Metagenomic Studies Research Articles

GutMetaNet: an integrated database for exploring horizontal gene transfer and functional redundancy in the human gut microbiome

Abstract Metagenomic studies have revealed the critical roles of complex microbial interactions, including horizontal gene transfer (HGT) and functional redundancy (FR), in shaping the gut microbiome’s functional capacity and resilience. However, the lack of comprehensive data integration and systematic analysis approaches has limited the in-depth exploration of HGT and FR dynamics across large-scale gut microbiome datasets. To address this gap, we present GutMetaNet (https://gutmetanet.deepomics.org/), a first-of-its-kind database integrating extensive human gut microbiome data with comprehensive HGT and FR analyses. GutMetaNet contains 21 567 human gut metagenome samples with whole-genome shotgun sequencing data related to various health conditions. Through systematic analysis, we have characterized the taxonomic profiles and FR profiles, and identified 14 636 HGT events using a shared reference genome database across the collected samples. These HGT events have been curated into 8049 clusters, which are annotated with categorized mobile genetic elements, including transposons, prophages, integrative mobilizable elements, genomic islands, integrative conjugative elements and group II introns. Additionally, GutMetaNet incorporates automated analyses and visualizations for the HGT events and FR, serving as an efficient platform for in-depth exploration of the interactions among gut microbiome taxa and their implications for human health.

Bacterial diversity in rice field soil and sludge soil samples: A comparative metagenomics-based study

Abstract Soil contains several organic and inorganic substances. It also has a large number of microbial diversity including bacteria. This microbial community affects the physicochemical properties of soil. This bacterial bacteria diversity is also involved in biomass degradation and plant growth promotion. Metagenomic sequencing is used to analyze microbial diversity in environmental samples. The metagenomic profiling of bacterial populations in the sludge and rice field soil was done at phylum, class, order, family, genus, and species levels. The metagenomic study revealed that Proteobacteria, Bacteroidetes, Chloroflexi, Acidobacteria and other unknown bacteria were reported in 16S rRNA Illumina MiSeq in the sludge and rice soil samples. We compared the taxonomic classification of bacterial diversity found in sewage soils with that of rice field soils using metagenomic sequencing analysis of the V3-V4 region of the 16S rRNA. In rice soil, the most abundant group was Proteobacteria followed by Chloroflexi, Acidobacteria, Actinobacteria, and Bacteroidetes which increase the soil nutrient and influence the production of the plant. Bacteroidetes are the most dominant group in sludge soil than rice soil. Consequently, the core microbiome analysis indicated that Sorangium, Geobacter, Azoarcus, and Anaeromyxobacter bacterial genera were more abundant in both soil samples.

Metagenomic Analysis of Microbial Diversity in the Moroccan Coastal Water of the Gibraltar Strait

Coastal waters are known for higher productivity and organic matter levels, which support a high diversity and abundance of microorganisms compared to some aquatic environments. The characterization of marine microbiomes can provide valuable information for evaluating the sustainability of coastal waters that are increasingly subjected to environmental and human impacts. Our study is the first metagenomic study realized on Moroccan Mediterranean coastal seawater. We analyzed and described the Gibraltar Detroit marine microbiome taxonomic and functional profiling using MG-RAST software. Shotgun sequencing revealed a predominance of bacterial taxa, particularly the Proteobacteria (57.29%) and Bacteroidetes (27.06%) phyla, alongside notable populations of eukaryotes, viruses, and archaea. Alphaproteobacteria and Gammaproteobacteria emerged as the dominant bacterial classes, while Flavobacteria represented a significant portion of Bacteroidetes. Functional profiling of the microbial community highlighted a wide array of metabolic pathways, emphasizing genes related to carbohydrate metabolism, amino acid synthesis, and protein processing. The marine microbiome exhibited essential biogeochemical activities, particularly in nitrogen, sulfur, and carbon cycles, with notable pathways including denitrification, thiosulfate oxidation, and carbon fixation. This functional diversity underlines the microbiome’s role in sustaining ecosystem health through nutrient cycling and organic matter degradation. Our findings offer a crucial baseline for understanding microbial community structure and resilience in Mediterranean coastal ecosystems, with implications for assessing future environmental and anthropogenic impacts on these microbial dynamics.

