Solving genomic puzzles: computational methods for metagenomic binning.

Metagenomics involves the study of genetic material obtained directly from communities of microorganisms living in natural environments. The field of metagenomics has provided valuable insights into the structure, diversity and ecology of microbial communities. Once an environmental sample is sequenced and processed, metagenomic binning clusters the sequences into bins representing different taxonomic groups such as species, genera, or higher levels. Several computational tools have been developed to automate the process of metagenomic binning. These tools have enabled the recovery of novel draft genomes of microorganisms allowing us to study their behaviors and functions within microbial communities. This review classifies and analyzes different approaches of metagenomic binning and different refinement, visualization, and evaluation techniques used by these methods. Furthermore, the review highlights the current challenges and areas of improvement present within the field of research.

AFITbin: a metagenomic contig binning method using aggregate l-mer frequency based on initial and terminal nucleotides.

BACKGROUND: Using next-generation sequencing technologies, scientists can sequence complex microbial communities directly from the environment. Significant insights into the structure, diversity, and ecology of microbial communities have resulted from the study of metagenomics. The assembly of reads into longer contigs, which are then binned into groups of contigs that correspond to different species in the metagenomic sample, is a crucial step in the analysis of metagenomics. It is necessary to organize these contigs into operational taxonomic units (OTUs) for further taxonomic profiling and functional analysis. For binning, which is synonymous with the clustering of OTUs, the tetra-nucleotide frequency (TNF) is typically utilized as a compositional feature for each OTU. RESULTS: In this paper, we present AFIT, a new l-mer statistic vector for each contig, and AFITBin, a novel method for metagenomic binning based on AFIT and a matrix factorization method. To evaluate the performance of the AFIT vector, the t-SNE algorithm is used to compare species clustering based on AFIT and TNF information. In addition, the efficacy of AFITBin is demonstrated on both simulated and real datasets in comparison to state-of-the-art binning methods such as MetaBAT 2, MaxBin 2.0, CONCOT, MetaCon, SolidBin, BusyBee Web, and MetaBinner. To further analyze the performance of the purposed AFIT vector, we compare the barcodes of the AFIT vector and the TNF vector. CONCLUSION: The results demonstrate that AFITBin shows superior performance in taxonomic identification compared to existing methods, leveraging the AFIT vector for improved results in metagenomic binning. This approach holds promise for advancing the analysis of metagenomic data, providing more reliable insights into microbial community composition and function. AVAILABILITY: A python package is available at: https://github.com/SayehSobhani/AFITBin .

Antimicrobial resistance and its risks evaluation in wetlands on the Qinghai-Tibetan Plateau.

Amidst the global antimicrobial resistance (AMR) crisis, antibiotic resistance has permeated even the most remote environments. To understand the dissemination and evolution of AMR in minimally impacted ecosystems, the resistome and mobilome of wetlands across the Qinghai-Tibetan Plateau and its marginal regions were scrutinized using metagenomic sequencing techniques. The composition of wetland microbiomes exhibits significant variability, with dominant phyla including Proteobacteria, Actinobacteria, Bacteroidetes, and Verrucomicrobia. Notably, a substantial abundance of Antibiotic Resistance Genes (ARGs) and Mobile Genetic Elements (MGEs) was detected, encompassing 17 ARG types, 132 ARG subtypes, and 5 types of MGEs (Insertion Sequences, Insertions Sequences, Genomic Islands, Transposons, and Integrative Conjugative Elements). No significant variance was observed in the prevalence of resistome and mobilome across different wetland types (i.e., the Yellow River, other rivers, lakes, and marshes) (R=-0.5882, P=0.607). The co-occurrence of 74 ARG subtypes and 22 MGEs was identified, underscoring the pivotal role of MGEs in shaping ARG pools within the Qinghai-Tibetan Plateau wetlands. Metagenomic binning and analysis of assembled genomes (MAGs) revealed that 93 out of 206 MAGs harbored ARGs (45.15 %). Predominantly, Burkholderiales, Pseudomonadales, and Enterobacterales were identified as the primary hosts of these ARGs, many of which represent novel species. Notably, a substantial proportion of ARG-carrying MAGs also contained MGEs, reaffirming the significance of MGEs in AMR dissemination. Furthermore, utilizing the arg_ranker framework for risk assessment unveiled severe contamination of high-risk ARGs across most plateau wetlands. Moreover, some prevalent human pathogens were identified as potential hosts for these high-risk ARGs, posing substantial transmission risks. This study aims to investigate the prevalence of resistome and mobilome in wetlands, along with evaluating the risk posed by high-risk ARGs. Such insights are crucial for informing environmental protection strategies and facilitating the management of water resources on the Qinghai-Tibetan Plateau.

