Shotgun Metagenomics Research Articles

The skin microbiome includes bacteria, fungi, and viruses, with composition varying significantly across body sites. Although 16S rRNA gene sequencing is common, it excludes non-prokaryotic taxa and offers limited functional data. Shotgun metagenomics provides broader taxonomic and functional insights but is challenging for low-biomass skin samples due to limited microbial DNA and high host contamination. In this study, we characterized the microbiome of the forehead and armpits in healthy individuals using shotgun metagenomics and assessed the strategies to improve sequencing success. We compared collection kits, DNA extraction protocols, and tested multiple displacement amplification (MDA). We found that sampling with D-Squame discs followed by an in-house DNA extraction protocol was the most effective combination to maximize DNA yields. MDA introduced significant compositional biases and is not recommended. Shotgun sequencing, without MDA, produced microbial compositions and diversity indices broadly consistent with 16S rRNA metabarcoding, although it showed discrepancies in the relative abundance of some genera. Consistent with prior studies, the armpit microbiome was dominated by Staphylococcus spp., whereas the forehead microbiome was dominated by Cutibacterium spp. Critically, shotgun sequencing provided additional insights into viral and eukaryotic microorganisms and revealed the functional potential of microbial communities, demonstrating its clear advantages over 16S rRNA metabarcoding for comprehensive skin microbiome research.IMPORTANCEWith growing evidence of the role of microorganisms in maintaining healthy skin, accurately characterizing the skin microbiome remains a significant challenge. In this study, we demonstrate that shotgun sequencing, carried out with adapted wet lab protocols, provides deep insights into the microbiome composition of specific areas, such as the forehead or the armpits. Notably, it enables the characterization of fungi and viruses while offering direct functional insights into microbial communities, providing a clear advantage over 16S ribosomal RNA gene sequencing. Our findings highlight the potential of shotgun metagenomics as a powerful tool for comprehensive skin microbiome analysis. They emphasize the importance of tailored protocols for low-biomass samples, improving the reliability of shotgun sequencing and paving the way for more robust clinical studies focused on the skin microbiome.

The human endometrium, previously considered a sterile environment, is now recognized as a low-biomass but biologically active microbial niche critical to reproductive health. Advances in sequencing technologies, particularly shotgun metagenomics, have provided unprecedented insights into the taxonomic and functional complexity of the endometrial microbiome. While 16S rRNA sequencing has delineated the distinction between Lactobacillus-dominant and non-dominant microbial communities, shotgun metagenomics has revealed additional diversity at the species and strain level, uncovering microbial signatures that remain undetected by amplicon-based approaches. Current evidence supports the association of Lactobacillus dominance with endometrial homeostasis and favorable reproductive outcomes. Dysbiosis, characterized by increased microbial diversity and enrichment of anaerobic taxa such as Gardnerella, Atopobium, Prevotella, and Streptococcus, is linked to chronic endometritis, implantation failure, and adverse IVF results. Beyond compositional differences, the endometrial microbiome interacts with the host through immunological, metabolic, and epigenetic mechanisms. These interactions modulate cytokine signaling, epithelial barrier integrity, and receptivity-associated gene expression, ultimately influencing embryo implantation. However, discrepancies between published studies reflect the lack of standardized protocols for sampling, DNA extraction, and bioinformatic analysis, as well as the inherent challenges of studying low-biomass environments. Factors such as geography, ethnicity, hormonal status, and antibiotic exposure further contribute to interindividual variability. Culturomics approaches complement sequencing by enabling the isolation of viable bacterial strains, offering perspectives for microbiome-based biotherapeutics. Emerging 3D endometrial models provide additional tools to dissect microbiome–host interactions under controlled conditions. Taken together, the growing body of data highlights the potential of endometrial microbiome profiling as a biomarker for reproductive success and as a target for personalized interventions. Future research should focus on integrating multi-omics approaches and functional analyses to establish causal relationships and translate findings into clinical practice. This review gives a new insight into current knowledge on the uterine microbiome and its impact on implantation success, analyzed through the lenses of microbiology, immunology, and oxidative stress.