Simple, reference-independent assessment to empirically guide correction and polishing of hybrid microbial community metagenomic assembly

Hybrid metagenomic assembly of microbial communities, leveraging both long- and short-read sequencing technologies, is becoming an increasingly accessible approach, yet its widespread application faces several challenges. High-quality references may not be available for assembly accuracy comparisons common for benchmarking, and certain aspects of hybrid assembly may benefit from dataset-dependent, empiric guidance rather than the application of a uniform approach. In this study, several simple, reference-free characteristics–particularly coding gene content and read recruitment profiles–were hypothesized to be reliable indicators of assembly quality improvement during iterative error-fixing processes. These characteristics were compared to reference-dependent genome- and gene-centric analyses common for microbial community metagenomic studies. Two laboratory-scale bioreactors were sequenced with short- and long-read platforms, and assembled with commonly used software packages. Following long read assembly, long read correction and short read polishing were iterated up to ten times to resolve errors. These iterative processes were shown to have a substantial effect on gene- and genome-centric community compositions. Simple, reference-free assembly characteristics, specifically changes in gene fragmentation and short read recruitment, were robustly correlated with advanced analyses common in published comparative studies, and therefore are suitable proxies for hybrid metagenome assembly quality to simplify the identification of the optimal number of correction and polishing iterations. As hybrid metagenomic sequencing approaches will likely remain relevant due to the low added cost of short-read sequencing for differential coverage binning or the ability to access lower abundance community members, it is imperative that users are equipped to estimate assembly quality prior to downstream analyses.

CRISPR spacers acquired from plasmids primarily target backbone genes, making them valuable for predicting potential hosts and host range.

In recent years, there has been a surge in metagenomic studies focused on identifying plasmids in environmental samples. Although these studies have unearthed numerous novel plasmids, enriching our understanding of their environmental roles, a significant gap remains: the scarcity of information regarding the bacterial hosts of these newly discovered plasmids. Furthermore, even when plasmids are identified within bacterial isolates, the reported host is typically limited to the original isolate, with no insights into alternative hosts or the plasmid's potential host range. Given that plasmids depend on hosts for their existence, investigating plasmids without the knowledge of potential hosts offers only a partial perspective. This study introduces a method for identifying potential hosts and host ranges for plasmids through alignment with CRISPR spacers. To validate the method, we compared the PLSDB plasmids database with the CRISPR spacers database, yielding host predictions for 46% of the plasmids. When compared with reported hosts, our predictions achieved 84% concordance at the family level and 99% concordance at the phylum level. Moreover, the method frequently identified multiple potential hosts for a plasmid, thereby enabling predictions of alternative hosts and the host range. Notably, we found that CRISPR spacers predominantly target plasmid backbone genes while sparing functional genes, such as those linked to antibiotic resistance, aligning with our hypothesis that CRISPR spacers are acquired from plasmid-specific regions rather than insertion elements from diverse sources. Finally, we illustrate the network of connections among different bacterial taxa through plasmids, revealing potential pathways for horizontal gene transfer.IMPORTANCEPlasmids are notorious for their role in distributing antibiotic resistance genes, but they may also carry and distribute other environmentally important genes. Since plasmids are not free-living entities and rely on host bacteria for survival and propagation, predicting their hosts is essential. This study presents a method for predicting potential hosts for plasmids and offers insights into the potential paths for spreading functional genes between different bacteria. Understanding plasmid-host relationships is crucial for comprehending the ecological and clinical impact of plasmids and implications for various biological processes.

Comparative genomic analysis and characterization of novel high-quality draft genomes from the coal metagenome.

