As microbial ecologists take advantage of high-throughput sequencing technologies to describe microbial communities across ever-increasing numbers of samples, new analysis tools are required to ...relate the distribution of microbes among larger numbers of communities, and to use increasingly rich and standards-compliant metadata to understand the biological factors driving these relationships. In particular, the Earth Microbiome Project drives these needs by profiling the genomic content of tens of thousands of samples across multiple environment types.
Features of EMPeror include: ability to visualize gradients and categorical data, visualize different principal coordinates axes, present the data in the form of parallel coordinates, show taxa as well as environmental samples, dynamically adjust the size and transparency of the spheres representing the communities on a per-category basis, dynamically scale the axes according to the fraction of variance each explains, show, hide or recolor points according to arbitrary metadata including that compliant with the MIxS family of standards developed by the Genomic Standards Consortium, display jackknifed-resampled data to assess statistical confidence in clustering, perform coordinate comparisons (useful for procrustes analysis plots), and greatly reduce loading times and overall memory footprint compared with existing approaches. Additionally, ease of sharing, given EMPeror's small output file size, enables agile collaboration by allowing users to embed these visualizations via emails or web pages without the need for extra plugins.
Here we present EMPeror, an open source and web browser enabled tool with a versatile command line interface that allows researchers to perform rapid exploratory investigations of 3D visualizations of microbial community data, such as the widely used principal coordinates plots. EMPeror includes a rich set of controllers to modify features as a function of the metadata. By being specifically tailored to the requirements of microbial ecologists, EMPeror thus increases the speed with which insight can be gained from large microbiome datasets.
Key points
Age‐related arterial dysfunction, characterized by oxidative stress‐ and inflammation‐mediated endothelial dysfunction and arterial stiffening, is the primary risk factor for ...cardiovascular diseases.
To investigate whether age‐related changes in the gut microbiome may mediate arterial dysfunction, we suppressed gut microbiota in young and old mice with a cocktail of broad‐spectrum, poorly‐absorbed antibiotics in drinking water for 3–4 weeks.
In old mice, antibiotic treatment reversed endothelial dysfunction and arterial stiffening and attenuated vascular oxidative stress and inflammation.
To provide insight into age‐related changes in gut microbiota that may underlie these observations, we show that ageing altered the abundance of microbial taxa associated with gut dysbiosis and increased plasma levels of the adverse gut‐derived metabolite trimethylamine N‐oxide.
The results of the present study provide the first proof‐of‐concept evidence that the gut microbiome is an important mediator of age‐related arterial dysfunction and therefore may be a promising therapeutic target for preserving arterial function with ageing, thereby reducing the risk of cardiovascular diseases.
Oxidative stress‐mediated arterial dysfunction (e.g. endothelial dysfunction and large elastic artery stiffening) is the primary mechanism driving age‐related cardiovascular diseases. Accumulating evidence suggests the gut microbiome modulates host physiology because dysregulation (‘gut dysbiosis’) has systemic consequences, including promotion of oxidative stress. The present study aimed to determine whether the gut microbiome modulates arterial function with ageing. We measured arterial function in young and older mice after 3–4 weeks of treatment with broad‐spectrum, poorly‐absorbed antibiotics to suppress the gut microbiome. To identify potential mechanistic links between the gut microbiome and age‐related arterial dysfunction, we sequenced microbiota from young and older mice and measured plasma levels of the adverse gut‐derived metabolite trimethylamine N‐oxide (TMAO). In old mice, antibiotics reversed endothelial dysfunction area‐under‐the‐curve carotid artery dilatation to acetylcholine in young: 345 ± 16 AU vs. old control (OC): 220 ± 34 AU, P < 0.01; vs. old antibiotic‐treated (OA): 334 ± 15 AU; P < 0.01 vs. OC and arterial stiffening (aortic pulse wave velocity in young: 3.62 ± 0.15 m s−1 vs. OC: 4.43 ± 0.38 m s−1; vs. OA: 3.52 ± 0.35 m s−1; P = 0.03). These improvements were accompanied by lower oxidative stress and greater antioxidant enzyme expression. Ageing altered the abundance of gut microbial taxa associated with gut dysbiosis. Lastly, plasma TMAO was higher with ageing (young: 2.6 ± 0.4 μmol L−1 vs. OC: 7.2 ± 2.0 μmol L−1; P < 0.0001) and suppressed by antibiotic treatment (OA: 1.2 ± 0.2 μmol L−1; P < 0.0001 vs. OC). The results of the present study provide the first evidence for the gut microbiome being an important mediator of age‐related arterial dysfunction and oxidative stress and suggest that therapeutic strategies targeting gut microbiome health may hold promise for preserving arterial function and reducing cardiovascular risk with ageing in humans.