Colitis-associated cancer (CAC) arises from a complex interplay between host and environmental factors. In this report, we investigated the role of the gut microbiome using Winnie mice, a UC-like model with a missense mutation in the Muc2 gene. Upon rederivation from a conventional (CONV) to a specific-pathogen-free (SPF) facility, Winnie mice developed severe colitis and, notably, spontaneous CAC that progressively worsened over time. In contrast, CONV Winnie showed only mild colitis but no tumorigenesis. By comparison, when rederived into germ-free (GF) conditions, SPF Winnie mice were protected from colitis and colon tumors, indicating an essential role for the gut microbiome in the development of CAC in these mice. Using shotgun metagenomics, metabolomics, and lipidomics, we identified a distinct pro-inflammatory microbial and metabolic signature that potentially drives the transition from colitis to CAC. Fecal microbiota transplantation (FMT), using either SPF Winnie or WT (Bl/6) donors into GF Winnie recipients, demonstrated that while colitis developed regardless of the donor, only FMT from SPF Winnie donors resulted in CAC. Our studies present a relevant model of CAC, providing strong evidence that the microbiome plays a key role in its pathogenesis, thereby challenging the concept of colon cancer as a strictly non-transmissible disease.

Damage induced by lung infections in people with cystic fibrosis (CF) is the leading factor to the mortality and morbidity of CF. To treat bacterial infections, people with CF are prescribed antibiotics. Routine antibiotic susceptibility (ABS) testing relies on culture-dependent, phenotypic techniques. These take several days up to more than a week, while timely intervention is key. To bridge this time gap, physicians in CF care use patient history of ABS data to start antibiotics, with risk of resistance to it. This pilot study explores a time saving alternative: the possibility to predict antibiotic resistance genes using shotgun metagenomics and artificial intelligence. By quicker prediction of ABS, people with CF can receive more adequate care, which results in the possible prevention of chronic infections and contributes to antibiotic stewardship.

Gut microbiota enable wood-feeding insects to digest recalcitrant diets. Two DNA-based analyses were performed. Amplicon sequencing of gut microbiota samples from Cryptocercus punctulatus showed inter-individual heterogeneity with visually distinct ordination patterns; however, no statistically significant differences were detected. Shotgun metagenomics was used to compare the taxonomic and functional profiles of C. punctulatus gut microbiota with those of other xylophagous Dictyoptera. Despite taxonomic differences, C. punctulatus microbiota revealed functional convergence with termites (Mastotermes darwiniensis and Nasutitermes sp.). Carbohydrate metabolism was performed by different bacterial phyla across all insects. All insect species possessed metabolic potential for cellulose, hemicellulose, pectin, and starch digestion, but lignin degradation capabilities were not detected. Termites showed higher abundance of chitin and xylan degradation pathways and nitrogen fixation genes, though nitrogen fixation was also present in Cryptocercus cockroaches. Genes for oxidative stress tolerance were present across all species but were most abundant in cockroaches, particularly, Cryptocercus. All insects harbored antibiotic resistance genes, with highest levels found in cockroaches. These findings indicate that metabolic requirements for wood digestion shape gut microbial community assembly across xylophagous insects, with distinct microbial taxa contributing to cellulose and hemicellulose breakdown. Moreover, the widespread presence of antibiotic resistance genes raises concerns about the potential transmission of antibiotic resistance within insect-associated microbiomes.

Neutral and metalliferous drainage (NMD) poses an environmental risk for both operating and legacy mine sites. Near-neutral pH distinguishes NMD from more acidic conditions of acid and metalliferous drainage (AMD), however NMD contains elevated levels of metals that necessitate strict management. Microbial communities are key indicators of ecological conditions and play important roles in NMD biogeochemical cycling, often exhibiting distinct dynamics compared to AMD. Shotgun sequencing and metagenome assembled genomes (MAGs) were used to characterize microbial diversity and functional potential across water, biofilm, and sediment microbiomes along a flow path at a historical lead-zinc mine in Western Australia. Zn levels peaked upstream and declined downstream, corresponding to shifts in microbial diversity. In water microbiomes, a Polynucleobacter MAG became dominant where Zn concentrations dropped below known toxicity thresholds. The genomic traits of Polynucleobacter, including a streamlined genome, Zn- (LpxC) and heat-responsive membrane genes, and enriched lipid metabolism pathways, enabled survival under metal and nutrient stress. Photosynthetic biofilms, dominated by cyanobacteria such as Synechococcaceae and Leptolyngbyaceae, played a central role in ecosystem function. These biofilms contained genes for photosynthesis, metal transport, and motility, and likely contributed organic carbon and sulfur intermediates that supported heterotrophs like Polynucleobacter and Sediminibacterium. Coordinated microbial sulfur metabolism across habitats was evident, with sulfur oxidation occurring in water and biofilms and sulfate reduction localized to sediment, evidenced with ZnS mineral phases associated with increased DsrMKJOP gene abundance. These findings are vital for mine closure and land reclamation, offering knowledge on key microbial adaption and syntrophy in NMD systems.

