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Biomolecules May 2024The understanding of the human genome has been greatly improved by the advent of next-generation sequencing technologies (NGS). Despite the undeniable advantages... (Review)
Review
The understanding of the human genome has been greatly improved by the advent of next-generation sequencing technologies (NGS). Despite the undeniable advantages responsible for their widespread diffusion, these methods have some constraints, mainly related to short read length and the need for PCR amplification. As a consequence, long-read sequencers, called third-generation sequencing (TGS), have been developed, promising to overcome NGS. Starting from the first prototype, TGS has progressively ameliorated its chemistries by improving both read length and base-calling accuracy, as well as simultaneously reducing the costs/base. Based on these premises, TGS is showing its potential in many fields, including the analysis of difficult-to-sequence genomic regions, structural variations detection, RNA expression profiling, DNA methylation study, and metagenomic analyses. Protocol standardization and the development of easy-to-use pipelines for data analysis will enhance TGS use, also opening the way for their routine applications in diagnostic contexts.
Topics: Humans; High-Throughput Nucleotide Sequencing; Sequence Analysis, DNA; Genome, Human; Metagenomics; DNA Methylation; Genomics
PubMed: 38785975
DOI: 10.3390/biom14050568 -
Nature Communications Jun 2024Serpentinization, a geochemical process found on modern and ancient Earth, provides an ultra-reducing environment that can support microbial methanogenesis and...
Serpentinization, a geochemical process found on modern and ancient Earth, provides an ultra-reducing environment that can support microbial methanogenesis and acetogenesis. Several groups of archaea, such as the order Methanocellales, are characterized by their ability to produce methane. Here, we generate metagenomic sequences from serpentinized springs in The Cedars, California, and construct a circularized metagenome-assembled genome of a Methanocellales archaeon, termed Met12, that lacks essential methanogenesis genes. The genome includes genes for an acetyl-CoA pathway, but lacks genes encoding methanogenesis enzymes such as methyl-coenzyme M reductase, heterodisulfide reductases and hydrogenases. In situ transcriptomic analyses reveal high expression of a multi-heme c-type cytochrome, and heterologous expression of this protein in a model bacterium demonstrates that it is capable of accepting electrons. Our results suggest that Met12, within the order Methanocellales, is not a methanogen but a CO-reducing, electron-fueled acetogen without electron bifurcation.
Topics: Methane; Genome, Archaeal; Archaeal Proteins; Oxidoreductases; Metagenome; Phylogeny; Acetyl Coenzyme A; Carbon Dioxide; Metagenomics
PubMed: 38871712
DOI: 10.1038/s41467-024-48185-5 -
Nature Protocols May 2024Deep investigation of the microbiome of food-production and food-processing environments through whole-metagenome sequencing (WMS) can provide detailed information on... (Review)
Review
Deep investigation of the microbiome of food-production and food-processing environments through whole-metagenome sequencing (WMS) can provide detailed information on the taxonomic composition and functional potential of the microbial communities that inhabit them, with huge potential benefits for environmental monitoring programs. However, certain technical challenges jeopardize the application of WMS technologies with this aim, with the most relevant one being the recovery of a sufficient amount of DNA from the frequently low-biomass samples collected from the equipment, tools and surfaces of food-processing plants. Here, we present the first complete workflow, with optimized DNA-purification methodology, to obtain high-quality WMS sequencing results from samples taken from food-production and food-processing environments and reconstruct metagenome assembled genomes (MAGs). The protocol can yield DNA loads >10 ng in >98% of samples and >500 ng in 57.1% of samples and allows the collection of, on average, 12.2 MAGs per sample (with up to 62 MAGs in a single sample) in ~1 week, including both laboratory and computational work. This markedly improves on results previously obtained in studies performing WMS of processing environments and using other protocols not specifically developed to sequence these types of sample, in which <2 MAGs per sample were obtained. The full protocol has been developed and applied in the framework of the European Union project MASTER (Microbiome applications for sustainable food systems through technologies and enterprise) in 114 food-processing facilities from different production sectors.
