Metagenomic sequencing has allowed for the recovery of previously unexplored microbial genomes. Whereas short-read sequencing platforms often result in highly fragmented metagenomes, nanopore-based ...sequencers could lead to more contiguous assemblies due to their potential to generate long reads. Nevertheless, there is a lack of updated and systematic studies evaluating the performance of different assembly tools on nanopore data. In this study, we have benchmarked the ability of different assemblers to reconstruct two different commercially-available mock communities that have been sequenced using Oxford Nanopore Technologies platforms. Among the tested tools, only metaFlye, Raven, and Canu performed well in all the datasets. These tools retrieved highly contiguous genomes (or even complete genomes) directly from the metagenomic data. Despite the intrinsic high error of nanopore sequencing, final assemblies reached high accuracy (~ 99.5 to 99.8% of consensus accuracy). Polishing strategies demonstrated to be necessary for reducing the number of indels, and this had an impact on the prediction of biosynthetic gene clusters. Correction with high quality short reads did not always result in higher quality draft assemblies. Overall, nanopore metagenomic sequencing data-adapted to MinION's current output-proved sufficient for assembling and characterizing low-complexity microbial communities.
Neonates on extracorporeal membrane oxygenation (ECMO) are at high risk of infection. Rapid and accurate identification of pathogens is essential to improve the prognosis of children on ECMO. ...Metagenome next-generation sequencing (mNGS) has been used in recent years to detect pathogenic bacteria, but evidence for its use in neonates on ECMO is lacking.
This retrospective study was conducted using an electronic medical record system. We analyzed the results of mNGS and conventional microbiological tests (CMTs) in bronchoalveolar lavage fluid of neonates receiving ECMO support with pulmonary infections in our hospital from July 2021 to January 2023.
We screened 18 ECMO-supported neonates with pneumonia for inclusion in the study. The median age of the included children was 2 (1-4) days, the median gestational age was 38.3 (33-40 +4 ) weeks, and the median weight was 3.3 (2.2-4.8) kg. The detection rate of mNGS was 77.8% (14/18), higher than the 44.4% (8/18) of CMT ( P = 0.04). A total of 20 pathogens were detected in mNGS, with the top 3 most common pathogens being Klebsiella pneumoniae , Acinetobacter baumannii and Escherichia coli . Mixed infections were found in 14 cases (77.8%), including 13 cases (72.2%) with mixed infections detected by mNGS and 7 cases (27.8%) with mixed infections detected by CMT. A total of 9 children underwent treatment changes based on mNGS results and all of them experienced relief of symptoms.
Compared with CMT, mNGS can detect pathogens earlier and more sensitively, and may play an important role in ECMO-supported neonatal pneumonia pathogen detection and optimization of antibiotic therapy.
Background:
Despite a reduction in stroke incidence and age-standardized death rates, stroke remains a leading cause of death and disability worldwide. Significant interest in recent years has ...focused on the microbiota-host interaction because accumulating evidence has revealed myriad ways in which bacteria may contribute to risk of stroke and adverse outcomes after stroke. The emergence of endovascular thrombectomy as a treatment provides a unique opportunity to utilize thrombus retrieved from cerebral arteries to fill knowledge gaps about the influence of bacteria on stroke pathophysiology. While bacterial signatures have been confirmed in cerebral thrombi, the exact nature of the pathogenesis has not been established.
Methods:
Thrombi were obtained from a cohort of adult ischemic stroke patients during standard of care thrombectomy. After DNA extraction and quantification, thrombi underwent 16S rRNA amplicon-based metagenomic sequencing, followed by bioinformatics processing. Taxonomic identification of bacterial colonies isolated on Agar plates from plated suspension was performed using DNA extraction and full length 16S Sanger sequencing.
Results:
A broad diversity of bacterial signatures was identified in specimens, primarily of cariogenic origin.
Conclusion:
In this small study, we demonstrate proof of concept and technical feasibility for amplicon-based metagenomic sequencing of arterial thrombi and briefly discuss preliminary findings, challenges, and near-term translational opportunities for thrombus genomics.