Oxygen mediated mobilization and co-occurrence of antibiotic resistance in lab-scale bioreactor using metagenomic binning.

Sub-lethal levels of antibiotic stimulate bacteria to generate reactive oxygen species (ROS) that promotes emergence and spread of antibiotic resistance mediated by mobile genetic elements (MGEs). Nevertheless, the influence of dissolved oxygen (DO) levels on mobility of antibiotic resistance genes (ARGs) in response to ROS-induced stress remains elusive. Thus, the study employs metagenomic assembly and binning approaches to decipher mobility potential and co-occurrence frequency of ARGs and MGEs under hyperoxic (5.5-7 mgL- 1), normoxic (2.5-4 mgL- 1), and hypoxic (0.5-1 mgL- 1) conditions in lab-scale bioreactor for 6 months. Among 163 high-quality metagenome-assembled genomes (MAGs) recovered from 13 metagenomes, 42 MAGs harboured multiple ARGs and were assigned to priority pathogen group. Total ARG count increased by 4.3 and 2.5% in hyperoxic and normoxic, but decreased by 0.53% in hypoxic conditions after 150 days. On contrary, MGE count increased by 7.3-1.3% in all the DO levels, with only two ARGs showed positive correlation with MGEs in hypoxic compared to 20 ARGs under hyperoxic conditions. Opportunistic pathogens (Escherichia, Klebsiella, Clostridium, and Proteus) were detected as potential hosts of ARGs wherein co-localisation of critical ARG gene cassette (sul1, dfr1,adeF, and qacC) were identified in class 1 integron/Tn1 family transposons. Thus, enhanced co-occurrence frequency of ARGs with MGEs in pathogens suggested promotion of ARGs mobility under oxidative stress. The study offers valuable insights into ARG dissemination and hosts dynamics that is essential for controlling oxygen-related stress for mitigating MGEs and ARGs in the environment.

Fairy: fast approximate coverage for multi-sample metagenomic binning.

BACKGROUND: Metagenomic binning, the clustering of assembled contigs that belong to the same genome, is a crucial step for recovering metagenome-assembled genomes (MAGs). Contigs are linked by exploiting consistent signatures along a genome, such as read coverage patterns. Using coverage from multiple samples leads to higher-quality MAGs; however, standard pipelines require all-to-all read alignments for multiple samples to compute coverage, becoming a key computational bottleneck. RESULTS: We present fairy ( https://github.com/bluenote-1577/fairy ), an approximate coverage calculation method for metagenomic binning. Fairy is a fast k-mer-based alignment-free method. For multi-sample binning, fairy can be > 250 × faster than read alignment and accurate enough for binning. Fairy is compatible with several existing binners on host and non-host-associated datasets. Using MetaBAT2, fairy recovers 98.5 % of MAGs with > 50 % completeness and < 5 % contamination relative to alignment with BWA. Notably, multi-sample binning with fairy is always better than single-sample binning using BWA ( > 1.5 × more > 50 % complete MAGs on average) while still being faster. For a public sediment metagenome project, we demonstrate that multi-sample binning recovers higher quality Asgard archaea MAGs than single-sample binning and that fairy's results are indistinguishable from read alignment. CONCLUSIONS: Fairy is a new tool for approximately and quickly calculating multi-sample coverage for binning, resolving a computational bottleneck for metagenomics. Video Abstract.

Metagenomic insights into the toxicity of carbamazepine to functional microorganisms in sludge anaerobic digestion.