Abstract The intratumoral microbiome is an emerging hallmark of cancer, yet its multi‐kingdom host–microbiome ecosystem in colorectal cancer (CRC) remains poorly characterized. Here, we conducted an integrated analysis using deep shotgun metagenomics and proteomics on 185 tissue samples, including adenoma (A), paired tumor (T), and para‐tumor (P). We identified 4057 bacterial, 61 fungal, 108 archaeal, and 374 viral species in tissues and revealed distinct intratumor microbiota dysbiosis, indicating a CRC‐specific multi‐kingdom microbial ecosystem. Proteomic profiling uncovered four CRC subtypes (C1–C4), each with unique clinical prognoses and molecular signatures. We further discovered that host‐microbiome interactions are dynamically reorganized during carcinogenesis, where different microbial taxa converge on common host pathways through distinct proteins. Leveraging this interplay, we identified 14 multi‐kingdom microbial and 8 protein markers that strongly distinguished A from T samples (area under the receiver operating characteristic curve (AUROC) = 0.962), with external validation in two independent datasets (AUROC = 0.920 and 0.735). Moreover, we constructed an early‐ versus advanced‐stage classifier using 8 microbial and 4 protein markers, which demonstrated high diagnostic accuracy (AUROC = 0.926) and was validated externally (AUROC = 0.659–0.744). Functional validation in patient‐derived organoids and murine allograft models confirmed that enterotoxigenic Bacteroides fragilis and Fusobacterium nucleatum promoted tumor growth by activating Wnt/β‐catenin and NF‐κB signaling pathways, corroborating the functional potential of these biomarkers. Together, these findings reveal dynamic host–microbiome interactions at the protein level, tracing the transition from adenoma to carcinoma and offering potential diagnostic and therapeutic targets for CRC.

Background: The microbially produced phenylalanine–derived metabolite Phenylacetic acid (PAA) contributes to endothelial senescence in aging. However, the effects of PAA on perivascular adipose tissue (PVAT) physiology and its role in atherosclerosis are unknown. Using both human and animal model studies, we investigated the potential contributory role of ImP in the development of atherosclerosis. Methods: To address the knowledge gap, plasma PAA levels were measured by LC-MS/MS, coupled with fecal shotgun metagenomics in the TwinsUK Aging Cohort (n=2,953) and Aging Heart Zurich Cohort (ASCVD patients; n=105). In a mouse model of chronological aging, the impact of PAA on PVAT senescence and dysfunction was examined. Additionally, atheroprone Ldlr −/− mice fed a high-fat diet were treated with and without PAA (50 mg/kg/d), and aortic atherosclerotic lesion area was evaluated after 12 weeks. Results: Here, we show that the gut microbial metabolite phenylacetic acid (PAA) drives aortic PVAT senescence and dysfunction in both chronological aging and atherosclerotic states. Our findings indicate that PAA production increases with age, which is positively associated with the abundance of gut bacterium Clostridium sp. ASF356 in aged mice. The bacterium- and PAA-induced senescence phenotype was verified in both cellular and mouse models. In atheroprone Ldlr -/- mice with an ABx-suppressed intestinal microbiota, daily supplementation with PAA increased atherosclerotic lesion size in vivo . Mechanistically, senescence-messaging secretome, including IL6, from senescent endothelial cells induces a senescence-like state and disrupted insulin and metabolic signaling through Notch1 activation in adipocytes, leading to vascular dysfunction. Our senolytic D+Q therapy impeded PAA-induced cellular senescence in vivo , restoring PVAT function. Conclusions: Our findings uncover a gut-vascular axis whereby gut microbiota metabolite PAA causally exacerbates aortic PVAT dysfunction through endothelial senescence. Furthermore, these mechansisms accelerate the development of atherosclerosis in aging, suggesting that PAA metabolism is a potential therapeutic target in atherosclerotic cardiovascular disease.

