembled, with over 65% of these MAGs corresponding to novel bacterial and archaeal species (including Firmicutes, Chloroflexota, Bacteroidia and Methanobacteriota), greatly enhancing our current comprehension anaerobic digestion process. Notably, functional annotation identified that 44_bin.2 (Methanothrix_A sp.001602645) harbors enzymes associated with hydrogenotrophic metabolism. Additionally, this microorganism exhibited diverse metabolic capabilities at 44℃, a temperature commonly employed in industrial practice yet less explored in bench studies. Consequently, it implies a promising potential for conducting anaerobic digestion at a moderate thermophilic temperature, as opposed to the conventional mesophilic range. The microorganism exhibited a variety of metabolic capabilities at 44℃, a temperature frequently employed in industrial applications but underexplored in laboratory investigations. The findings suggest that anaerobic digestion carried out at moderate thermophilic temperatures may have a higher potential for methane production.

The metagenomic landscape of a high-altitude geothermal spring in Tajikistan reveals a novel Desulfurococcaceae member, Zestomicrobium tamdykulense gen. nov., sp. nov.

Metagenomic analysis was conducted to assess the microbial community in the high-altitude Tamdykul geothermal spring in Tajikistan. This analysis yielded six high-quality bins from the members of Thermaceae, Aquificaceae, and Halothiobacillaceae, with a 41.2%, 19.7%, and 18.1% share in the total metagenome, respectively. Minor components included Schleiferia thermophila (1.6%) and members of the archaeal taxa Pyrobaculum (1.2%) and Desulfurococcaceae (0.7%). Further analysis of the metagenome-assembled genome (MAG) from the Desulfurococcaceae family (MAG002) revealed novel taxonomy with only 80.95% closest placement average nucleotide identity value to its most closely related member of the Desulfurococcaceae family, which is part of the Thermoproteota phylum comprising hyperthermophilic members widespread in geothermal environments. MAG002 consisted of 1.3 Mbp, distributed into 48 contigs with 1504 predicted coding sequences, had an average GC content of 41.3%, a completeness and contamination rate of 98.7% and 2.6%, respectively, and branched phylogenetically between the Ignisphaera and Zestosphaera lineages. Digital DNA-DNA hybridization values compared with Ignisphaera aggregans and Zestosphaera tikiterensis were 33.7% and 19.4%, respectively, suggesting that this MAG represented a novel species and genus. Its 16S rRNA gene contained a large 421 bp intron. It encodes a complete gluconeogenesis pathway involving a bifunctional fructose-1,6-bisphosphate phosphatase/aldolase; however, the glycolysis pathway is incomplete. The ribulose monophosphate pathway enzymes could be used for pentose synthesis. MAG002 encodes several hydrogen-evolving hydrogenases, with possible roles as hydrogen sinks during fermentation. We propose the name Zestomicrobium tamdykulense gen. nov. sp. nov. for this organism; it is the first thermophilic genome reported from Tajikistan.

Feedstock-dependent antibiotic resistance gene patterns and expression profiles in industrial scale biogas plants revealed by meta-omics technology

This study investigated antimicrobial resistance in the anaerobic digesters of two industrial-scale biogas plants processing agricultural biomass and municipal wastewater sludge. A combination of deep sequencing and genome-centric workflow was implemented for metagenomic and metatranscriptomics data analysis to comprehensively examine potential antimicrobial resistance in microbial communities. Anaerobic microbes were found to harbour numerous antibiotic resistance genes (ARGs), with 58.85% of the metagenome-assembled genomes (MAGs) harbouring antibiotic resistance. A moderately positive correlation was observed between the abundance and expression of ARGs. ARGs were located primarily on bacterial chromosomes. A higher expression of resistance genes was observed on plasmids than on chromosomes. Risk index assessment suggests that most ARGs identified posed a significant risk to human health. However, potentially pathogenic bacteria showed lower ARG expression than non-pathogenic ones, indicating that anaerobic treatment is effective against pathogenic microbes. Resistomes at the gene category level were associated with various antibiotic resistance categories, including multidrug resistance, beta-lactams, glycopeptides, peptides, and macrolide-lincosamide-streptogramin (MLS). Differential expression analysis revealed specific genes associated with potential pathogenicity, emphasizing the importance of active gene expression in assessing the risks associated with ARGs.