Coal, a sedimentary rock harbours a complex microbial community that plays a significant role in its formation and characteristics. However, coal metagenome sequencing and studies were less, limiting our understanding of this complex ecosystem. This study aimed to reconstruct high-quality metagenome-assembled genomes (MAGs) from the coal sample collected in the Neyveli mine to explore the unrevealed diversity of the coal microbiome. Using Illumina sequencing, we obtained high-quality raw reads in FASTQ format. Subsequently, de novo assembly and binning with metaWRAP software facilitated the reconstruction of coal MAGs. Quality assessment using CheckM identified 10 High-Quality MAGs (HQ MAGs), 7 medium-quality MAGs (MQ MAGs), and 6 low-quality MAGs (LQ MAGs). Further analysis using GTDB-Tk revealed four HQ MAGs as known species like Dermacoccus abyssi, Sphingomonas aquatilis, Acinetobacter baumannii, and Burkholderia cenocepacia. The remaining six HQ MAGs were classified as Comamonas, Arthrobacter, Noviherbaspirillum, Acidovorax, Oxalicibacterium, and Bordetella and designated as novel genomes by the validation of digital DNA-DNA hybridization (dDDH). Phylogenetic analysis and further pangenome analysis across the phylogenetic groups revealed a similar pattern with a high proportion of cloud genes. We further analysed the functional potential of these MAGs and closely related genomes using COG. The comparative functional genomics revealed that novel genomes are highly versatile, potentially reflecting adaptations to the coal environment. BlastKOALA was used to conduct a detailed analysis of the metabolic pathways associated with the MAGs. This study highlights the comparative genomic analysis of novel coal genomes with their closely related genomes to understand the evolutionary relationships and functional properties.

Adenosine regulates depressive behavior in mice with chronic social defeat stress through gut microbiota

Major depressive disorder (MDD) is recognized as the most prevalent affective disorder worldwide. Metagenomic studies increasingly support a critical role for dysbiosis of gut microbiota in the development of depression. Previous studies have demonstrated that adenosine alleviates gut dysbiosis, suggesting that elevating adenosine levels could be a novel intervention for MDD; however, the mechanisms underlying this effect remain unclear. This study utilized 16S rRNA gene sequencing, fecal microbiota transplantation (FMT) and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to test the hypothesis that increased adenosine alleviates depressive behaviors in male mice subjected to chronic social defeat stress (CSDS) through alterations to gut microbiota. The data showed that depression-susceptible (SUS) mice exhibited gut dysbiosis, and FMT from SUS mice increased depression-like behaviors in healthy recipients. In SUS mice, adenosine supplementation ameliorated both depression-like behaviors and abnormalities in gut microbiota, and co-administration of probiotics and adenosine not only mitigated depression-like behaviors but also enhanced gut barrier integrity. By including 83 depressed adolescents and 67 healthy controls, this study found that the level of short-chain fatty acids (SCFAs) in the depression group was reduced, this finding parallels reductions seen in SUS mice and in recipient mice after FMT from SUS donors. Conversely, supplementation with either adenosine or probiotics led increased SCFAs concentrations in the serum of SUS mice. These findings suggest that adenosine may alleviate depression-like behaviors in CSDS mice by modulating the gut microbiota. This effect is likely associated with increased serum SCFAs, metabolites produced by the gut microbiota, following adenosine supplementation.

Lymphadenopathy in systemic lupus erythematosus: no microbial trigger found by shotgun metagenomics in a retrospective study on 38 patients.

Lymphadenopathy is a classical manifestation of systemic lupus erythematosus (SLE) flare, occurring in approximately half of patients during the course of the disease. Lymphadenopathy in SLE is frequently associated with fever. Microbial infection may play a role in SLE onset and flares. Objectives of this study were to describe lymphadenopathy in the course of SLE and identify potential infectious triggers using microbial metagenomic analysis. We performed a retrospective monocentric study of 38 patients with SLE who had lymph node biopsy at baseline or during follow-up. Shotgun metagenomics were performed in patient's lymph node biopsy to look for microbial RNA and/or DNA. Lymph node pathological analyses revealed follicular and/or paracortical hyperplasia 73.7% of patients and histiocytic necrotizing lymphadenitis 23.7%. At the time of biopsy, SLE patients exhibited fever in 29%, splenomegaly in 10%, cutaneous manifestations in 47%, polyarthritis in 32%, seritis in 13% and lupus nephritis in 18%. Half of patients (50%) had increased CRP level, 35% had low C3, 65% had hypergammaglobulinemia. Microbial metagenomic analysis of lymph node biopsy did not reveal the presence of microbial DNA in 92% of patients, the presence of CMV in very small quantities in 2 patients, and the presence of HHV-7 in low quantities in a single patient. Despite suggestion that certain microorganisms may play a role in the pathogenesis and flares of SLE, our microbial metagenomic analysis study did not highlight possible infectious triggering factors. Further and better-designed studies are needed to confirm these results.