Contaminants of emerging concern (CECs) contained in sludge, such as carbamazepine, may be toxic to microorganisms and affect the biogenesis of methane during anaerobic digestion. In this study, different scales of anaerobic digesters were constructed to investigate the inhibitory effect of carbamazepine. Results showed that carbamazepine reduced methane production by 11.3 % and 62.1 % at concentrations of 0.4 and 2 mg/g TS, respectively. Carbamazepine hindered the dissolution of organic matter and the degradation of protein. Carbamazepine inhibited some fermentative bacteria, especially uncultured Aminicenantales, whose abundance decreased by 9.5-93.4 % under carbamazepine stress. It is worth noting that most prior studies investigated the effects of CECs only based on well-known microorganisms, ignoring the metabolisms of uncultured microorganisms. Genome-predicted metabolic potential suggested that 54 uncultured metagenome-assembled genomes (MAGs) associated with acidogenesis or acetogenesis. Therein, uncultured Aminicenantales related MAGs were proved to be acetogenic fermenters, their significant reduction may be an important reason for the decrease of methane production under carbamazepine stress. The toxicity of carbamazepine to microorganisms was mainly related to the overproduction of reactive oxygen species. This study elucidates the inhibition mechanism of carbamazepine and emphasizes the indispensable role of uncultured microorganisms in anaerobic digestion.

A mixed blessing of influent leachate microbes in downstream biotreatment systems of a full-scale landfill leachate treatment plant.

In landfill leachate treatment plants (LLTPs), the microbiome plays a pivotal role in the decomposition of organic compounds, reduction in nutrient levels, and elimination of toxins. However, the effects of microbes in landfill leachate influents on downstream treatment systems remain poorly understood. To address this knowledge gap, we collected 23 metagenomic and 12 metatranscriptomic samples from landfill leachate and activated sludge from various treatment units in a full-scale LLTP. We successfully recovered 1,152 non-redundant metagenome-assembled genomes (MAGs), encompassing a wide taxonomic range, including 48 phyla, 95 classes, 166 orders, 247 families, 238 genera, and 1,152 species. More diverse microbes were observed in the influent leachate than in the downstream biotreatment systems, among which, an unprecedented ∼30 % of microbes with transcriptional expression migrated from the influent to the biological treatment units. Network analysis revealed that 399 shared MAGs across the four units exhibited high node centrality and degree, thus supporting enhanced interactions and increased stability of microbial communities. Functional reconstruction and genome characterization of MAGs indicated that these shared MAGs possessed greater capabilities for carbon, nitrogen, sulfur, and arsenic metabolism compared to non-shared MAGs. We further identified a novel species of Zixibacteria in the leachate influent with discrete lineages from those in other environments that accounted for up to 17 % of the abundance of the shared microbial community and exhibited notable metabolic versatility. Meanwhile, we presented groundbreaking evidence of the involvement of Zixibacteria-encoded genes in the production of harmful gas emissions, such as N2O and H2S, at the transcriptional level, thus suggesting that influent microbes may pose safety risks to downstream treatment systems. In summary, this study revealed the complex impact of the influent microbiome on LLTP and emphasizes the need to consider these microbial characteristics when designing treatment technologies and strategies for landfill leachate management.

Co-occurrence of dominant bacteria and methanogenic archaea and their metabolic traits in a thermophilic anaerobic digester.

Thermophilic anaerobic digestion (TAD) represents a promising biotechnology for both methane energy production and waste stream treatment. However, numerous critical microorganisms and their metabolic characteristics involved in this process remain unidentified due to the limitations of culturable isolates. This study investigated the phylogenetic composition and potential metabolic traits of bacteria and methanogenic archaea in a TAD system using culture-independent metagenomics. Predominant microorganisms identified in the stable phase of TAD included hydrogenotrophic methanogens (Methanothermobacter and Methanosarcina) and hydrogen-producing bacteria (Coprothermobacter, Acetomicrobium, and Defluviitoga). Nine major metagenome-assembled genomes (MAGs) associated with the dominant genera were selected to infer their metabolic potentials. Genes related to thermal resistance were widely found in all nine major MAGs, such as the molecular chaperone genes, Clp protease gene, and RNA polymerase genes, which may contribute to their predominance under thermophilic condition. Thermophilic temperatures may increase the hydrogen partial pressure of Coprothermobacter, Acetomicrobium, and Defluviitoga, subsequently altering the primary methanogenesis pathway from acetoclastic pathway to hydrogenotrophic pathway in the TAD. Consequently, genes encoding the hydrogenotrophic methanogenesis pathway were the most abundant in the recovered archaeal MAGs. The potential interaction between hydrogen-producing bacteria and hydrogenotrophic methanogens may play critical roles in TAD processes.