Introduction Despite durable viral suppression, neuroinflammation and neurocognitive complications remain common yet poorly understood in people with HIV (PWH). HIV alters human viromes, and virome perturbations have been linked to neurocognitive issues in people without HIV. Recently characterized, the brain and cerebrospinal fluid (CSF) viromes represent a new avenue to understand brain and mental health in PWH. Methods This cross-sectional study analyzed 85 CSF samples (74 from PWH on suppressive antiretroviral therapy, and 11 from controls without HIV, CWH) through shotgun metagenomics for DNA and RNA viruses. Taxonomic composition (reads and contigs), diversity, and relative abundance (RA) of prokaryotic (PV), human eukaryotic (hEV), and non-human eukaryotic viruses (nhEV) were evaluated in relation to HIV status, markers of neuroinflammation/neurodegeneration, cognitive functions, and depressive symptoms. Sensitivity analyses and post-hoc cluster analysis on the RA of hEV, non-human viruses (NHV) and blood–brain barrier permeability were performed. Multivariable models assessed the relationship between cognition and clusters. Results Of 46 read-positive CSF samples, 93.5% contained PV sequences, 47.8% hEV, and 45.6% nhEV. PWH displayed lower α diversity, although p > 0.05. At β diversity analysis, HIV status explained 3.4% of the variation in viral composition ( p = 0.016). Contigs assembly yielded 13 samples positive for 8 hEV, 2 nhEV, and 6 PV. Higher RA of PV was correlated with higher CSF S100β (rho 0.36, p = 0.002) and β-Amyloid 1–42 fragment (βA-42, rho 0.27, p = 0.026), whereas higher RA of nhEV with poorer cognitive performance (rho 0.26, p = 0.022). Conversely, higher RA of hEV correlated with better cognition (rho −0.38, p = 0.003) and lower βA-42 (rho −0.30, p = 0.012). Sensitivity analyses restricted to only CSF samples with detectable reads confirmed these findings. Three CSF clusters were identified and showed differences in astrocytosis, βA-42, tau protein, and cognitive functions. Participants with hEV-enriched CSF showed better cognitive performance compared to those with virus-devoid and NHV-enriched CSF (all p < 0.05). Conclusion This study provides the first comprehensive description of the CSF virome in PWH, revealing associations with neuroinflammation and cognition. These findings highlight the potential involvement of the CSF virome in brain health and inform about its composition, origin, and potential clinical implications in people with and without HIV.

Resistant starch (RS), a major category of dietary fiber, promotes healthy lipid and glucose metabolism. These effects are partly mediated by gut microbes. RS is known to influence microbial bile acid metabolism and, in turn, can lower serum cholesterol. We hypothesized that RS may promote another microbial process that affects serum cholesterol levels – the cholesterol:coprostanol transformation pathway, which has not been fully characterized. Individuals who harbor gut microbes encoding a recently identified gene in the pathway, intestinal sterol metabolism A (ismA), can transform cholesterol to less absorbable coprostanol. Individuals with microbial ismA genes have lower serum cholesterol than people without microbial ismA genes. We conducted a seven-week crossover study with 59 individuals completing three treatments: dietary supplementation with RS type 2, RS type 4, and a digestible starch, for ten days each with five-day washout periods in between. We collected fecal samples at the beginning and end of each treatment and extracted DNA to generate a shotgun metagenomics dataset from the gut microbiomes. We also measured the concentrations of bile acids and the sterols, cholesterol and coprostanol, in the stool. RS supplementation caused interindividual variability in gut microbiome response. Also, during the RS type 2 treatment, we observed a decrease in taurocholic acid, a bile acid that may promote colon cancer (q < 0.05). RS supplementation did not universally affect sterol levels. However, we observed consistent correlations between the abundances of some microbes, or species level genome bins (SGBs), and sterol concentrations longitudinally within subjects. We identified 24 SGBs, including three Oscillibacter species, which were negatively correlated with cholesterol and positively correlated with coprostanol (Spearman's rho ≥ 0.4). In addition to its ability to transform cholesterol, Oscillibacter has previously been implicated as benefiting lipid metabolism and cardiovascular health. We also identified 12 unclassified SGBs that may be involved in the transformation of cholesterol to coprostanol in the human gut. These SGBs may represent previously undiscovered species that participate in the cholesterol:coprostanol transformation pathway, and these species may possess unknown genes in the pathway. Comprehensive characterization of this pathway could lead to novel microbiome-based therapeutics to treat hypercholesterolemia.