Viral pathogen surveillance in wastewater using metagenomic approaches: obstacles and prospects

Abstract Background Wastewater monitoring offers an important tool for pathogen detection and tracking within large populations at a minimized cost and labor. Untargeted metagenomic approaches for the detection and sequencing of viruses still face limitations. In this study we tested the application of the sequence-independent single-primer amplification (SISPA) protocol in conjunction with nanopore sequencing to the study of viromes in wastewater samples. Methods Influent wastewater was collected from four Wastewater Treatment Plants located in the Lisbon region and Alentejo, Portugal in 2019 and 2023. Samples were processed with different methods including filtration, flocculation, Nanotraps or absorvent tampons. We applied the SISPA protocol, that includes a random amplification step, followed by nanopore sequencing on flow cells R9.4.1. We used the CZ ID (https://czid.org) pipeline for metagenomic analysis and taxonomic reporting. Results A large heterogeneity was found between samples in sequencing output (ranging from 17 thousand to 12 million reads) and the proportion of sequence reads classified as viral (ranging from 0.06% to 4% per sample). Viral taxa with higher number of reads were Picornavirales, dsDNA and ssDNA phages, Mimiviridae and Circoviridae. Known viral pathogens included Rotavirus A, Orthopoxvirus, Human betaherpesvirus and Avian orthoavulavirus. Conclusions Metagenomic studies of environmental samples generally exhibit a low proportion of viral reads, even after viral enrichment. A substantial sequencing effort is thus required to achieve a comprehensive virome characterization. As alternative, a more targeted approach, e.g. to target families of viruses of interest, may be considered. Funding: Fundação para a Ciência e Tecnologia (FCT): GHTM - UID/04413/2020; LA-REAL - LA/P/0117/2020; https://doi.org/10.54499/CEECINST/00102/2018/CP1567/CT0040 to SGS. Research council of the Vrije Universiteit Brussel (OZR-VUB): OZR3863BOF to PL. Key messages • Enrichment for viruses in metagenomic approaches remain challenging in very complex samples as are influent wastewaters. • Virome characterization still necessitates high sequencing efforts but may be used as a complementary environmental survey tool integrated with more targeted approaches.

Metagenomic study of the microbiome and key geochemical potentials associated with architectural heritage sites: a case study of the Song Dynasty city wall in Shou County, China

Historical cultural heritage sites are valuable for all of mankind, as they reflect the material and spiritual wealth of by nations, countries, or specific groups during the development of human civilization. The types and functions of microorganisms that form biofilms on the surfaces of architectural heritage sites influence measures to preserve and protect these sites. These microorganisms contribute to the biocorrosion of architectural heritage structures through the cycling of chemical elements. The ancient city wall of Shou County is a famous architectural and cultural heritage site from China’s Song Dynasty, and the protection and study of this site have substantial historical and cultural significance. In this study, we used metagenomics to study the microbial diversity and taxonomic composition of the Song Dynasty city wall in Shou County, a tangible example of Chinese cultural heritage. The study covered three main topics: (1) examining the distribution of bacteria in the biofilm on the surfaces of the Song Dynasty city wall in Shou County; (2) predicting the influence of bacteria involved in the C, N, and S cycles on the corrosion of the city wall via functional gene analysis; and (3) discussing cultural heritage site protection measures for biocorrosion-related bacteria to investigate the impact of biocorrosion on the Song Dynasty city wall in Shou County, a tangible example of Chinese cultural heritage. The study revealed that (1) the biofilm bacteria mainly belonged to Proteobacteria, Actinobacteria, Cyanobacteria, Bacteroidetes, and Firmicutes, which accounted for more than 70% of the total bacteria in the biofilms. The proportion of fungi in the microbial community of the well-preserved city wall was greater than that in the damaged city wall. The proportion of archaea was low—less than 1%. (2) According to the Shannon diversity index, the well-preserved portion of the ancient city wall had the highest diversity of bacteria, fungi, and archaea, and bacterial diversity on the good city wall was greater than that on the corroded city wall. (3) Bray–Curtis distances revealed that the genomes of the two good city walls were similar and that the genomes of the corroded city wall portions were similar. Researchers also detected human intestine-related bacteria in four locations on the city walls, with the proportion of these bacteria in the microbial community being greater on good city walls than on bad city walls. (4) KEGG functional analysis revealed that the energy metabolism and inorganic ion transport activities of the bacterial community on the corroded city wall were greater than those of the good city wall. (5) In the carbon cycle, the absence of active glycolysis, the ED pathway, and the TCA cycle played significant roles in the collapse of the east city wall. (6) The nitrogen cycling processes involved ammonia oxidation and nitrite reduction to nitrate. (7) In the sulfur cycle, researchers discovered a crucial differential functional gene, SoxY, which facilitates the conversion of thiosulfate to sulfate. This study suggests that, in the future, biological approaches can be used to help cultural heritage site protectors achieve targeted and precise protection of cultural relics through the use of microbial growth inhibition technology. The results of this study serve as a guide for the protection of cultural heritage sites in other parts of China and provide a useful supplement to research on the protection of world cultural heritage or architectural heritage sites.

