A guide for comparing microbial co‐occurrence networks

The article provides a pipeline for comparing microbial co-occurrence networks based on the R microeco package and meconetcomp package. It has high flexibility and expansibility and can help users efficiently compare networks built from different groups of samples or different construction approaches. Microorganisms are ubiquitous in diverse environments of the earth and play important roles in ecosystem functions ranging from biogeochemical cycles 1 to the maintenance of host health 2, 3. Microbial assemblages are generally comprised of a large number of species, which is represented as a “microbial community” defined in the context of different spatiotemporal scales. Identifying microbial members and their abundance in a community is a basic task in microbial ecology studies. Over the past two decades, a giant leap forward in sequencing techniques has made this task possible, leading to a rapid increase in data size. Furthermore, the advances of bioinformatic softwares (e. g. , QIIME2 4) have profoundly improved the speed and convenience of sequence analysis. After obtaining operational taxonomic units (OTU), amplicon sequence variants (ASV) or species and their abundances (sequence counts or estimated abundance) in the bioinformatic analysis process, the following statistics and visualizations can be performed using R language 5 and related packages, which have grown up to be a cutting-edge system in the recent decades 6. Many statistical approaches in ecology can be used in microbiome data analysis benefiting from the similarities between macro- and micro-ecosystems. However, there are also some dissimilarities in the research methods and routes between these two ecosystems 2. The invisibility of microbes, a high proportion of uncultured species, and huge diversity in ecosystems lead to the difficulty of hypothesis-driven studies, especially those referring to microbial interactions and functions. Researchers usually need to try a series of statistical tools and approaches and find the suitable one to verify a hypothesis. To maximize the accessibility of the R language and provide a good user experience, code must be organized in a manner that adheres to both conciseness and functionality. Based on this background, the R microeco package 7 was developed using the R6 class to make the customized pipeline easier and faster. In addition, the file2meco package 8 was also developed to facilitate the conversion of data files from other software, such as QIIME2 4 and HUMAnN 9. The co-occurrence network analysis is often used to decipher the hidden patterns of complex microbial consortia in a wide range of spatiotemporal studies (see 10 and the references therein). In contrast to networks built for macro-organisms (mainly based on the observations of species interactions 11, 12), microbial co-occurrence networks are mostly constructed from count tables obtained from amplicon or metagenomic sequencing data. The sequencing data have several general issues rendering network construction a challenge, including compositionality (sequence counts denote proportions instead of absolute abundances), sparsity (a large number of zeros), and difficulty for the inference of direct associations between paired taxa. Another challenge is how to explain the edges and their signs of the co-occurrence network given that the edges are not recommended to be interpreted as microbial interactions for the cross-sectional data 13. To the best of our knowledge, the correlation-based (Pearson or Spearman correlation) network may be the earliest and widely used approach in diverse studied habitats, such as soil 14. To address the issues existed in the correlation network, a range of approaches have been developed, including Sparse Correlations for Compositional data (SparCC) 15, Compositionally Corrected by REnormalization and PErmutation (CCREPE) 16, and Correlation inference for Compositional data through Lasso (CCLasso) 17. Further, several approaches based on graphical model are created to robustly infer the direct associations between taxa and optimize network structure. For example, SParse InversE Covariance Estimation for Ecological Association Inference (SPIEC-EASI) 18 combines data transformations developed for compositional data and algorithms for sparse neighborhood and inverse covariance selection to reconstruct network. FlashWeave approach 19 adopts a local-to-global learning framework to infer the directly associated neighbors (i. e. , Markov blanket) of a taxon and has good scalability and high speed on large heterogenous data sets. The studies on network comparisons 20 and approach reviews 10 have thoroughly discussed the robustness of different approaches and particularly recommended suitable network approaches depending upon different challenges. In addition, BEEM-static method 21 is dedicated to seek out the interactions for cross-sectional microbiome data with the generalized Lotka-Volterra (gLV) model and an expectation-maximization algorithm, offering a directed network to gain insight into microbial co-occurrence in communities. Along with the network approach development, some controversial voices worry about the misuse of network-related methods (especially correlation network) on the inference of biotic interactions 13, 22, 23. The main reason is that species