Key points
Age‐related arterial dysfunction, characterized by oxidative stress‐ and inflammation‐mediated endothelial dysfunction and arterial stiffening, is the primary risk factor for cardiovascular diseases.
To investigate whether age‐related changes in the gut microbiome may mediate arterial dysfunction, we suppressed gut microbiota in young and old mice with a cocktail of broad‐spectrum, poorly‐absorbed antibiotics in drinking water for 3–4 weeks.
In old mice, antibiotic treatment reversed endothelial dysfunction and arterial stiffening and attenuated vascular oxidative stress and inflammation.
To provide insight into age‐related changes in gut microbiota that may underlie these observations, we show that ageing altered the abundance of microbial taxa associated with gut dysbiosis and increased plasma levels of the adverse gut‐derived metabolite trimethylamine N‐oxide.
The results of the present study provide the first proof‐of‐concept evidence that the gut microbiome is an important mediator of age‐related arterial dysfunction and therefore may be a promising therapeutic target for preserving arterial function with ageing, thereby reducing the risk of cardiovascular diseases.
Inflammatory bowel diseases, which include Crohn's disease and ulcerative colitis, affect several million individuals worldwide. Crohn's disease and ulcerative colitis are complex diseases that are ...heterogeneous at the clinical, immunological, molecular, genetic, and microbial levels. Individual contributing factors have been the focus of extensive research. As part of the Integrative Human Microbiome Project (HMP2 or iHMP), we followed 132 subjects for one year each to generate integrated longitudinal molecular profiles of host and microbial activity during disease (up to 24 time points each; in total 2,965 stool, biopsy, and blood specimens). Here we present the results, which provide a comprehensive view of functional dysbiosis in the gut microbiome during inflammatory bowel disease activity. We demonstrate a characteristic increase in facultative anaerobes at the expense of obligate anaerobes, as well as molecular disruptions in microbial transcription (for example, among clostridia), metabolite pools (acylcarnitines, bile acids, and short-chain fatty acids), and levels of antibodies in host serum. Periods of disease activity were also marked by increases in temporal variability, with characteristic taxonomic, functional, and biochemical shifts. Finally, integrative analysis identified microbial, biochemical, and host factors central to this dysregulation. The study's infrastructure resources, results, and data, which are available through the Inflammatory Bowel Disease Multi'omics Database ( http://ibdmdb.org ), provide the most comprehensive description to date of host and microbial activities in inflammatory bowel diseases.
Data from 16S ribosomal RNA (rRNA) amplicon sequencing present challenges to ecological and statistical interpretation. In particular, library sizes often vary over several ranges of magnitude, and ...the data contains many zeros. Although we are typically interested in comparing relative abundance of taxa in the ecosystem of two or more groups, we can only measure the taxon relative abundance in specimens obtained from the ecosystems. Because the comparison of taxon relative abundance in the specimen is not equivalent to the comparison of taxon relative abundance in the ecosystems, this presents a special challenge. Second, because the relative abundance of taxa in the specimen (as well as in the ecosystem) sum to 1, these are compositional data. Because the compositional data are constrained by the simplex (sum to 1) and are not unconstrained in the Euclidean space, many standard methods of analysis are not applicable. Here, we evaluate how these challenges impact the performance of existing normalization methods and differential abundance analyses.
Effects on normalization: Most normalization methods enable successful clustering of samples according to biological origin when the groups differ substantially in their overall microbial composition. Rarefying more clearly clusters samples according to biological origin than other normalization techniques do for ordination metrics based on presence or absence. Alternate normalization measures are potentially vulnerable to artifacts due to library size. Effects on differential abundance testing: We build on a previous work to evaluate seven proposed statistical methods using rarefied as well as raw data. Our simulation studies suggest that the false discovery rates of many differential abundance-testing methods are not increased by rarefying itself, although of course rarefying results in a loss of sensitivity due to elimination of a portion of available data. For groups with large (~10×) differences in the average library size, rarefying lowers the false discovery rate. DESeq2, without addition of a constant, increased sensitivity on smaller datasets (<20 samples per group) but tends towards a higher false discovery rate with more samples, very uneven (~10×) library sizes, and/or compositional effects. For drawing inferences regarding taxon abundance in the ecosystem, analysis of composition of microbiomes (ANCOM) is not only very sensitive (for >20 samples per group) but also critically the only method tested that has a good control of false discovery rate.