LukProt: A Database of Eukaryotic Predicted Proteins Designed for Investigations of Animal Origins.

The origins and early evolution of animals are subjects with many outstanding questions. One problem faced by researchers trying to answer them is the absence of a comprehensive database with sequences from nonbilaterians. Publicly available data are plentiful but scattered and often not associated with proper metadata. A new database presented in this paper, LukProt, is an attempt at solving this issue. The database contains protein sequences obtained mostly from genomic, transcriptomic, and metagenomic studies and is an extension of EukProt (Richter DJ, Berney C, Strassert JFH, Poh Y-P, Herman EK, Muñoz-Gómez SA, Wideman JG, Burki F, de Vargas C. EukProt: a database of genome-scale predicted proteins across the diversity of eukaryotes. Peer Community J. 2022:2:e56. https://doi.org/10.24072/pcjournal.173). LukProt adopts the EukProt naming conventions and includes data from 216 additional animals. The database is associated with a taxonomic grouping (taxogroup) scheme suitable for studying early animal evolution. Minor updates to the database will contain species additions or metadata corrections, whereas major updates will synchronize LukProt to each new version of EukProt, and releases are permanently stored on Zenodo (https://doi.org/10.5281/zenodo.7089120). A BLAST server to search the database is available at: https://lukprot.hirszfeld.pl/. Users are invited to participate in maintaining and correcting LukProt. As it can be searched without downloading locally, the database aims to be a convenient resource not only for evolutionary biologists, but for the broader scientific community as well.

Metagenomic Study Reveals Phage–Bacterial Interactome Dynamics in Gut and Oral Microbiota in Pancreatic Diseases

Individuals with pancreatic-related health conditions usually show lower diversity and different composition of bacterial and viral species between the gut and oral microbiomes compared to healthy individuals. We performed a thorough microbiome analysis, using deep shotgun sequencing of stool and saliva samples obtained from patients with chronic pancreatitis (CP), pancreatic ductal adenocarcinoma (PDAC), and healthy controls (HCs).We observed similar microbiota composition at the species level in both the gut and oral samples in PDAC patients compared to HCs, among which the most distinctive finding was that the abundance of oral-originated Fusobacterium nucleatum species did not differ between the oral and the gut samples. Moreover, comparing PDAC patients with HCs, Klebsiella oxytoca was significantly more abundant in the stool samples of PDAC patients, while Streptococcus spp. showed higher abundance in both the oral and stool samples of PDAC patients. Finally, the most important finding was the distinctive gut phage–bacterial interactome pattern among PDAC patients. CrAssphages, particularly Blohavirus, showed mutual exclusion with K. oxytoca species, while Burzaovirus showed co-occurrence with Enterobacteriaceae spp., which have been shown to be capable of inducing DNA damage in human pancreatic cells ex vivo. The interactome findings warrant further mechanistic studies, as our findings may provide new insights into developing microbiota-based diagnostic and therapeutic methods for pancreatic diseases.

Microbial Gatekeepers of Fertility in the Female Reproductive Microbiome of Cattle.

