Genomic representativeness and chimerism in large collections of SAGs and MAGs of marine prokaryoplankton

Microbiome, Journal Year: 2024, Volume and Issue: 12(1)

Published: July 15, 2024

Abstract Background Single amplified genomes (SAGs) and metagenome-assembled (MAGs) are the predominant sources of information about coding potential uncultured microbial lineages, but their strengths limitations remain poorly understood. Here, we performed a direct comparison two previously published collections thousands SAGs MAGs obtained from same, global environment. Results We found that were less prone to chimerism more accurately reflected relative abundance pangenome content lineages inhabiting epipelagic tropical subtropical ocean, as compared MAGs. also better suited link genome with taxa discovered through 16S rRNA amplicon analyses. Meanwhile, had advantage readily recovering rare lineages. Conclusions Our analyses revealed weaknesses most commonly used recovery approaches in environmental microbiology. These considerations, well need for tools quality assessment, should be taken into account when designing studies interpreting data involve or

Language: Английский

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Long-read epicPCR enhances species-level host identification of clinically relevant antibiotic resistance genes in environmental microbial communities

Environment International, Journal Year: 2025, Volume and Issue: 197, P. 109337 - 109337

Published: Feb. 16, 2025

Identifying clinically relevant antibiotic resistance gene (ARG) hosts in complex microbial communities is crucial for environmental health. EpicPCR (emulsion, paired isolation, and concatenation PCR), a single-cell technology, has advanced this field. However, its traditional format, which links target genes to the V4 region of 16S rRNA (∼300 bp), limits species-level identification. To overcome this, we developed "long-read" epicPCR, segments spanning V4-V9 regions (∼1000 bp) by refining primer pairing strategies balance amplification length specificity. We validated approach targeting seven ARGs (optrA, tet(X4), mcr-3, NDM-5, KPC-2, IMP-4, VIM-1), an efflux pump (tmexD), insertion sequence (IS1216E), all confirming correct fusion. Using optrA as model target, long-read epicPCR demonstrated greater precision fewer false positives than short-read method mock communities. It also significantly improved identification rate host species from 29.0 % 54.4 anaerobic digestion reactors, while maintaining consistency with profiling bacterial Moreover, identified two novel species, Lactobacillus amylotrophicus Streptococcus alactolyticus, effluents, highlighting potential dissemination risks. Notably, versatile envisioned enhance targeted antimicrobial surveillance functional dynamics monitoring environment.

Language: Английский

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Dynamics of gut resistome and mobilome in early life: a meta-analysis

EBioMedicine, Journal Year: 2025, Volume and Issue: 114, P. 105630 - 105630

Published: March 5, 2025

Language: Английский

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Small amounts of misassembly can have disproportionate effects on pangenome-based metagenomic analyses

mSphere, Journal Year: 2025, Volume and Issue: unknown

Published: April 29, 2025

ABSTRACT Individual genes from microbiomes can drive host-level phenotypes. To help identify such candidate genes, several recent tools estimate microbial gene copy numbers directly metagenomes. These rely on alignments to pangenomes, which, in turn, are derived the set of all individual genomes one species. While large-scale metagenomic assembly efforts have made pangenome estimates more complete, mixed communities also introduce contamination into assemblies, and it is unknown how robust pangenome-based analyses these errors. gain insight this problem, we re-analyzed a case-control study gut microbiome cirrhosis, focusing commensal Clostridia previously implicated disease. We tested for differentially prevalent Lachnospiraceae then investigated which were likely be contaminants using sequence similarity searches. Out 86 found that 33 (38%) probably originating taxa as Veillonella Haemophilus , unrelated genera independently correlated with disease status. Our results demonstrate even small amounts metagenome below typical quality thresholds, threaten overwhelm gene-level analyses. However, show accurately identified method based gene-to-species correlation. After removing contaminants, observe flagellar motility clusters Lachnospira eligens associated cirrhosis integrated our an analysis visualization pipeline, PanSweep, automatically cases where may bias gene-resolved IMPORTANCE Metagenome-assembled genomes, or MAGs, constructed without pure cultures microbes. Large-scale build MAGs yielded complete pangenomes (i.e., sets species). Pangenomes allow us measure strain variation content, strongly affect phenotype. because come communities, they contaminate DNA; much impacts downstream has not been studied. Using microbes test case, investigate affects content. Surprisingly, small, MAG (<5%) result disproportionately high levels false positive associations (38%). Fortunately, most flagged provide simple doing so. Furthermore, applying reveals new association between motility.