interactions hold hub roles in answering many questions in community ecology. Another reason is that, no matter which network method is used, the edges can contain more or less information of species interactions depending on the approaches, parameters, and data features themselves. There is also another case that some actual species interactions may not be captured because of the approach itself or biological system characteristics (e. g. , higher-order biological interaction 24, 25). Broadly speaking, the interpretations on how network edges represent have been largely overlooked mainly due to its difficulty. Although recently developed approaches are dedicated to reveal the direct interactions among microbes 19, 21, the applicability of these methods is still not clear when heterogeneous data sets are applied. So it is not recommended to explain co-occurrence network as the interaction network 13, 26, producing a dilemma for users on how to explain the co-occurrence network and to what extent the co-occurrence network can represent the species interactions. Researchers have appealed to explain the inferred networks with biological background knowledge in previous reviews 13, 26. Even in macroecology with empirical species interaction information, a study has revealed that associations among taxa rarely matched empirical net or direct species interactions 27, highlighting the inadvisable practice of coupling species co-occurrences to species interactions. In terms of the ecological hypothesis, currently frequently used network analysis is largely different with other ecological approaches, such as null model and constrained ordination analysis. The direct or conditional associations among taxa represent the deterministic patterns in microbial consortia, as random assemblages (such as drift and dispersal in neutral process) cannot generate strong co-occurrence patterns. So the network approach can be understood as a new application of the classic “checkerboard distribution” approach. The co-occurrence patterns are derived from the joint species distribution and historical legacy. Various layers of complexity inherent to ecological systems (e. g. , the variability of multiple abiotic factors and the interactions between abiotic and biotic factors) blur the inference that whether an association is a real interaction and how abiotic factors influence the association. Moreover, different network construction approaches correspond to dependent models in the underlying algorithms, also generating a problem in the data interpretation. Although the prevalence of network approach has boosted a burst of research on various microbial fields, it is far from enough to provide an ecological hypotheses and explain the mechanisms behind the descriptive metrics. The methods implemented in software are generally a “black-box” and lack the flexibility to combine the network analysis and other types of analysis methods. To reasonably use the network approaches, researchers need a thorough understanding of the analysis flows and the details of the network construction. In practice, it is important to combine multiple network approaches to improve the robustness of the edge finding. Moreover, in research with complicated experimental design, network construction may be performed for different treatments or groups 28, highlighting the urgency of the user-friendly pipeline for network comparison. There are already several integrated online tools (e. g. , MetagenoNets 29 and iNAP 30) and R packages (e. g. , igraph 31, NetCoMi 32, and ggClusterNet 33) devoted to network analysis and visualizations. But the comparison of multiple networks is still a daunting challenge. In this protocol, to facilitate network comparison, R package meconetcomp (https: //github. com/ChiLiubio/meconetcomp) was created (Figure 1A). The protocol mainly introduces the usage of the transₙetwork class of microeco packages and as much as possible combines other classes to show the power of the microeco package for network analysis (Figure 1B). A microbial co-occurrence network is characterized as an edge-weighted graph G = (V, E), where V (node) represents a feature (ASV/OTU/species) and E (edge) encodes the inferred connection in the network. The weight of E denotes the strength of the connection. The signs (+ or −) of edges represent the inferred positive or negative associations. The data set soilₐmp and stoolₘet are both microtable objects that have been prepared. How to import the own data of users? Please read the help document and the tutorial of the microtable class of microeco package (https: //chiliubio. github. io/microecoₜutorial/basic-class. html#microtable-class). For Pearson or Spearman correlation of large datasets, adding parameter “useWGCNAₚearsonₛpearman = TRUE” can largely speed up the calculation. Note that using this parameter requires the WGCNA package to be installed (https: //cran. r-project. org/web/packages/WGCNA/). For more parameters or approaches of network construction, please see the function calₙetwork of transₙetwork class with the command: help (transₙetwork). Module hubs (nodes with highly links within their own module, Zi > 2. 5 and Pi ≤ 0. 62) ; (2) Connectors (nodes that connect different modules, Zi ≤ 2. 5 and Pi > 0. 62) ; (3) Network hubs (nodes that act as both module hubs and connectors, Zi > 2. 5 and Pi > 0. 