This review paper delves into the intricate relationship between the genital microbiome and fertility outcomes in livestock, with a specific focus on cattle. Drawing upon insights derived from culture-independent metagenomics studies, the paper meticulously examines the composition and dynamics of the genital microbiome. Through advanced techniques such as high-throughput sequencing, the review illuminates the temporal shifts in microbial communities and their profound implications for reproductive health. The analysis underscores the association between dysbiosis-an imbalance in microbial communities-and the development of reproductive diseases, shedding light on the pivotal role of microbial gatekeepers in livestock fertility. Furthermore, the paper emphasizes the need for continued exploration of uncharted dimensions of the female reproductive microbiome to unlock new insights into its impact on fertility. By elucidating the complex interplay between microbial communities and reproductive health, this review underscores the importance of innovative strategies aimed at enhancing fertility and mitigating reproductive diseases in livestock populations.

Oxygen exposure decreases the yield of high-molecular-weight DNA from some anaerobic bacteria and bacterial communities during DNA extraction

ObjectivesThe central challenge in third-generation sequencing lies in meeting the requirements for DNA quality (integrity and purity) and quantity. Therefore, novel improvements in DNA extraction methods are needed to satisfy these requirements. We reasoned that in anaerobic microbial communities, the presence of certain strict anaerobes containing oxygen-activated DNase activity might contribute substantially to the poor integrity of extracted metagenomic DNA (or genomic DNA from some pure cultures) if exposed to air. MethodsTo test this hypothesis, we developed an enhanced genomic and metagenomic DNA isolation technique that we applied to a specifically chosen set of both strict and aerotolerant anaerobes, as well as to the hindgut microbiota of a herbivorous marine fish. ResultsConsidering the quality (or degradation) of extracted DNA obtained under anaerobic versus aerobic conditions, we found that DNA extracted aerobically from cells of some strict anaerobes showed more degradation of high molecular weight DNA than analogous preparations under anaerobic conditions. In contrast, with the selected aerotolerant anaerobes, no discernible difference was found between the molecular sizes of DNA extracted aerobically and anaerobically. Metagenomic DNA extracted from the fish hindgut microbiota showed higher yields and better quality under anaerobic conditions compared to aerobic conditions. ConclusionOur study effectively demonstrates the advantages of our improved extraction protocol in anaerobic conditions. This is evident through the improved quality of extracted DNA. Such findings may be valuable for studies, especially metagenomic studies, where the quality and quantity of DNA are crucial for downstream analysis.

Characterisation of the bacteriomes harboured by major wireworm pest species in the Canadian Prairies.

Nearly all insects harbour bacterial communities that can have a profound effect on their life history, including regulating and shaping host metabolism, development, immunity and fitness. The bacteriomes of several coleopterans have been described; however, very little has been reported for wireworms. These long-lived larvae of click beetles (Coleoptera: Elateridae) are major agricultural pests of a variety of crops grown in the Canadian Prairies. Consequently, the goal of this study was to characterise the bacteriomes of five of the most significant pest species within the region: Limonius californicus, Hypnoidus abbreviatus, H. bicolor, Aeolus mellillus and Dalopius spp. To do this, we collected larvae from southern Manitoba fields (pre-seeding) and carried out 16S rRNA sequencing on individual specimens. Our results indicate wireworms have diverse and taxon-rich bacterial communities, with over 400 genera identified predominately from the phyla Proteobacteria, Actinobacteriota, Bacteroidota and Firmicutes. However, each species had nine or fewer genera comprising >80% of their bacteriome. Network analyses revealed some community structuring consistent among species, which may culminate in shaping/regulating host biology. Moreover, the microbial signatures were influenced by both ontogeny (early vs. late stage larvae) and reproductive strategy (sexual vs. parthenogenetic), with a myriad of other factors likely contributing to bacterial diversity that are impossible to resolve from our study. Overall, this metagenomics study represents the first to characterise the bacteriomes of wireworms in the Canadian Prairies and the findings could assist in the development of sustainable management strategies for these important agricultural pests.

Assessment of Skimmed Milk Flocculation for Bacterial Enrichment from Water Samples, and Benchmarking of DNA Extraction and 16S rRNA Databases for Metagenomics.