Seasonal dynamics in microbial trace metals transporters during phytoplankton blooms in the Southern Ocean.

Trace metals are required as cofactors in metalloproteins that are essential in microbial metabolism and growth. The microbial requirements of diverse metals and the capabilities of prokaryotic taxa to acquire these metals remain poorly understood. We present here results from metagenomic observations over an entire productive season in the region off Kerguelen Island (Indian Sector of the Southern Ocean). We observed seasonal patterns in the abundance of prokaryotic transporters of seven trace elements (zinc [Zn], manganese [Mn], nickel [Ni], molybdenum [Mo], tungsten [W], copper [Cu] and cobalt [Co]) and the consecutive spring and summer phytoplankton blooms were strong drivers of these temporal trends. Taxonomic affiliation of the functional genes revealed that Rhodobacteraceae had a broad repertoire of trace metal transporters (Mn, Zn, Ni, W and Mo) and a more restricted set was observed for other prokaryotic groups, such as Flavobacteriaceae (Zn), Nitrincolaceae (Ni and W) and Thioglobaceae (Mo). The prevalence of trace metal transporters within a prokaryotic group, as determined on the family level, was overall confirmed in representative metagenome-assembled genomes. We discuss the potential involvement of prokaryotic groups in processes related to organic matter utilisation that require these metals and the consequences on carbon and trace metal cycling in surface waters of the Southern Ocean.

Microbial acidification by N, S, Fe and Mn oxidation as a key mechanism for deterioration of subsea tunnel sprayed concrete

The deterioration of fibre-reinforced sprayed concrete was studied in the Oslofjord subsea tunnel (Norway). At sites with intrusion of saline groundwater resulting in biofilm growth, the concrete exhibited significant concrete deterioration and steel fibre corrosion. Using amplicon sequencing and shotgun metagenomics, the microbial taxa and surveyed potential microbial mechanisms of concrete degradation at two sites over five years were identified. The concrete beneath the biofilm was investigated with polarised light microscopy, scanning electron microscopy and X-ray diffraction. The oxic environment in the tunnel favoured aerobic oxidation processes in nitrogen, sulfur and metal biogeochemical cycling as evidenced by large abundances of metagenome-assembled genomes (MAGs) with potential for oxidation of nitrogen, sulfur, manganese and iron, observed mild acidification of the concrete, and the presence of manganese- and iron oxides. These results suggest that autotrophic microbial populations involved in the cycling of several elements contributed to the corrosion of steel fibres and acidification causing concrete deterioration.

Assessment of health risk of antibiotics resistance genes from human disturbed habitat to wild animals: Metagenomic insights into availability and functional changes of gut microbiome

Not all antibiotic resistance genes (ARGs) pose an ecological risk to their host animals. A standard should be developed to study which types of ARGs posed an ecological risk to wild animals under human disturbances (HDs). In this study, the golden snub-nosed monkeys (Rhinopithecus roxellana) were used as sentinel species. According to the animals-associated enrichment, mobility, and pathogenicity, the ARGs in habitat of sentinel species were divided into four levels. If the mobile and pathogenic ARGs that could be collinear with the metagenome-assembled genome (MAGs) in the gut of the sentinel species, the ARGs were defined as Rank I ARGs and they were considered to have ecological risk to sentinel species. Functional genes in the MAGs that collinear with the Rank I ARGs were used to predict the health risks of sentinel species. The ecological risk to sentinel species was present in 0.158 % of the ARGs-contigs in the habitat. Cultivation and villages, but not grazing, agriculture and ecotourism, increased the ecological risk of the ARGs to wild animals, The ability of gut microbiome to acquire mobile and pathogenic ARGs increased, as did the collinear functional genes, and the health risks of the wild animals also enhanced by the disturbances of cultivation and villages. Cultivation and villages increased the nutrient content of the soil, and they had a positive effect on the ecological risk of Rank I ARGs by affecting the mobile genetic elements (MGEs), microbiome and the resistant group in the habitat, which was why the cultivation and villages increased the health risks of wild animals. We proposed that cultivation and living should be controlled, while grazing, agriculture and ecotourism could be developed in nature reserves of wild animals, but the nutrients in the wild animals' habitat should be monitored.