Optimizing anaerobic digestion: Benefits of mild temperature transition from thermophilic to mesophilic conditions.

Anaerobic digestion (AD) plays a significant role in renewable energy recovery. Upgrading AD from thermophilic (50-57 °C) to mesophilic (30-38 °C) conditions to enhance process stability and reduce energy input remains challenging due to the high sensitivity of thermophilic microbiomes to temperature fluctuations. Here we compare the effects of two decreasing-temperature modes from 55 to 35 °C on cell viability, microbial dynamics, and interspecies interactions. A sharp transition (ST) is a one-step transition by 20 °C d-1, while a mild transition (MT) is a stepwise transition by 1 °C d-1. We find a greater decrease in methane production with ST (88.8%) compared to MT (38.9%) during the transition period. ST mode overproduced reactive oxygen species by 1.6-fold, increased membrane permeability by 2.2-fold, and downregulated microbial energy metabolism by 25.1%, leading to increased apoptosis of anaerobes by 1.9-fold and release of intracellular substances by 2.9-fold, further constraining methanogenesis. The higher (1.6 vs. 1.1 copies per gyrA) metabolic activity of acetate-dependent methanogenesis implied more efficient methane production in a steady mesophilic, MT-mediated system. Metagenomic binning and network analyses indicated that ST induced dysbiosis in keystone species and greatly enhanced microbial functional redundancy, causing loss of microbial syntrophic interactions and redundant metabolic pathways. In contrast, the greater microbial interconnections (average degrees 44.9 vs. 22.1) in MT at a steady mesophilic state suggested that MT could better maintain necessary system functionality and stability through microbial syntrophy or specialized pathways. Adopting MT to transform thermophilic digesters into mesophilic digesters is feasible and could potentially enhance the further optimization and broader application of practical anaerobic engineering.

Composition and metabolic flexibility of hydrocarbon-degrading consortia in oil reservoirs.

Hydrocarbon-degrading consortia (HDC) play an important role in petroleum exploitation. However, the real composition and metabolic mechanism of HDC in the microbial enhanced oil recovery (MEOR) process remain unclear. By combining 13C-DNA stable isotope probing microcosms with metagenomics, some newly reported phyla, including Chloroflexi, Synergistetes, Thermotogae, and Planctomycetes, dominated the HDC in the oil reservoirs. In the field trials, the HDC in the aerobic-facultative-anaerobic stage of oilfields jointly promoted the MEOR process, with monthly oil increments of up to 189 tons. Pseudomonas can improve oil recovery by producing rhamnolipid in the facultative condition. Roseovarius was the novel taxa potentially oxidizing alkane and producing acetate to improve oil porosity and permeability in the aerobic condition. Ca. Bacteroidia were the new members potentially degrading hydrocarbons by fumarate addition in the anaerobic environment. Comprehensive identification of the active HDC in oil reservoirs provides a novel theoretical basis for oilfield regulatory scheme.

Functional traits and health implications of the global household drinking-water microbiome retrieved using an integrative genome-centric approach.