62) ; (4) Peripherals (nodes that have only a few links and almost always to the nodes within their own modules, Zi ≤ 2. 5 and Pi < 0. 62). In this protocol, we mainly introduced the pipeline for the analysis and comparisons of multiple co-occurrence networks constructed from different groups of samples or different network approaches. Different from the network comparison approach in NetCoMi package 32, we take full advantage of the R list class, transₙetwork class of microeco package and the meconetcomp package to perform each part of network analysis. The most noteworthy advantage is the number of networks for comparison has no limitation in the practice. Furthermore, the pipeline has high flexibility and expansibility to be applied in other network analysis not covered in this protocol. To date, no standards for network approach selection are available in microbial ecology. Nor is there a “one size fits all” tool that can automatically match a set of network methods or parameters for the user's data set. Similar to the results from a benchmark study that correlation detection strategies in microbial data sets vary widely in sensitivity and precision 20, we also found the results across network methods vary at each aspect (Figure 2B and other saved figures using the stoolₘetₙetwork data set). We argued that, except for the empirical selections of network approaches 10, 20, 25, the user's comparison on the network approaches is also feasible and valuable to learn more on what network approach can reflect and explain by integrating other ecological and statistical methods (such as null model) implemented in the microeco package and other R packages. Disentangling the environmental effects on microbial association networks is another attractive topic 39. Yet the lack of gold standard data may diminish power and feasibility when studying these questions. A recent report shows that rare positive interactions are likely caused by metabolic cross-feeding among microbes in soil 40. So in heterogeneous habitats like soil, co-occurrence network results should be directed more to the ecological patterns instead of the interpretation of how species interacts. Even though the lack of standard data set with determinate species interactions and ecological patterns has largely impede methodological development and limit our ability to broadly link network results to ecological patterns, it is still feasible to compare different networks built from different groups of samples to reveal the ecological patterns and infer the assembly processes by combining network method and other ecological approaches. For instance, differential phylogenetic distance (Figure 2C) across different groups in soilₐmp data set showed that taxa associations (strong positive correlations) might be caused by different abiotic factors and varying extent of deterministic processes. Furthermore, different factors might affect the topological structure of networks in the soils from different categories of Chinese wetlands (Figure 2E). With the increasing number of cultured species and metagenome-assembled genomes, it is possible to verify the biological meaning of the shared edges (Figure 2B) among different network approaches, and figure out how and to what extent the paired species interact 41 in the studies with important biological mechanisms in explaining the results. While a full analysis of all the network related approaches is beyond the scope of this study 28, these examples in the protocol demonstrated the power of the pipeline mainly generated by the combination of the R list, “for” loop, transₙetwork class and the meconetcomp package. To make the pipeline of the protocol easy to use, we did not consider too many factors on data manipulations, such as rarefaction, sparse data filtering, and network construction parameters. These factors and operations may be critical in customized data analysis in the sense that they can affect the robustness of results expected. In addition, since the R6 object has the special attribute of binding functions to the objects, the loop approach in this protocol can also be applied in other data analysis with microeco package, such as comparisons of different groups of samples in other classes. All authors contributed to the protocol development. The initial idea was conceived by Chi Liu, Minjie Yao, and Xiangzhen Li. During the protocol development, all the authors contributed to the test and improvements. The original manuscript was written by Chi Liu, Minjie Yao, and Xiangzhen Li, and revised by Chaonan Li, Yanqiong Jiang, and Raymond J. Zeng. This work was supported by the National Natural Science Foundation of China (42077206 and 32070548). We thank four anonymous reviewers for their helpful suggestions on the original manuscript. We also thank the users of the R microeco package for reporting bugs, suggestions, and usability problems. The authors declare no conflict of interest. Besides the datasets loaded from meconetcomp package, the data sets used in the protocol are also deposited in GitHub (https: //github. com/ChiLiubio/networkₚrotocol) and Gitee (https: //gitee. com/chiliubio/networkₚrotocol). Supporting Information materials (figures, tables, scripts, graphical abstracts, slides, videos, Chinese translated versions, and update materials) may be found in the online DOI or iMeta Science http: //www. imeta. science/.