Metagenomic analyses of a consortium for the bioremediation of hydrocarbons polluted soils.

A bacterial consortium was isolated from a soil in Noblejas (Toledo, Spain) with a long history of mixed hydrocarbons pollution, by enrichment cultivation. Serial cultures of hydrocarbons polluted soil samples were grown in a minimal medium using diesel (1mL/L) as the sole carbon and energy source. The bacterial composition of the Noblejas Consortium (NC) was determined by sequencing 16S rRNA gene amplicon libraries. The consortium contained around 50 amplicon sequence variants (ASVs) and the major populations belonged to the genera Pseudomonas, Enterobacter, Delftia, Stenotrophomonas, Achromobacter, Acinetobacter, Novosphingobium, Allorhizobium-Neorhizobium-Rhizobium, Ochrobactrum and Luteibacter. All other genera were below 1%. Metagenomic analysis of NC has shown a high abundance of genes encoding enzymes implicated in aliphatic and (poly) aromatic hydrocarbons degradation, and almost all pathways for hydrocarbon degradation are represented. Metagenomic analysis has also allowed the construction of metagenome assembled genomes (MAGs) for the major players of NC. Metatranscriptomic analysis has shown that several of the ASVs are implicated in hydrocarbon degradation, being Pseudomonas, Acinetobacter and Delftia the most active populations.

Biotransformations of arsenic in marine sediments across marginal slope to hadal zone

Arsenic compounds are accumulating in deep ocean, but their ecological impacts on deep-sea ecosystem remain elusive. We studied 32 sediment cores (101 layers for metagenomes, along with 41 global reference sediment metagenomes) collected from the South China Sea and the Mariana Trench (MT), characterized with high arsenic accumulation in MT. In these metagenomes we revealed a significantly positive correlation between relative abundance of arsenite methyltransferase gene (arsM) and sampling depth, which suggests that arsenic methylation is the most prevalent arsenic biotransformation process in the deep sea. Lower relative abundance of arsenic efflux gene, compared with arsM, indicates that microbes in deep-sea sediments were prone to methylate arsenite and retain it rather than efflux it. Phylogenetic analysis identified seven clades of ArsM proteins, including two new clades derived primarily from deep-sea microorganisms. Five metagenome-assembled genomes containing aioA for arsenite oxidation also harbor carbon fixation genes in the deep-sea sediment layers, suggesting previously unnoticed contribution of arsenite-oxidizing autotrophic bacteria to the carbon cycle. Therefore, deep-sea microorganisms adopt different detoxification and transformation strategies in response to arsenic compounds, which renews our understanding of arsenic in their ecological impacts and potential contribution in deep ocean.

Deep longitudinal lower respiratory tract microbiome profiling reveals genome-resolved functional and evolutionary dynamics in critical illness

