Explore the vast range of titles available.

Microbial network construction is a popular explorative data analysis technique in microbiome research. Although a large number of microbial network construction tools has been developed to date, there are several issues concerning the construction and interpretation of microbial networks that have received less attention. The purpose of this perspective is to draw attention to these underexplored challenges of microbial network construction and analysis.

Microbial networks are an increasingly popular tool to investigate microbial community structure, as they integrate multiple types of information and may represent systems-level behaviour. Interpreting these networks is not straightforward, and the biological implications of network properties are unclear. Analysis of microbial networks allows researchers to predict hub species and species interactions. Additionally, such analyses can help identify alternative community states and niches. Here, we review factors that can result in spurious predictions and address emergent properties that may be meaningful in the context of the microbiome. We also give an overview of studies that analyse microbial networks to identify new hypotheses. Moreover, we show in a simulation how network properties are affected by tool choice and environmental factors. For example, hub species are not consistent across tools, and environmental heterogeneity induces modularity. We highlight the need for robust microbial network inference and suggest strategies to infer networks more reliably.

Key Points Microorganisms form various ecological relationships, ranging from mutualism to competition, that in addition to other factors (such as niche preferences and random processes) shape microbial abundances. Recently, network inference techniques have frequently been applied to microbial presence–absence or abundance data to detect significant patterns of co-presence and mutual exclusion between taxa and to represent them as a network. In addition to predicting links between taxa and between environmental traits and taxa, the analysis of microbial association networks reveals niches, points out keystone species and indicates alternative community configurations. However, several pitfalls in the construction and interpretation of these networks exist, ranging from data normalization to multiple test correction. Thorough evaluation is needed to determine the best-performing network inference technique. Recent advances in the cultivation of unknown microorganisms, combinatorial labelling and parallel cultivation may soon allow systematic co-culturing and perturbation (that is, species removal) experiments. Interaction strengths that have been obtained from static networks or that have been measured experimentally can serve as inputs for dynamic models of microbial communities, which in turn can simulate the behaviour of the system in various conditions. In the long run, dynamic models could help to engineer microbial communities. The theory of dynamic systems can contribute to our understanding of microbial communities. For instance, alternative community states can arise as a consequence of system dynamics without being driven by environmental differences. Correlation and co-occurrence patterns found in metagenomic and phylogenetic data sets are increasingly being used to predict species interactions in the environment. Here, Faust and Raes describe the range of approaches for predicting microbial network models, the pitfalls that are associated with each approach and the future for developing ecosystem-wide models. Metagenomics and 16S pyrosequencing have enabled the study of ecosystem structure and dynamics to great depth and accuracy. Co-occurrence and correlation patterns found in these data sets are increasingly used for the prediction of species interactions in environments ranging from the oceans to the human microbiome. In addition, parallelized co-culture assays and combinatorial labelling experiments allow high-throughput discovery of cooperative and competitive relationships between species. In this Review, we describe how these techniques are opening the way towards global ecosystem network prediction and the development of ecosystem-wide dynamic models.

Here we present the Cytoscape app version of our association network inference tool CoNet. Though CoNet was developed with microbial community data from sequencing experiments in mind, it is designed to be generic and can detect associations in any data set where biological entities (such as genes, metabolites or species) have been observed repeatedly. The CoNet app supports Cytoscape 2.x and 3.x and offers a variety of network inference approaches, which can also be combined. Here we briefly describe its main features and illustrate its use on microbial count data obtained by 16S rDNA sequencing of arctic soil samples. The CoNet app is available at: http://apps.cytoscape.org/apps/conet .

While clinical gut microbiota research is ever-expanding, extending reference knowledge of healthy between- and within-subject gut microbiota variation and its drivers remains essential; in particular, temporal variability is under-explored, and a comparison with cross-sectional variation is missing. Here, we perform daily quantitative microbiome profiling on 713 fecal samples from 20 Belgian women over six weeks, combined with extensive anthropometric measurements, blood panels, dietary data, and stool characteristics. We show substantial temporal variation for most major gut genera; we find that for 78% of microbial genera, day-to-day absolute abundance variation is substantially larger within than between individuals, with up to 100-fold shifts over the study period. Diversity, and especially evenness indicators also fluctuate substantially. Relative abundance profiles show similar but less pronounced temporal variation. Stool moisture, and to a lesser extent diet, are the only significant host covariates of temporal microbiota variation, while menstrual cycle parameters did not show significant effects. We find that the dysbiotic Bact2 enterotype shows increased between- and within-subject compositional variability. Our results suggest that to increase diagnostic as well as target discovery power, studies could adopt a repeated measurement design and/or focus analysis on community-wide microbiome descriptors and indices. Here, the authors report quantitative daily gut microbiome variation of individual gut bacterial abundances in healthy individuals, linked to changes in transit time and diet, highlighting the potential need for multiple samplings for microbiome target identification and the development and application of reliable microbiome diagnostics.