Taxonomic and functional characteristics of the gut microbiota in obesity: A systematic review.

Exploring the complexities of intestinal and pulmonary microbiota in cystic fibrosis: A multi-omics approach.

BackgroundKhecheopalri Lake, a sacred freshwater body and recently recognized Ramsar Wetland site in Sikkim, India, holds both ecological and cultural significance. The ecological health of this lake is influenced by elemental inputs and environmental parameters, yet its microbial and functional diversity remain poorly characterized. In this study, we employed a multi-omics approach combining shotgun metagenomics, inductively coupled plasma mass spectrometry (ICP-MS), and culture-dependent analyses to provide an integrated understanding of the lake’s microbial ecosystem. Shotgun metagenomics revealed taxonomic diversity and functional gene profiles, ICP-MS quantified elemental composition and its potential role in shaping microbial communities, while culture-dependent methods complemented metagenomic insights by isolating representative taxa. Together, these approaches highlight the interactions between microbes and elemental dynamics, offering new perspectives on the ecological functioning of this Himalayan wetland and its potential vulnerability to environmental change.ResultsICP-MS analysis revealed phosphorus (P) as the most abundant element, followed by iron (Fe), sodium (Na), magnesium (Mg), and potassium (K). Elevated BOD and COD levels in sample KES4 indicated organic pollution and coincided with the dominance of Microcystis aeruginosa, a cyanobacterium indicative of eutrophication. Shotgun metagenomic sequencing generated approximately 213 million reads, with bacteria constituting 98.85% of the community. Dominant phyla included Pseudomonadota and Cyanobacteria. Culturable isolates confirmed the presence of genera such as Limnohabitans, Microcystis, and Mycolicibacterium. Functional gene profiling showed that metabolism was the most enriched category (71.64%), with several genes (e.g., xylB, pchF, clcD) associated with xenobiotic degradation pathways.ConclusionThis first comprehensive metagenomic assessment of Khecheopalri Lake reveals diverse microbial populations involved in nutrient cycling and pollutant detoxification. The presence of genes linked to aromatic hydrocarbon degradation highlights the ecological potential of native microbes in mitigating environmental stress.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12866-025-04450-1.

Background/Objectives: Gallstone disease is a prevalent digestive disorder worldwide, with incidence increasingly linked to obesity, insulin resistance, and dyslipidemia. Emerging evidence suggests that intestinal microbial communities and their bioactive compounds play a central role in pathogenesis. Here, we aimed to clarify whether diet-related microbial alterations and circulating metabolites contribute to gallstone formation. Methods: We integrated dietary inflammatory index (DII) evaluation, genetic analyses of large-scale cohorts, and a cholesterol gallstone mouse model induced by a lithogenic diet (LD). Serum and fecal samples were subjected to metabolomic and metagenomic profiling, followed by multi-omics integration to identify links between microbial taxa, metabolites, and gallstone risk. Results: Higher DII scores were associated with increased gallstone risk. Genetic evidence supported bile acid and amino acid metabolism as potential mediating pathways, with Akkermansia muciniphila linked to decreased N-acetylarginine levels and CAG-448 showing an inverse association with glycodeoxycholate (GDCA). In LD-fed mice, shotgun metagenomics revealed enrichment of lithogenic taxa such as Bacteroides stercorirosoris and Enterocloster, whereas protective taxa, including Akkermansia muciniphila and CAG-448, were markedly depleted. Untargeted metabolomics confirmed elevations of GDCA and N-acetylarginine together with broader bile acid imbalance, amino acid stress, and long-chain acylcarnitine accumulation. Correlation analyses further showed that protective taxa were inversely associated with risk metabolites, whereas gallstone-enriched microbes displayed the opposite pattern. Conclusions: This study provides evidence consistent with a contributory role of gut microbiota–metabolite dysregulation in gallstone pathogenesis. Specific taxa (A. muciniphila, CAG-448) and metabolites (GDCA, N-acetylarginine) may serve as potential biomarkers or targets for microbiota- and diet-based prevention strategies.