Topics: Microbiota; Food Handling; DNA, Bacterial; Metagenome; Metagenomics; DNA; Sequence Analysis, DNA; Food Microbiology
PubMed: 38267717
DOI: 10.1038/s41596-023-00949-x -
Journal of Zhejiang University.... May 2024Infectious diseases are a great threat to human health. Rapid and accurate detection of pathogens is important in the diagnosis and treatment of infectious diseases.... (Review)
Review
Infectious diseases are a great threat to human health. Rapid and accurate detection of pathogens is important in the diagnosis and treatment of infectious diseases. Metagenomics next-generation sequencing (mNGS) is an unbiased and comprehensive approach for detecting all RNA and DNA in a sample. With the development of sequencing and bioinformatics technologies, mNGS is moving from research to clinical application, which opens a new avenue for pathogen detection. Numerous studies have revealed good potential for the clinical application of mNGS in infectious diseases, especially in difficult-to-detect, rare, and novel pathogens. However, there are several hurdles in the clinical application of mNGS, such as: (1) lack of universal workflow validation and quality assurance; (2) insensitivity to high-host background and low-biomass samples; and (3) lack of standardized instructions for mass data analysis and report interpretation. Therefore, a complete understanding of this new technology will help promote the clinical application of mNGS to infectious diseases. This review briefly introduces the history of next-generation sequencing, mainstream sequencing platforms, and mNGS workflow, and discusses the clinical applications of mNGS to infectious diseases and its advantages and disadvantages.
Topics: Metagenomics; Humans; High-Throughput Nucleotide Sequencing; Communicable Diseases; Computational Biology; Workflow
PubMed: 38910493
DOI: 10.1631/jzus.B2300029 -
Biomedicine & Pharmacotherapy =... Jun 2024Recent advances in metagenomic testing opened a new window into the mammalian blood virome. Comprised of well-known viruses like human immunodeficiency virus, hepatitis... (Review)
Review
Recent advances in metagenomic testing opened a new window into the mammalian blood virome. Comprised of well-known viruses like human immunodeficiency virus, hepatitis C virus, and hepatitis B virus, the virome also includes many other eukaryotic viruses and phages whose medical significance, lifecycle, epidemiology, and impact on human health are less well known and thus regarded as commensals. This review synthesizes available information for the so-called commensal virome members that circulate in the blood of humans considering their restriction to and interaction with the human host, their natural history, and their impact on human health and physiology.
Topics: Humans; Virome; Animals; Viruses; Metagenomics; Virus Diseases
PubMed: 38703502
DOI: 10.1016/j.biopha.2024.116608 -
Clinical Chemistry Sep 2023
Topics: Humans; Metagenomics; Respiratory Tract Infections; High-Throughput Nucleotide Sequencing; Reference Standards
PubMed: 37534530
DOI: 10.1093/clinchem/hvad101 -
Molecular Ecology Resources Oct 2023At the genome level, microorganisms are highly adaptable both in terms of allele and gene composition. Such heritable traits emerge in response to different...
At the genome level, microorganisms are highly adaptable both in terms of allele and gene composition. Such heritable traits emerge in response to different environmental niches and can have a profound influence on microbial community dynamics. As a consequence, any individual genome or population will contain merely a fraction of the total genetic diversity of any operationally defined "species", whose ecological potential can thus be only fully understood by studying all of their genomes and the genes therein. This concept, known as the pangenome, is valuable for studying microbial ecology and evolution, as it partitions genomes into core (present in all the genomes from a species, and responsible for housekeeping and species-level niche adaptation among others) and accessory regions (present only in some, and responsible for intra-species differentiation). Here we present SuperPang, an algorithm producing pangenome assemblies from a set of input genomes of varying quality, including metagenome-assembled genomes (MAGs). SuperPang runs in linear time and its results are complete, non-redundant, preserve gene ordering and contain both coding and non-coding regions. Our approach provides a modular view of the pangenome, identifying operons and genomic islands, and allowing to track their prevalence in different populations. We illustrate this by analysing intra-species diversity in Polynucleobacter, a bacterial genus ubiquitous in freshwater ecosystems, characterized by their streamlined genomes and their ecological versatility. We show how SuperPang facilitates the simultaneous analysis of allelic and gene content variation under different environmental pressures, allowing us to study the drivers of microbial diversification at unprecedented resolution.
Topics: Phylogeny; Bacteria; Metagenome; Algorithms; Microbiota; Metagenomics
PubMed: 37382302
DOI: 10.1111/1755-0998.13826 -
Viruses May 2024Routinely used metagenomic next-generation sequencing (mNGS) techniques often fail to detect low-level viremia (<10 copies/mL) and appear biased towards viruses with...