The study of host-associated microbial community composition has suggested the presence of alternative community types. We discuss three mechanisms that could explain these observations. The most commonly invoked mechanism links community types to a response to environmental change; alternatively, community types were shown to emerge from interactions between members of local communities sampled from a metacommunity. Here, we emphasize multi-stability as a third mechanism, giving rise to different community types in the same environmental conditions. We illustrate with a toy model how multi-stability can generate community types and discuss the consequences of multi-stability for data interpretation.

Mako is a software tool that converts microbiome data and networks into a graph database and visualizes query results, thus allowing users without programming knowledge to carry out network-based queries. Mako is accompanied by a database compiled from 60 microbiome studies that is easily extended with the user’s own data. We illustrate mako’s strengths by enumerating association partners linked to propionate production and comparing frequencies of different network motifs across habitat types. This work describes mako, a tool for efficiently constructing and querying network databases with large-scale microbiome data.

Background Given a genome-scale metabolic model (GEM) of a microorganism and criteria for optimization, flux balance analysis (FBA) predicts the optimal growth rate and its corresponding flux distribution for a specific medium. FBA has been extended to microbial consortia and thus can be used to predict interactions by comparing in-silico growth rates for co- and monocultures. Although FBA-based methods for microbial interaction prediction are becoming popular, a systematic evaluation of their accuracy has not yet been performed. Results Here, we evaluate the accuracy of FBA-based predictions of human and mouse gut bacterial interactions using growth data from the literature. For this, we collected 26 GEMs from the semi-curated AGORA database as well as four previously published curated GEMs. We tested the accuracy of three tools (COMETS, Microbiome Modeling Toolbox and MICOM) by comparing growth rates predicted in mono- and co-culture to growth rates extracted from the literature and also investigated the impact of different tool settings and media. We found that except for curated GEMs, predicted growth rates and their ratios (i.e. interaction strengths) do not correlate with growth rates and interaction strengths obtained from in vitro data. Conclusions Prediction of growth rates with FBA using semi-curated GEMs is currently not sufficiently accurate to predict interaction strengths reliably.

PlasticEnz is a new open-source tool for detecting plastic-degrading enzymes (plastizymes) in metagenomic data by combining sequence homology-based search with machine learning techniques. It integrates custom Hidden Markov Models, DIAMOND alignments, and polymer-specific classifiers trained on ProtBERT embeddings to identify candidate depolymerases from user-provided contigs, genomes, or protein sequences. PlasticEnz supports 11 plastic polymers with ML classifiers for PET and PHB, achieving F1 > 0.7 on an independent test set. Applied to plastic-exposed microcosms and field metagenomes, the tool recovered known PETases and PHBases, distinguished plastic-contaminated from pristine environments, and clustered predictions with validated reference enzymes. PlasticEnz is fast, scalable, and user-friendly, providing a robust framework for exploring microbial plastic degradation potential in complex communities.

Despite recent advances in sequencing and culturing, a deep knowledge of the wiring and functioning of the human gut ecosystem and its microbiota as a community is still missing. A holistic mechanistic understanding will require study of the gut microbiota as an interactive and spatially organized biological system, which is difficult to do in complex natural communities. Synthetic gut microbial ecosystems can function as model systems to further current understanding of the composition, stability and functional activities of the microbiota. In this Review, we provide an overview of the current synthetic ecology strategies that can be used towards a more comprehensive understanding of the human gut ecosystem. Such approaches that integrate in vitro experiments using cultured isolates with mathematical modelling will enable the ultimate goal: translating mechanistic and ecological knowledge into novel and effective therapies.