The biological safety of drinking water plays a crucial role in public health protection. However, research on the drinking water microbiome remains in its infancy, especially little is known about the potentially pathogenic bacteria in and functional characteristics of the microbiome in household tap water that people are directly exposed to. In this study, we used a genomic-centric approach to construct a genetic catalogue of the drinking water microbiome by analysing 116 metagenomic datasets of household tap water worldwide, spanning nine countries/regions on five continents. We reconstructed 859 high-quality metagenome-assembled genomes (MAGs) spanning 27 bacterial and 2 archaeal phyla, and found that the core MAGs belonging to the phylum Proteobacteria encoded the highest metabolic functional diversity of the 33 key complete metabolic modules. In particular, we found that two core MAGs of Brevibacillus and Methylomona encoded genes for methane metabolism, which may support the growth of heterotrophic organisms observed in the oligotrophic ecosystem. Four MAGs of complete ammonia oxidation (comammox) Nitrospira were identified and functional metabolic analysis suggested these may enable mixotrophic growth and encode genes for reactive oxygen stress defence and arsenite reduction that could aid survival in the environment of oligotrophic drinking water systems. Four MAGs were annotated as potentially pathogenic bacteria (PPB) and thus represented a possible public health concern. They belonged to the genera Acinetobacter (n = 3) and Mycobacterium (n = 1), with a total relative abundance of 1.06 % in all samples. The genomes of PPB A. junii and A. ursingii were discovered to contain antibiotic resistance genes and mobile genetic elements that could contribute to antimicrobial dissemination in drinking water. Further network analysis suggested that symbiotic microbes which support the growth of pathogenic bacteria can be targets for future surveillance and removal.

Unveiling degradation mechanism of PAHs by a Sphingobium strain from a microbial consortium.

Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent pollutants with adverse biological effects and pose a serious threat to ecological environments and human health. The previously isolated phenanthrene-degrading bacterial consortium (PDMC) consists of the genera Sphingobium and Pseudomonas and can degrade a wide range of PAHs. To identify the degradation mechanism of PAHs in the consortium PDMC, metagenomic binning was conducted and a Sphingomonadales assembly genome with 100% completeness was obtained. Additionally, Sphingobium sp. SHPJ-2, an efficient degrader of PAHs, was successfully isolated from the consortium PDMC. Strain SHPJ-2 has powerful degrading abilities and various degradation pathways of high-molecular-weight PAHs, including fluoranthene, pyrene, benzo[a]anthracene, and chrysene. Two ring-hydroxylating dioxygenases, five cytochrome P450s, and a pair of electron transfer chains associated with PAH degradation in strain SHPJ-2, which share 83.0%-99.0% similarity with their corresponding homologous proteins, were identified by a combination of Sphingomonadales assembly genome annotation, reverse-transcription quantitative polymerase chain reaction and heterologous expression. Furthermore, when coexpressed in Escherichia coli BL21(DE3) with the appropriate electron transfer chain, PhnA1B1 could effectively degrade chrysene and benzo[a]anthracene, while PhnA2B2 degrade fluoranthene. Altogether, these results provide a comprehensive assessment of strain SHPJ-2 and contribute to a better understanding of the molecular mechanism responsible for the PAH degradation.

Identification of novel Carnobacterium maltaromaticum strains in bone marrow samples of patients with acute myeloid leukemia using a metagenomic binning approach / Identificación de nuevas cepas de Carnobacterium maltaromaticum en muestras de médula ósea de pacientes con leucemia mieloide aguda mediante un enfoque de combinación metagenómica

The aim of this study aimed to examine the existence of a bacterial metagenome in the bone marrow of patients with acute myeloid leukemia (AML). We re-examined whole-genome sequencing data from the bone marrow samples of seven patients with AML, four of whom were remitted after treatment, for metagenomic analysis. After the removal of human reads, unmapped reads were used to profile the species-level composition. We used the metagenomic binning approach to confirm whether the identified taxon was a complete genome of known or novel strains. We observed a unique and novel microbial signature in which Carnobacterium maltaromaticum was the most abundant species in five patients with AML or remission. The complete genome of C. maltaromaticum “BMAML_KR01,” which was observed in all samples, was 100% complete with 8.5% contamination and closely clustered with C. maltaromaticum strains DSM20730 and SF668 based on single nucleotide polymorphism variations. We identified five unique proteins that could contribute to cancer progression and 104 virulent factor proteins in the BMAML_KR01 genome. To our knowledge, this is the first report of a new strain of C. maltaromaticum in patients with AML. The presence of C. maltaromaticum and its new strain in patients indicates an urgent need to validate the existence of this bacterium and evaluate its pathophysiological role.(AU)