Water samples for bacterial microbiome studies undergo biomass concentration, DNA extraction, and taxonomic identification steps. Through benchmarking, we studied the applicability of skimmed milk flocculation (SMF) for bacterial enrichment, an adapted in-house DNA extraction protocol, and six 16S rRNA databases (16S-DBs). Surface water samples from two rivers were treated with SMF and vacuum filtration (VF) and subjected to amplicon or shotgun metagenomics. A microbial community standard underwent five DNA extraction protocols, taxonomical identification with six different 16S-DBs, and evaluation by the Measurement Integrity Quotient (MIQ) score. In SMF samples, the skimmed milk was metabolized by members of lactic acid bacteria or genera such as Polaromonas, Macrococcus, and Agitococcus, resulting in increased relative abundance (p < 0.5) up to 5.0 log fold change compared to VF, rendering SMF inapplicable for bacterial microbiome studies. The best-performing DNA extraction protocols were FastSpin Soil, the in-house method, and EurX. All 16S-DBs yielded comparable MIQ scores within each DNA extraction kit, ranging from 61-66 (ZymoBIOMICs) up to 80-82 (FastSpin). DNA extraction kits exert more bias toward the composition than 16S-DBs. This benchmarking study provided valuable information to inform future water metagenomic study designs.

C/N ratio effect on oily wastewater treatment using column type SBR: machine learning prediction and metagenomics study

The sequencing batch reactor has emerged as a promising technology in treating wastewater; however, its application in the treatment of generated water still needs to be explored. This research gap led to the investigation of various carbon-to-nitrogen (C/N) ratios in a column-type sequencing batch reactor (cSBR). The resulting data and model demonstrated that augmenting the SND process with an external carbon source is effective until the C/N ratio reaches 15, ultimately eliminating nitrogen in the produced water. Conversely, a reduced C/N ratio can limit the ability of polyphosphate-accumulating organisms to incorporate carbon into polyphosphate synthesis, thereby decreasing phosphorus removal efficiency within the cSBR. When the C/N ratio ranged from 6 to 8, and the mixed liquor suspended solids concentration was high, the average phosphate removal was approximately 55%, compared to only around 25% when the C/N ratio was less than 6.

"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.

TimeNorm: a novel normalization method for time course microbiome data.

Metagenomic time-course studies provide valuable insights into the dynamics of microbial systems and have become increasingly popular alongside the reduction in costs of next-generation sequencing technologies. Normalization is a common but critical preprocessing step before proceeding with downstream analysis. To the best of our knowledge, currently there is no reported method to appropriately normalize microbial time-series data. We propose TimeNorm, a novel normalization method that considers the compositional property and time dependency in time-course microbiome data. It is the first method designed for normalizing time-series data within the same time point (intra-time normalization) and across time points (bridge normalization), separately. Intra-time normalization normalizes microbial samples under the same condition based on common dominant features. Bridge normalization detects and utilizes a group of most stable features across two adjacent time points for normalization. Through comprehensive simulation studies and application to a real study, we demonstrate that TimeNorm outperforms existing normalization methods and boosts the power of downstream differential abundance analysis.

Viral metagenomic survey of Caspian seals.

Viral diseases of pinnipeds cause substantial mortality and morbidity and can influence population demography. Viral metagenomic studies can therefore play an important role in pinniped health assessments and disease surveillance relevant to both individual species and in a "One Health" context. This study used a metagenomic approach with high throughput sequencing to make the first assessment of viral diversity in Caspian seals (Pusa caspica), the only marine mammal species endemic to the Caspian Sea. Sequencing libraries from 35 seals sampled 2009-2020 were analysed, finding sequences from the viral families Circoviridae, Parvoviridae, Herpesviridae, Papillomaviridae, Picornaviridae, Caliciviridae, Cruciviridae, Anelloviridae, Smacoviridae, and Orthomyxoviridae, with additional detection of Adenoviridae via PCR. The similarity of viral contigs from Caspian seal to sequences recovered from other pinnipeds ranged from 63.74% (San Miguel sea lion calicivirus) to 78.79% (Seal anellovirus 4). Some findings represent novel viral species, but overall, the viral repertoire of Caspian seals is similar to available viromes from other pinnipeds. Among the sequences recovered were partial contigs for influenza B, representing only the second such molecular identification in marine mammals. This work provides a foundation for further studies of viral communities in Caspian seals, the diversity of viromes in pinnipeds more generally, and contributes data relevant for disease risk assessments in marine mammals.