Routinely used metagenomic next-generation sequencing (mNGS) techniques often fail to detect low-level viremia (<10 copies/mL) and appear biased towards viruses with linear genomes. These limitations hinder the capacity to comprehensively characterize viral infections, such as those attributed to the family. These near ubiquitous non-pathogenic components of the human virome have circular single-stranded DNA genomes that vary in size from 2.0 to 3.9 kb and exhibit high genetic diversity. Hence, species identification using short reads can be challenging. Here, we introduce a rolling circle amplification (RCA)-based metagenomic sequencing protocol tailored for circular single-stranded DNA genomes, utilizing the long-read Oxford Nanopore platform. The approach was assessed by sequencing anelloviruses in plasma drawn from people who inject drugs (PWID) in two geographically distinct cohorts. We detail the methodological adjustments implemented to overcome difficulties inherent in sequencing circular genomes and describe a computational pipeline focused on anellovirus detection. We assessed our protocol across various sample dilutions and successfully differentiated anellovirus sequences in conditions simulating mixed infections. This method provides a robust framework for the comprehensive characterization of circular viruses within the human virome using the Oxford Nanopore.
Topics: Anelloviridae; Humans; High-Throughput Nucleotide Sequencing; Metagenomics; Genome, Viral; Nanopore Sequencing; Nanopores; DNA, Viral; Virome; Sequence Analysis, DNA
PubMed: 38793605
DOI: 10.3390/v16050723 -
Food Research International (Ottawa,... Nov 2023An insightful exploration of the fermented vegetable microbiome is the key to improving food quality and sustainability. Based on 57 fermented vegetable samples from... (Meta-Analysis)
Meta-Analysis
An insightful exploration of the fermented vegetable microbiome is the key to improving food quality and sustainability. Based on 57 fermented vegetable samples from China, Ireland, the UK, and Germany retrieved from public genome databases, we conducted a high-resolution meta-analysis of the fermented vegetable microbiomes. There were significant differences in the microbiota composition and functional pathway diversity of the tested samples, as reflected by the differences in their geographical origins. Metagenomic analysis also revealed the metagenomic features of carbohydrate-active enzymes and antibiotic resistance genes in the fermented vegetable metagenomes. Five putative new species were detected by recovering 221 metagenome-assembled genomes belonging to the genera Rubrobacteraceae, Bifidobacteriaceae, and Ruminococcaceae. Our results provide new ecological insights into the implications of fermented vegetable microbiota composition and functional potential and highlight the importance of high-resolution metagenomic analysis to further investigate the fermented food microbiome.
Topics: Metagenome; Vegetables; Microbiota; Lactobacillales; Drug Resistance, Microbial
PubMed: 37803564
DOI: 10.1016/j.foodres.2023.113248 -
Frontiers in Cellular and Infection... 2023The advent of metagenomics next-generation sequencing (mNGS) has garnered attention as a novel method for detecting pathogenic infections, including Non-Tuberculous...
INTRODUCTION
The advent of metagenomics next-generation sequencing (mNGS) has garnered attention as a novel method for detecting pathogenic infections, including Non-Tuberculous Mycobacterial (NTM) and tuberculosis (TB).However, the robustness and specificity of mNGS in NTM diagnostics have not been fully explored.
METHODS
In this retrospective study, we enrolled 27 patients with NTM genomic sequences via mNGS and conducted a comprehensive clinical evaluation.
RESULTS
Pulmonary NTM disease was the most commonly observed presentation, with a subset of patients also presenting with extrapulmonary NTM infections.mNGS analysis identified six distinct NTM species, primarily Mycobacteriumavium complex (MAC), followed by Mycobacterium intracellulare andMycobacterium abscessus. Conventional routine culture methods encountered challenges, resulting in negative results for all available 22 samples. Among the 10 patients who underwent quantitative polymerase chain reaction (qPCR) testing, five tested positive for NTM.
DISCUSSION
It is important to note that further species typing is necessary to determine the specific NTM type, as traditional pathogen detection methods serve as an initial step. In contrast, when supplemented with pathogen data, enables the identification of specific species, facilitating precise treatment decisions. In conclusion, mNGS demonstrates significant potential in aidingthe diagnosis of NTMdisease by rapidly detecting NTM pathogens and guiding treatment strategies. Its enhanced performance, faster turnaround time (TAT), and species identification capabilities make mNGS a promising tool for managing NTM infections.
Topics: Humans; Nontuberculous Mycobacteria; Metagenomics; Retrospective Studies; Metagenome; High-Throughput Nucleotide Sequencing; Mycobacterium abscessus
PubMed: 37719673
DOI: 10.3389/fcimb.2023.1253020