Language: Английский

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Reference-free Structural Variant Detection in Microbiomes via Long-read Coassembly Graphs

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Jan. 30, 2024

Bacterial genome dynamics are vital for understanding the mechanisms underlying microbial adaptation, growth, and their broader impact on host phenotype. Structural variants (SVs), genomic alterations of 10 base pairs or more, play a pivotal role in driving evolutionary processes maintaining heterogeneity within bacterial populations. While SV detection isolate genomes is relatively straightforward, metagenomes present challenges due to absence clear reference presence mixed strains. In response, our proposed method rhea, forgoes metagenome-assembled (MAGs) by encompassing single metagenome coassembly graph constructed from all samples series. The log fold change coverage between subsequent then calculated call SVs that thriving declining throughout We show rhea outperform existing methods horizontal gene transfer (HGT) two simulated mock metagenomes, which particularly noticeable as reads diverge an increase strain diversity incorporated. additionally demonstrate use cases series metagenomic data environmental fermented food microbiomes detect specific sequence time temperature samples, suggesting advantage. Our innovative approach leverages raw read patterns rather than references MAGs include sequencing analysis, thus provide versatility studying across diverse poorly characterized communities more comprehensive insights into dynamics.

Language: Английский

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Reference-free structural variant detection in microbiomes via long-read co-assembly graphs

Bioinformatics, Journal Year: 2024, Volume and Issue: 40(Supplement_1), P. i58 - i67

Published: April 18, 2024

The study of bacterial genome dynamics is vital for understanding the mechanisms underlying microbial adaptation, growth, and their impact on host phenotype. Structural variants (SVs), genomic alterations 50 base pairs or more, play a pivotal role in driving evolutionary processes maintaining heterogeneity within populations. While SV detection isolate genomes relatively straightforward, metagenomes present broader challenges due to absence clear reference presence mixed strains. In response, our proposed method rhea, forgoes metagenome-assembled (MAGs) by encompassing all metagenomic samples series (time other metric) into single co-assembly graph. log fold change graph coverage between successive then calculated call SVs that are thriving declining.

Language: Английский

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Small amounts of misassembly can have disproportionate effects on pangenome-based metagenomic analyses

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Oct. 13, 2024

Abstract Individual genes from microbiomes can drive host-level phenotypes. To help identify such candidate genes, several recent tools estimate microbial gene copy numbers directly metagenomes. These rely on alignments to pangenomes, which in turn are derived the set of all individual genomes one species. While large-scale metagenomic assembly efforts have made pangenome estimates more complete, mixed communities also introduce contamination into assemblies, and it is unknown how robust pangenome-based analyses these errors. gain insight this problem, we re-analyzed a case-control study gut microbiome cirrhosis, focusing commensal Clostridia previously implicated disease. We tested for differentially prevalent Lachnospiraceae , then investigated were likely be contaminants using sequence similarity searches. Out 86 found that 33 (38%) probably originating taxa as Veillonella Haemophilus unrelated genera independently correlated with disease status. Our results demonstrate even small amounts metagenome below typical quality thresholds, threaten overwhelm gene-level analyses. However, show accurately identified method based gene-to-species correlation. After removing contaminants, observe flagellar motility clusters Lachnospira eligens associated cirrhosis integrated our an analysis visualization pipeline, PanSweep, automatically cases where may bias gene-resolved Importance Metagenome-assembled genomes, or MAGs, constructed without pure cultures microbes. Large scale build MAGs yielded complete pangenomes (i.e., sets species). Pangenomes allow us measure strain variation content, strongly affect phenotype. because come communities, they contaminate DNA, much impacts downstream has not been studied. Using microbes test case, investigate affects content. Surprisingly, small, MAG (<5%) result disproportionately high levels false positive associations (38%). Fortunately, most flagged, provide simple doing so. Furthermore, applying reveals new association between motility.

Language: Английский

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Using genomics to explore the epidemiology of vancomycin resistance in a sewage system

Microbiology Spectrum, Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 10, 2024

ABSTRACT VanHAX -mediated glycopeptide resistance has been consistently high in one of the three main sewer systems Copenhagen, Lynetten, for +20 years. To explore this other genes, and whether colonization resulted establishment multiple bacterial taxa, we mapped 505 shotgun metagenomic data sets from inlet sewage treatment plants to 831 different genes. Only vanHAX vanHBX genes were differentially abundant Lynetten. Analyses eight contigs suggested limited variations flanking regions. Proximity ligation analysis 12 samples Lynetten identified 441 5 paired reads mapping , respectively. The end these was generated metagenomic-assembled genomes NCBI using BLAST. could only be linked phylum level (Bacillota). Plasmid Hi-C showed that mainly located on plasmids reported found enterococci species. Most -linked class level, but some assigned Enterococcus faecium (7 reads), faecalis (4 Paenibacillus apiarius (2 thiaminolyticus (27 reads). Ten 20 contigs-containing annotated as plasmid, all This study shows while technology is valuable linking antimicrobial it suffers challenges reliably read a genomic region with sufficient taxonomic information. Our results also suggest over years colonizing system, not become widespread across remaining primarily E. exception . IMPORTANCE Long-term microbial communities antimicrobial-resistant bacteria expected result sharing between several taxa communities. We investigated microbiomes sewer, which have colonized glycopeptide-resistant harboring mobile gene cluster minimum years, metagenomics sequencing Hi-C. despite long-term presence seemingly disseminated widely.

Language: Английский

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