Landfills are important sources of microorganisms associated with anaerobic digestion. However, the knowledge on microbiota along with their functional potential in this special habitat are still ...lacking. In this study, we recovered 1168 non-redundant metagenome-assembled genomes (MAGs) from nine landfill leachate samples collected from eight cities across China, spanning 42 phyla, 73 classes, 114 orders, 189 families, and 267 genera. Totally, 74.1% of 1168 MAGs could not be classified to any known species and 5.9% of these MAGs belonged to microbial dark matter phyla. Two putative novel classes were discovered from landfill leachate samples. The identification of thousands of novel carbohydrate-active enzymes showed similar richness level compared to the cow rumen microbiota. The methylotrophic methanogenic pathway was speculated to contribute significantly to methane production in the landfill leachate because of its co-occurrence with the acetoclastic and hydrogenotrophic methanogenic pathways. The genetic potential of dissimilatory nitrate reduction to ammonium (DNRA) was observed, implying DNRA may play a role in ammonium generation in landfill leachate. These findings implied that landfill leachate might be a valuable microbial resource repository and filled the previous understanding gaps for both methanogenesis and nitrogen cycling in landfill leachate microbiota. Our study provides a comprehensive genomic catalog and substantially provides unprecedented taxonomic and functional profiles of the landfill leachate microbiota.
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•Metagenomic binning identified 74% of MAGs belonging to unknown species.•Thousands of novel carbohydrate-active enzymes were discovered in the MAGs.•Hydrogenotrophic/acetoclastic/methylotrophic methanogens widely co-occur in leachate.•Dissimilatory nitrate reduction to ammonia might contribute to ammonia production.
Although immunomodulatory activities of Dendrobium officinale polysaccharide has been investigated for many years, yet the potential contribution of its metabolite derived from intestinal microbes on ...immunoregulation effect has not been reported. In this study, polysaccharide DOW-5B with average molecular weight of 39.4 kDa was isolated from the stem of Dendrobium officinale Kimura et Migo. The carbohydrate content was 91.97% and no protein was detected. The monosaccharide analysis showed this polysaccharide was composed of glucuronic acid and glucose at a molar ratio (M/G) of 1.2:19.4. Animal test indicated DOW-5B increased the diversity of gut microbiota on mice. Beneficial microbes such as Ruminococcus, Eubacterium, Clostridium, Bifidobacterium, Parabacteroides and Akkermansiamuciniphila increased while harmful bacteria in Proteobacteria decreased. Surprisingly, DOW-5B promoted gut microbes to generate more butyrate and mainly produced by Parabacteroides_sp_HGS0025. Further, we found the health of large intestine as well as immunity response of mice was improved. In addition, Parabacteroides_sp_HGS0025 positively correlated with butyrate, IgM, IL-10, and TNF-α products in intestine and mice blood, respectively. The data suggested that Dendrobium officinale polysaccharide has function on immunity may be mediated by butyrate. It adds new evidence to support the basis of how herbal polysaccharides affect immunity.
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Many of the Earth's microbes remain uncultured and understudied, limiting our understanding of the functional and evolutionary aspects of their genetic material, which remain largely overlooked in ...most metagenomic studies
. Here we analysed 149,842 environmental genomes from multiple habitats
and compiled a curated catalogue of 404,085 functionally and evolutionarily significant novel (FESNov) gene families exclusive to uncultivated prokaryotic taxa. All FESNov families span multiple species, exhibit strong signals of purifying selection and qualify as new orthologous groups, thus nearly tripling the number of bacterial and archaeal gene families described to date. The FESNov catalogue is enriched in clade-specific traits, including 1,034 novel families that can distinguish entire uncultivated phyla, classes and orders, probably representing synapomorphies that facilitated their evolutionary divergence. Using genomic context analysis and structural alignments we predicted functional associations for 32.4% of FESNov families, including 4,349 high-confidence associations with important biological processes. These predictions provide a valuable hypothesis-driven framework that we used for experimental validatation of a new gene family involved in cell motility and a novel set of antimicrobial peptides. We also demonstrate that the relative abundance profiles of novel families can discriminate between environments and clinical conditions, leading to the discovery of potentially new biomarkers associated with colorectal cancer. We expect this work to enhance future metagenomics studies and expand our knowledge of the genetic repertory of uncultivated organisms.