The alternative pathway (AP) plays a crucial role in triggering complement activation and promoting chronic inflammation. This study aims to investigate the longitudinal association between AP and atherosclerosis, and explore the potential role of gut microbiota and inflammatory factors in their association. This study was based on a 9-year prospective cohort of 3382 participants from Guangzhou, China (mean age±SD, 57.75±5.85 years; 68.8% female), with data on serum APACPs (AP-associated complement proteins) and carotid plaque (measured by ultrasound) repeatedly measured up to 3×. Baseline inflammatory markers were evaluated in 923 participants, and gut shotgun metagenome data were obtained from 1567 participants. Mendelian randomization analysis was performed using genome-wide significant genetic variants as instrumental variables to suggest potential causal associations. Both longitudinal and prospective analyses consistently demonstrated positive associations between carotid plaque and 3 complement components: C3 (odds ratios [95% Cl] for the highest versus lowest quartiles, 1.36 [1.07-1.74] in longitudinal analysis and 1.29 [1.06-1.56] in prospective analysis), CFB (complement factor B; 1.36 [1.07-1.72] in longitudinal analysis and 1.39 [1.15-1.69] in prospective analysis), and CFH (complement factor H; 1.39 [1.10-1.76] in longitudinal analysis and 1.31 [1.07-1.61] in prospective analysis). Mendelian randomization analysis suggested a potential causal association between CFB and carotid plaque. Inflammatory factors (CRP [C-reactive protein] and IL-6 [interleukin-6]) and microbial species (Ruminococcus bromii, Roseburia hominis, Rothia mucilaginosa, Collinsella stercoris, Olsenella scatoligenes, and Bacteroides massiliensis) were significantly associated with both APACPs and carotid plaque (P<0.05). For example, butyrate-producing bacterium R bromii was inversely associated with CFB and carotid plaque (odds ratios [95% CI], 0.83 [0.79-0.88]) and may mediate the CFB-carotid plaque association (proportion mediated, 13.5%; P=0.005). Microbial risk score (weighted sum of selected microbial species; proportion mediated, 42.6%; P<0.001) and total immune factors (the sum of all inflammatory factors; proportion mediated, 19.0%; P=0.002) mediated the association between Total-APACPs (sum of standardized carotid plaque-related APACPs [C3, CFB, and CFH]) and carotid plaque. Our study showed a negative association between the AP and carotid plaque in a longitudinal cohort. Gut microbiota and inflammatory biomarkers may provide mechanistic insights into the association between the AP and atherosclerosis. Our findings pave the way for the development of new therapeutic targets for atherosclerosis.