The lower respiratory tract (LRT) microbiome impacts human health, especially among critically ill patients. However, comprehensive characterizations of the LRT microbiome remain challenging due to low microbial mass and host contamination. We develop a chelex100-based low-biomass microbial-enrichment method (CMEM) that enables deep metagenomic profiling of LRT samples to recover near-complete microbial genomes. We apply the method to 453 longitudinal LRT samples from 157 intensive care unit (ICU) patients in three geographically distant hospitals. We recover 120 high-quality metagenome-assembled genomes (MAGs) and associated plasmids without culturing. We detect divergent longitudinal microbiome dynamics and hospital-specific dominant opportunistic pathogens and resistomes in pneumonia patients. Diagnosed pneumonia and the ICU stay duration were associated with the abundance of specific antibiotic-resistance genes (ARGs). Moreover, CMEM can serve as a robust tool for genome-resolved analyses. MAG-based analyses reveal strain-specific resistome and virulome among opportunistic pathogen strains. Evolutionary analyses discover increased mobilome in prevailing opportunistic pathogens, highly conserved plasmids, and new recombination hotspots associated with conjugative elements and prophages. Integrative analysis with epidemiological data reveals frequent putative inter-patient strain transmissions in ICUs. In summary, we present a genome-resolved functional, transmission, and evolutionary landscape of the LRT microbiota in critically ill patients.

Metagenomics-based insights into the microbial community dynamics and flavor development potentiality of artificial and natural pit mud

Strong-flavor Baijiu (SFB) production has relied on pit mud (PM) as a starter culture. The maturation time of natural PM (NPM) is about 30 years, so artificial PM (APM) with a shorter maturation time has attracted widespread attention. This study reveals the microbial and functional dissimilarities of APM and NPM, and helps to elucidate the different metabolic roles of microbes during substrate degradation and flavor formation. Significant differences in the microbial community were observed between APM and NPM, manifesting as variations in the abundance of core microorganisms. Total of 187 high-quality metagenome-assembled genomes (MAGs) were obtained based on the metagenomic binning technology, mainly including Firmicutes (n = 106), Bacteroidota (n = 15) and Chloroflexota (n = 14). Furthermore, the relative concentration of flavor compounds in 4-year APM was similar to those in 30-year NPM, but different from those in 100-year NPMs. Methanosarcina, Methanobacterium, Methanoculleus, Anaerolineae bacterium and Aminobacterium were the key bacteria responsible for the flavor differences. From a functional perspective, amino acid and carbohydrate metabolism were key functions of PM microbial, and showed differences between APM and NPM. Finally, substrate degradation and flavor generation pathways were found to exist in multiple microorganisms. Combine the relative abundance of microorganisms with the absolute abundance of enzymes, Clostridium, Lactobacillus, Petrimonas, Methanoculleus, Prevotella, Methanobacterium, Methanosarcina, Methanothrix, Proteiniphilum, Bellilinea, Anaerolinea, Anaeromassilibacillus, Syntrophomonas and Brevefilum were identified as the key microorganisms in APM and NPM.

Investigating drivers of free-living diazotroph activity in paddy soils across China

Microbially mediated N fixation is widespread in rice paddy ecosystems and crucial in maintaining soil fertility. However, our understanding of the factors determining the distribution of free-living diazotrophic microorganisms that perform this process in paddy fields is limited. This study investigated the spatial distribution and factors influencing presence and potential activity of free-living microorganisms capable of N2 fixation in addition to dissimilatory nitrate reduction to ammonium (DNRA), anaerobic ammonium oxidation (anammox), and denitrification in 50 paddy soils across China. Using 15N isotope tracing in laboratory incubations and microbial community analysis via metagenomics, we demonstrate that paddy soils may represent a previously underappreciated hotspot for N2 fixation with mean potential rates of 24.4 ± 17.8 nmol N g−1 h−1, 10-fold higher than DNRA (2.55 ± 0.4 nmol N g−1 h−1), and could counterbalance a portion of N2 losses through anammox and denitrification (9.24 ± 1.1 nmol N g−1 h−1). Site longitude and organic carbon (C) concentrations, as well as the diazotrophic community composition, were the dominant abiotic and biotic factors accounting for regional variations in potential N2 fixation rates. The N2 metabolic pathways predicted from the metagenome-assembled genomes (MAGs) revealed significant co-occurrence of the diazotroph marker gene nifH with denitrification-associated genes (nirS/K and nosZ) and organic C oxidation-related genes (yiaY and galM). Furthermore, enzymes involved in organic C oxidation, particularly glycoside hydrolases and glycosyltransferases, were not only phenotypically correlated with free-living N2 fixation rates but were also identified in nifH-containing MAGs, indicating the heterotrophic capabilities of diazotrophs in paddy soils. Collectively, our results underscore the substantial contribution of free-living N2 fixation to soil N fertility in paddy fields, and highlight the importance of coupling organic C oxidation with nitrate reduction to enhance N2 fixation.