Waterborne human enteric viruses, such as noroviruses and adenoviruses, are excreted in the feces of infected individuals and transmitted via the fecal-oral route including contaminated food and ...water. Since viruses are normally present at low concentrations in aquatic environments, they should be concentrated into smaller volumes prior to downstream molecular biological applications, such as quantitative polymerase chain reaction (qPCR). This review describes recent progress made in the development of concentration and detection methods of human enteric viruses in water, and discusses their applications for providing a better understanding of the prevalence of the viruses in various types of water worldwide. Maximum concentrations of human enteric viruses in water that have been reported in previous studies are summarized to assess viral abundances in aquatic environments. Some descriptions are also available on recent applications of sequencing analyses used to determine the genetic diversity of viral genomes in water samples, including those of novel viruses. Furthermore, the importance and significance of utilizing appropriate process controls during viral analyses are discussed, and three types of process controls are considered: whole process controls, molecular process controls, and (reverse transcription (RT)-)qPCR controls. Although no standards have been established for acceptable values of virus recovery and/or extraction-(RT-)qPCR efficiency, use of at least one of these appropriate control types is highly recommended for more accurate interpretation of observed data.
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•Recent progress on research regarding waterborne human enteric viruses is summarized.•Methods for concentrating and detecting viruses in water are described.•Existing data on abundance and genetic diversity of viruses in water are compiled.•The characteristics of three types of process controls are introduced.•The inclusion of process control(s) is recommended for better data interpretation.
Bioinformatics tools available for metagenomic sequencing analysis are principally devoted to the identification of microorganisms populating an ecological niche, but they usually do not consider ...viruses. Only some software have been designed to profile the viral sequences, however they are not efficient in the characterization of viruses in the context of complex communities, like the intestinal microbiota, containing bacteria, archeabacteria, eukaryotic microorganisms and viruses. In any case, a comprehensive description of the host-microbiota interactions can not ignore the profile of eukaryotic viruses within the virome, as viruses are definitely critical for the regulation of the host immunophenotype.
ViromeScan is an innovative metagenomic analysis tool that characterizes the taxonomy of the virome directly from raw data of next-generation sequencing. The tool uses hierarchical databases for eukaryotic viruses to unambiguously assign reads to viral species more accurately and >1000 fold faster than other existing approaches. We validated ViromeScan on synthetic microbial communities and applied it on metagenomic samples of the Human Microbiome Project, providing a sensitive eukaryotic virome profiling of different human body sites.
ViromeScan allows the user to explore and taxonomically characterize the virome from metagenomic reads, efficiently denoising samples from reads of other microorganisms. This implies that users can fully characterize the microbiome, including bacteria and viruses, by shotgun metagenomic sequencing followed by different bioinformatic pipelines.
Computational methods are key in microbiome research, and obtaining a quantitative and unbiased performance estimate is important for method developers and applied researchers. For meaningful ...comparisons between methods, to identify best practices and common use cases, and to reduce overhead in benchmarking, it is necessary to have standardized datasets, procedures and metrics for evaluation. In this tutorial, we describe emerging standards in computational meta-omics benchmarking derived and agreed upon by a larger community of researchers. Specifically, we outline recent efforts by the Critical Assessment of Metagenome Interpretation (CAMI) initiative, which supplies method developers and applied researchers with exhaustive quantitative data about software performance in realistic scenarios and organizes community-driven benchmarking challenges. We explain the most relevant evaluation metrics for assessing metagenome assembly, binning and profiling results, and provide step-by-step instructions on how to generate them. The instructions use simulated mouse gut metagenome data released in preparation for the second round of CAMI challenges and showcase the use of a repository of tool results for CAMI datasets. This tutorial will serve as a reference for the community and facilitate informative and reproducible benchmarking in microbiome research.
We describe a method that adds long-read sequencing to a mix of technologies used to assemble a highly complex cattle rumen microbial community, and provide a comparison to short read-based methods. ...Long-read alignments and Hi-C linkage between contigs support the identification of 188 novel virus-host associations and the determination of phage life cycle states in the rumen microbial community. The long-read assembly also identifies 94 antimicrobial resistance genes, compared to only seven alleles in the short-read assembly. We demonstrate novel techniques that work synergistically to improve characterization of biological features in a highly complex rumen microbial community.
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