These findings guide which normalization and differential abundance techniques to use based on the data characteristics of a given study.
Advances in technical capabilities for reading complex human microbiomes are leading to an explosion of microbiome research, leading in turn to intense interest among clinicians in applying these ...techniques to their patients. In this review, we discuss the content of the human microbiome, including intersubject and intrasubject variability, considerations of study design including important confounding factors, and different methods in the laboratory and on the computer to read the microbiome and its resulting gene products and metabolites. We highlight several common pitfalls for clinicians, including the expectation that an individual's microbiome will be stable, that diet can induce rapid changes that are large compared with the differences among subjects, that everyone has essentially the same core stool microbiome, and that different laboratory and computational methods will yield essentially the same results. We also highlight the current limitations and future promise of these techniques, with the expectation that an understanding of these considerations will help accelerate the path toward routine clinical application of these techniques developed in research settings.
Accurate microbial identification and abundance estimation are crucial for metagenomics analysis. Various methods for classification of metagenomic data and estimation of taxonomic profiles, broadly ...referred to as metagenomic profilers, have been developed. Nevertheless, benchmarking of metagenomic profilers remains challenging because some tools are designed to report relative sequence abundance while others report relative taxonomic abundance. Here we show how misleading conclusions can be drawn by neglecting this distinction between relative abundance types when benchmarking metagenomic profilers. Moreover, we show compelling evidence that interchanging sequence abundance and taxonomic abundance will influence both per-sample summary statistics and cross-sample comparisons. We suggest that the microbiome research community pay attention to potentially misleading biological conclusions arising from this issue when benchmarking metagenomic profilers, by carefully considering the type of abundance data that were analyzed and interpreted and clearly stating the strategy used for metagenomic profiling.
The bacteria that colonize humans and our built environments have the potential to influence our health. Microbial communities associated with seven families and their homes over 6 weeks were ...assessed, including three families that moved their home. Microbial communities differed substantially among homes, and the home microbiome was largely sourced from humans. The microbiota in each home were identifiable by family. Network analysis identified humans as the primary bacterial vector, and a Bayesian method significantly matched individuals to their dwellings. Draft genomes of potential human pathogens observed on a kitchen counter could be matched to the hands of occupants. After a house move, the microbial community in the new house rapidly converged on the microbial community of the occupants’ former house, suggesting rapid colonization by the family’s microbiota.
The translational power of human microbiome studies is limited by high interindividual variation. We describe a dimensionality reduction tool, compositional tensor factorization (CTF), that ...incorporates information from the same host across multiple samples to reveal patterns driving differences in microbial composition across phenotypes. CTF identifies robust patterns in sparse compositional datasets, allowing for the detection of microbial changes associated with specific phenotypes that are reproducible across datasets.
Inflammatory bowel diseases (IBDs), including Crohn’s disease (CD), are genetically linked to host pathways that implicate an underlying role for aberrant immune responses to intestinal microbiota. ...However, patterns of gut microbiome dysbiosis in IBD patients are inconsistent among published studies. Using samples from multiple gastrointestinal locations collected prior to treatment in new-onset cases, we studied the microbiome in the largest pediatric CD cohort to date. An axis defined by an increased abundance in bacteria which include Enterobacteriaceae, Pasteurellacaea, Veillonellaceae, and Fusobacteriaceae, and decreased abundance in Erysipelotrichales, Bacteroidales, and Clostridiales, correlates strongly with disease status. Microbiome comparison between CD patients with and without antibiotic exposure indicates that antibiotic use amplifies the microbial dysbiosis associated with CD. Comparing the microbial signatures between the ileum, the rectum, and fecal samples indicates that at this early stage of disease, assessing the rectal mucosal-associated microbiome offers unique potential for convenient and early diagnosis of CD.
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•Microbiomes from multiple GI locations in new-onset Crohn’s disease (CD) cases analyzed•Co-occurring and coexcluded CD-associated organisms identified•Rectal mucosa-associated, but not fecal, microbiome is a robust disease predictor•Antibiotics amplify the dysbiosis associated with CD
Gevers et al. examined the microbiome in the largest pediatric cohort for Crohn’s disease (CD), with samples collected prior to treatment. Irrespective of the location sampled along the gastrointestinal tract, biopsies were found to be a robust disease predictor. Further, antibiotic use amplified the dysbiotic state associated with CD.
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