Two distinct Dehalobacter metagenome-assembled genomes from anaerobic chloroform and dichloromethane degrading consortia.

Here we present two metagenomes and two Dehalobacter metagenome-assembled genomes from subcultures of an anaerobic chloroform and dichloromethane degrading microbial community used for bioremediation. Our objective was to assemble and curate the genome(s) of Dehalobacter, key biodegraders in the culture, through repeated sequencing and joint assembly with previous datasets.

Metagenome quality metrics and taxonomical annotation visualization through the integration of MAGFlow and BIgMAG.

Building Metagenome-Assembled Genomes (MAGs) from highly complex metagenomics datasets encompasses a series of steps covering from cleaning the sequences, assembling them to finally group them into bins. Along the process, multiple tools aimed to assess the quality and integrity of each MAG are implemented. Nonetheless, even when incorporated within end-to-end pipelines, the outputs of these pieces of software must be visualized and analyzed manually lacking integration in a complete framework. We developed a Nextflow pipeline (MAGFlow) for estimating the quality of MAGs through a wide variety of approaches (BUSCO, CheckM2, GUNC and QUAST), as well as for annotating taxonomically the metagenomes using GTDB-Tk2. MAGFlow is coupled to a Python-Dash application (BIgMAG) that displays the concatenated outcomes from the tools included by MAGFlow, highlighting the most important metrics in a single interactive environment along with a comparison/clustering of the input data. By using MAGFlow/BIgMAG, the user will be able to benchmark the MAGs obtained through different workflows or establish the quality of the MAGs belonging to different samples following the divide and rule methodology. MAGFlow/BIgMAG represents a unique tool that integrates state-of-the-art tools to study different quality metrics and extract visually as much information as possible from a wide range of genome features.

INAP 2.0: Harnessing metabolic complementarity in microbial network analysis.

With the widespread adoption of metagenomic sequencing, new perspectives have emerged for studying microbial ecological networks, yielding metabolic evidence of interspecies interactions that traditional co-occurrence networks cannot infer. This protocol introduces the integrated Network Analysis Pipeline 2.0 (iNAP 2.0), which features an innovative metabolic complementarity network for microbial studies from metagenomics sequencing data. iNAP 2.0 sets up a four-module process for metabolic interaction analysis, namely: (I) Prepare genome-scale metabolic models; (II) Infer pairwise interactions of genome-scale metabolic models; (III) Construct metabolic interaction networks; and (IV) Analyze metabolic interaction networks. Starting from metagenome-assembled or complete genomes, iNAP 2.0 offers a variety of methods to quantify the potential and trends of metabolic complementarity between models, including the PhyloMint pipeline based on phylogenetic distance-adjusted metabolic complementarity, the SMETANA (species metabolic interaction analysis) approach based on cross-feeding substrate exchange prediction, and metabolic distance calculation based on parsimonious flux balance analysis (pFBA). Notably, iNAP 2.0 integrates the random matrix theory (RMT) approach to find the suitable threshold for metabolic interaction network construction. Finally, the metabolic interaction networks can proceed to analysis using topological feature analysis such as hub node determination. In addition, a key feature of iNAP 2.0 is the identification of potentially transferable metabolites between species, presented as intermediate nodes that connect microbial nodes in the metabolic complementarity network. To illustrate these new features, we use a set of metagenome-assembled genomes as an example to comprehensively document the usage of the tools. iNAP 2.0 is available at https://inap.denglab.org.cn for all users to register and use for free.