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Methods in Molecular Biology (Clifton,... 2022Microbial communities' taxonomic and functional diversity has been broadly studied since sequencing technologies enabled faster and cheaper data obtainment....
Microbial communities' taxonomic and functional diversity has been broadly studied since sequencing technologies enabled faster and cheaper data obtainment. Nevertheless, the programming skills needed and the amount of software available may be overwhelming to someone trying to analyze these data. Here, we present a comprehensive and straightforward pipeline that takes shotgun metagenomics data through the needed steps to obtain valuable results. The raw data goes through a quality control process, metagenomic assembly, binning (the obtention of single genomes from a metagenome), taxonomic assignment, and taxonomic diversity analysis and visualization.
Topics: Computational Biology; Metagenome; Metagenomics; Microbiota; Sequence Analysis, DNA; Software
PubMed: 35818005
DOI: 10.1007/978-1-0716-2429-6_10 -
Cell Aug 2019Metagenomic sequencing is revolutionizing the detection and characterization of microbial species, and a wide variety of software tools are available to perform... (Review)
Review
Metagenomic sequencing is revolutionizing the detection and characterization of microbial species, and a wide variety of software tools are available to perform taxonomic classification of these data. The fast pace of development of these tools and the complexity of metagenomic data make it important that researchers are able to benchmark their performance. Here, we review current approaches for metagenomic analysis and evaluate the performance of 20 metagenomic classifiers using simulated and experimental datasets. We describe the key metrics used to assess performance, offer a framework for the comparison of additional classifiers, and discuss the future of metagenomic data analysis.
Topics: Bacteria; Benchmarking; Databases, Genetic; Fungi; Metagenome; Metagenomics; Phylogeny; Polymerase Chain Reaction; Sequence Analysis, DNA; Software; Viruses
PubMed: 31398336
DOI: 10.1016/j.cell.2019.07.010 -
Nature Protocols Dec 2022Metagenomic experiments expose the wide range of microscopic organisms in any microbial environment through high-throughput DNA sequencing. The computational analysis of... (Review)
Review
Metagenomic experiments expose the wide range of microscopic organisms in any microbial environment through high-throughput DNA sequencing. The computational analysis of the sequencing data is critical for the accurate and complete characterization of the microbial community. To facilitate efficient and reproducible metagenomic analysis, we introduce a step-by-step protocol for the Kraken suite, an end-to-end pipeline for the classification, quantification and visualization of metagenomic datasets. Our protocol describes the execution of the Kraken programs, via a sequence of easy-to-use scripts, in two scenarios: (1) quantification of the species in a given metagenomics sample; and (2) detection of a pathogenic agent from a clinical sample taken from a human patient. The protocol, which is executed within 1-2 h, is targeted to biologists and clinicians working in microbiome or metagenomics analysis who are familiar with the Unix command-line environment.
Topics: Humans; Metagenome; Software; Metagenomics; High-Throughput Nucleotide Sequencing; Microbiota; Sequence Analysis, DNA
PubMed: 36171387
DOI: 10.1038/s41596-022-00738-y -
Current Opinion in Biotechnology Feb 2021Ability to directly sequence DNA from the environment permanently changed microbial ecology. Here, we review the new insights to microbial life gleaned from the... (Review)
Review
Ability to directly sequence DNA from the environment permanently changed microbial ecology. Here, we review the new insights to microbial life gleaned from the applications of metagenomics, as well as the extensive set of analytical tools that facilitate exploration of diversity and function of complex microbial communities. While metagenomics is shaping our understanding of microbial functions in ecosystems via gene-centric and genome-centric methods, annotating functions, metagenome assembly and binning in heterogeneous samples remains challenging. Development of new analysis and sequencing platforms generating high-throughput long-read sequences and functional screening opportunities will aid in harnessing metagenomes to increase our understanding of microbial taxonomy, function, ecology, and evolution in the environment.
Topics: Ecology; High-Throughput Nucleotide Sequencing; Metagenome; Metagenomics; Microbiota; Sequence Analysis, DNA
PubMed: 33592536
DOI: 10.1016/j.copbio.2021.01.019 -
International Journal of Molecular... Aug 2022Whole genome sequencing (WGS) provides the highest resolution for genome-based species identification and can provide insight into the antimicrobial resistance and... (Review)
Review
Whole genome sequencing (WGS) provides the highest resolution for genome-based species identification and can provide insight into the antimicrobial resistance and virulence potential of a single microbiological isolate during the diagnostic process. In contrast, metagenomic sequencing allows the analysis of DNA segments from multiple microorganisms within a community, either using an amplicon- or shotgun-based approach. However, WGS and shotgun metagenomic data are rarely combined, although such an approach may generate additive or synergistic information, critical for, e.g., patient management, infection control, and pathogen surveillance. To produce a combined workflow with actionable outputs, we need to understand the pre-to-post analytical process of both technologies. This will require specific databases storing interlinked sequencing and metadata, and also involves customized bioinformatic analytical pipelines. This review article will provide an overview of the critical steps and potential clinical application of combining WGS and metagenomics together for microbiological diagnosis.
Topics: Computational Biology; Humans; Metagenome; Metagenomics; Whole Genome Sequencing; Workflow
PubMed: 36077231
DOI: 10.3390/ijms23179834 -
Nature Communications Feb 2021Gut microbiota plays an important role in pig health and production. Still, availability of sequenced genomes and functional information for most pig gut microbes...