BackgroundThe aerial surface of plants, known as the phyllosphere, hosts a complex and dynamic microbiome that plays essential roles in plant health and environmental processes. While research has focused on root-associated microbiomes, the phyllosphere remains comparatively understudied, especially in forest ecosystems. Despite the global ecological dominance and importance of conifers, no previous study has applied shotgun metagenomics to their phyllosphere microbiomes.ResultsThis study uses metagenomic sequencing to explore the microbial phyllosphere communities of subalpine Western conifer needle surfaces from 67 trees at six sites spanning the Rocky Mountains, including 31 limber pine, 18 Douglas fir, and 18 Engelmann spruce. Sites span ~ 1,075 km and nearly 10° latitude, from Glacier National Park to Rocky Mountain Biological Laboratory, capturing broad environmental variation. Metagenomes were generated for each of the 67 samples, for which we produced individual assemblies, along with three large coassemblies specific to each conifer host. From these datasets, we reconstructed 447 metagenome-assembled genomes (MAGs), 417 of which are non-redundant at the species level. Beyond increasing the total number of extracted MAGs from 153 to 294, the three coassemblies yielded three large MAGs, representing partial sequences of host genomes. Phylogenomics of all microbial MAGs revealed communities predominantly composed of bacteria (n = 327) and fungi (n = 117). We show that both microbial community composition and metabolic potential differ significantly across host tree species and geographic sites, with site exerting a stronger influence than host.ConclusionsThis dataset offers new insights into the microbial communities inhabiting the conifer needle surface, laying the foundation for future research on needle microbiomes across temporal and spatial scales. Variation in functional capabilities, such as volatile organic compound (VOC) degradation and polysaccharide metabolism, closely tracks shifts in taxonomic composition, indicating that host-specific chemistry, local environmental factors, and regional microbial source pools jointly shape ecological roles. Moreover, the observed patterns of mobile genetic elements and horizontal gene transfer suggest that gene exchange predominantly occurs within microbial lineages, with occasional broader transfers dispersing key functional genes (e.g., those involved in polysaccharide metabolism), which may facilitate microbiome adaptation.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40168-025-02271-y.

High-protein diet (HPD) and high-fiber diet (HFiD) are two common types of diet, and the transition from HPD to HFiD frequently happens in people who perform diet control. Gut microbiota provide a bridge between diet and health. However, most of the research left out the metabolic landscape change and gut microbial influence under this kind of altered diet. Here, we examined gut microbiome and metabolites change using multi-omics (16S rRNA gene sequencing, shotgun metagenomics, LC-MS/MS metabolomics), and machine-learning methods were introduced to capture the response of gut microbiota to altered diet and associate the variable metabolites and microbial factors. The results suggest that the conversion from HPD to HFiD improves the gut microbial diversity and promotes the pathways of tryptophan, galactose, fructose, and mannose metabolism, which are associated with different gut microbiota and special metabolites. Among them, Faecalibacterium rodentium and Akkermansia muciniphila indicate potential roles in the multi-metabolism. Although these findings are exploratory and hypothesis-generating, they provide a foundation for future mechanistic investigations and emphasize the importance of diverse diets.

This study explored the metagenomic sequencing methodology for analyzing the breast milk microbiome and elucidated its composition. Twenty-two breast milk samples were collected from 11 healthy lactating women. By optimizing microbial cell wall disruption parameters and developing a nucleic acid extraction method, microbial DNA/RNA libraries were constructed and subjected to metagenomic next-generation sequencing (mNGS), microbial standards spiked into breast milk at serial dilutions served to validate the method's reliability. The sequencing data underwent rigorous quality control and classification using the Kraken2 software and a self-generated database. The breast milk microbiome was found to comprise 21 phyla, 234 genera, and 487 species, with Firmicutes and Proteobacteria being the dominant phyla. At the genus level, Staphylococcus and Streptococcus were the most abundant, while at the species level, Staphylococcus aureus, Streptococcus bradystis, and Staphylococcus epidermidis were the most prevalent. The microbial profiles of the left and right breast milk samples were consistent at the phylum, genus, and species levels. Besides common bacteria, diverse viral, eukaryotic, and archaeal sequences were also detected. Functional profiling revealed that the "lactose and galactose degradation I" pathway accumulated the highest read count, whereas the L-valine biosynthesis pathway was detected most frequently. This study provides a comprehensive understanding of the healthy breast milk microbiome, highlighting the presence of specific flora colonization and the distinct yet correlated microbial environments in bilateral breast milk, laying the groundwork for future research into the interactions between breast milk microbiota and maternal and infant health outcomes.

The progressive shift in anaerobic digestion communities under extreme propionate levels led to a redundant microbiome capable of producing methane.

Shotgun metagenomics holds great potential for identifying parasite DNA in biological samples, but its effectiveness is limited by widespread contamination in publicly available reference genomes, which hinders accurate detection. In this study, we systematically quantify and remove contamination from 831 published endoparasite genomes to create ParaRef, a curated reference database for species-level parasite detection. We show that decontamination significantly reduces false detection rates and improves overall detection accuracy. Our study highlights the pervasive issue of contamination in public databases and offers a resource that will enhance the reliability of parasite detection using metagenomics.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13059-025-03818-w.