Gut microbiota plays an important role in pig health and production. Still, availability of sequenced genomes and functional information for most pig gut microbes remains limited. Here we perform a landscape survey of the swine gut microbiome, spanning extensive sample sources by deep metagenomic sequencing resulting in an expanded gene catalog named pig integrated gene catalog (PIGC), containing 17,237,052 complete genes clustered at 90% protein identity from 787 gut metagenomes, of which 28% are unknown proteins. Using binning analysis, 6339 metagenome-assembled genomes (MAGs) were obtained, which were clustered to 2673 species-level genome bins (SGBs), among which 86% (2309) SGBs are unknown based on current databases. Using the present gene catalog and MAGs, we identified several strain-level differences between the gut microbiome of wild boars and commercial Duroc pigs. PIGC and MAGs provide expanded resources for swine gut microbiome-related research.
Topics: Animals; Bacteria; Female; Gastrointestinal Microbiome; Genes, Microbial; High-Throughput Nucleotide Sequencing; Metagenome; Metagenomics; Phylogeny; Species Specificity; Swine
PubMed: 33597514
DOI: 10.1038/s41467-021-21295-0 -
International Journal of Molecular... Feb 2022Whole genome metagenomic sequencing is a powerful platform enabling the simultaneous identification of all genes from entirely different kingdoms of organisms in a... (Review)
Review
Whole genome metagenomic sequencing is a powerful platform enabling the simultaneous identification of all genes from entirely different kingdoms of organisms in a complex sample. This technology has revolutionised multiple areas from microbiome research to clinical diagnoses. However, one of the major challenges of a metagenomic study is the overwhelming non-microbial DNA present in most of the host-derived specimens, which can inundate the microbial signals and reduce the sensitivity of microorganism detection. Various host DNA depletion methods to facilitate metagenomic sequencing have been developed and have received considerable attention in this context. In this review, we present an overview of current host DNA depletion approaches along with explanations of their underlying principles, advantages and disadvantages. We also discuss their applications in laboratory microbiome research and clinical diagnoses and, finally, we envisage the direction of the further perfection of metagenomic sequencing in samples with overabundant host DNA.
Topics: DNA; High-Throughput Nucleotide Sequencing; Humans; Metagenome; Metagenomics; Sequence Analysis, DNA; Technology
PubMed: 35216302
DOI: 10.3390/ijms23042181 -
Nature Oct 2023Metagenomes encode an enormous diversity of proteins, reflecting a multiplicity of functions and activities. Exploration of this vast sequence space has been limited to...
Metagenomes encode an enormous diversity of proteins, reflecting a multiplicity of functions and activities. Exploration of this vast sequence space has been limited to a comparative analysis against reference microbial genomes and protein families derived from those genomes. Here, to examine the scale of yet untapped functional diversity beyond what is currently possible through the lens of reference genomes, we develop a computational approach to generate reference-free protein families from the sequence space in metagenomes. We analyse 26,931 metagenomes and identify 1.17 billion protein sequences longer than 35 amino acids with no similarity to any sequences from 102,491 reference genomes or the Pfam database. Using massively parallel graph-based clustering, we group these proteins into 106,198 novel sequence clusters with more than 100 members, doubling the number of protein families obtained from the reference genomes clustered using the same approach. We annotate these families on the basis of their taxonomic, habitat, geographical and gene neighbourhood distributions and, where sufficient sequence diversity is available, predict protein three-dimensional models, revealing novel structures. Overall, our results uncover an enormously diverse functional space, highlighting the importance of further exploring the microbial functional dark matter.
Topics: Cluster Analysis; Metagenome; Metagenomics; Proteins; Databases, Protein; Protein Conformation; Microbiology
PubMed: 37821698
DOI: 10.1038/s41586-023-06583-7 -
Nature Biotechnology Jun 2020
Topics: Computational Biology; Genes, Bacterial; Metagenome; Metagenomics; RNA, Ribosomal, 16S; Software
PubMed: 32483366
DOI: 10.1038/s41587-020-0548-6 -
Advanced Science (Weinheim,... Dec 2021Fast and accurate identification of microbial pathogens is critical for the proper treatment of infections. Traditional culture-based diagnosis in clinics is...
Fast and accurate identification of microbial pathogens is critical for the proper treatment of infections. Traditional culture-based diagnosis in clinics is increasingly supplemented by metagenomic next-generation-sequencing (mNGS). Here, RNA/cDNA-targeted sequencing (meta-transcriptomics using NGS (mtNGS)) is established to reduce the host nucleotide percentage in clinic samples and by combining with Oxford Nanopore Technology (ONT) platforms (meta-transcriptomics using third-generation sequencing, mtTGS) to improve the sequencing time. It shows that mtNGS improves the ratio of microbial reads, facilitates bacterial identification using multiple-strategies, and discovers fungi, viruses, and antibiotic resistance genes, and displaying agreement with clinical findings. Furthermore, longer reads in mtTGS lead to additional improvement in pathogen identification and also accelerate the clinical diagnosis. Additionally, primary tests utilizing direct-RNA sequencing and targeted sequencing of ONT show that ONT displays important potential but must be further developed. This study presents the potential of RNA-targeted pathogen identification in clinical samples, especially when combined with the newest developments in ONT.
Topics: Aged; Bronchoalveolar Lavage; Bronchoalveolar Lavage Fluid; Female; High-Throughput Nucleotide Sequencing; Humans; Infections; Male; Metagenome; Metagenomics; Middle Aged; RNA; Sequence Analysis, RNA
PubMed: 34687159
DOI: 10.1002/advs.202102593