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Single-cell data is increasingly used for gene regulatory network (GRN) inference, and benchmarks for this have been developed based on simulated data. However, existing single-cell simulators cannot model the effects of gene perturbations. A further challenge lies in generating large-scale GRNs that often struggle with computational and stability issues. We present GeneSPIDER2, an update of the GeneSPIDER MATLAB toolbox for GRN benchmarking, inference, and analysis. Several software modules have improved capabilities and performance, and new functionalities have been added. A major improvement is the ability to generate large GRNs with biologically realistic topological properties in terms of scale-free degree distribution and modularity. Another major addition is a simulation of single-cell data, which is becoming increasingly popular as input for GRN inference. Specifically, we introduced the unique feature to generate single-cell data based on genetic perturbations. Finally, the simulated single-cell data was compared to real single-cell Perturb-seq data from two cell lines, showing that the synthetic and real data exhibit similar properties. Graphical Abstract

Prediction of orthologs is an important bioinformatics pursuit that is frequently used for inferring protein function and evolutionary analyses. The InParanoid database is a well known resource of ortholog predictions between a wide variety of organisms. Although orthologs have historically been inferred at the level of full-length protein sequences, many proteins consist of several independent protein domains that may be orthologous to domains in other proteins in a way that differs from the full-length protein case. To be able to capture all types of orthologous relations, conventional full-length protein orthologs can be complemented with orthologs inferred at the domain level. We here present InParanoiDB 9, covering 640 species and providing orthologs for both protein domains and full-length proteins. InParanoiDB 9 was built using the faster InParanoid-DIAMOND algorithm for orthology analysis, as well as Domainoid and Pfam to infer orthologous domains. InParanoiDB 9 is based on proteomes from 447 eukaryotes, 158 bacteria and 35 archaea, and includes over one billion predicted ortholog groups. A new website has been built for the database, providing multiple search options as well as visualization of groups of orthologs and orthologous domains. This release constitutes a major upgrade of the InParanoid database in terms of the number of species as well as the new capability to operate on the domain level. InParanoiDB 9 is available at https://inparanoidb.sbc.su.se/.

The need for systematic drug repurposing has seen a steady increase over the past decade and may be particularly valuable to quickly remedy unexpected pandemics. The abundance of functional interaction data has allowed mapping of substantial parts of the human interactome modeled using functional association networks, favoring network-based drug repurposing. Network crosstalk-based approaches have never been tested for drug repurposing despite their success in the related and more mature field of pathway enrichment analysis. We have, therefore, evaluated the top performing crosstalk-based approaches for drug repurposing. Additionally, the volume of new interaction data as well as more sophisticated network integration approaches compelled us to construct a new benchmark for performance assessment of network-based drug repurposing tools, which we used to compare network crosstalk-based methods with a state-of-the-art technique. We find that network crosstalk-based drug repurposing is able to rival the state-of-the-art method and in some cases outperform it., For corrigendum, see (DOI):https://doi.org/10.3389/fgene.2022.921286

Accurate inference of gene regulatory networks (GRNs) is important to unravel unknown regulatory mechanisms and processes, which can lead to the identification of treatment targets for genetic diseases. A variety of GRN inference methods have been proposed that, under suitable data conditions, perform well in benchmarks that consider the entire spectrum of false-positives and -negatives. However, it is very challenging to predict which single network sparsity gives the most accurate GRN. Lacking criteria for sparsity selection, a simplistic solution is to pick the GRN that has a certain number of links per gene, which is guessed to be reasonable. However, this does not guarantee finding the GRN that has the correct sparsity or is the most accurate one. In this study, we provide a general approach for identifying the most accurate and sparsity-wise relevant GRN within the entire space of possible GRNs. The algorithm, called SPA, applies a “GRN information criterion” (GRNIC) that is inspired by two commonly used model selection criteria, Akaike and Bayesian Information Criterion (AIC and BIC) but adapted to GRN inference. The results show that the approach can, in most cases, find the GRN whose sparsity is close to the true sparsity and close to as accurate as possible with the given GRN inference method and data. The datasets and source code can be found at https://bitbucket.org/sonnhammergrni/spa/.

The molecular mechanisms underlying lethal castration-resistant prostate cancer remain poorly understood, with intratumoral heterogeneity a likely contributing factor. To examine the temporal aspects of resistance, we analyze tumor heterogeneity in needle biopsies collected before and after treatment with androgen deprivation therapy. By doing so, we are able to couple clinical responsiveness and morphological information such as Gleason score to transcriptome-wide data. Our data-driven analysis of transcriptomes identifies several distinct intratumoral cell populations, characterized by their unique gene expression profiles. Certain cell populations present before treatment exhibit gene expression profiles that match those of resistant tumor cell clusters, present after treatment. We confirm that these clusters are resistant by the localization of active androgen receptors to the nuclei in cancer cells post-treatment. Our data also demonstrates that most stromal cells adjacent to resistant clusters do not express the androgen receptor, and we identify differentially expressed genes for these cells. Altogether, this study shows the potential to increase the power in predicting resistant tumors.

Predicting orthologs, genes in different species having shared ancestry, is an important task in bioinformatics. Orthology prediction tools are required to make accurate and fast predictions, in order to analyze large amounts of data within a feasible time frame. InParanoid is a well-known algorithm for orthology analysis, shown to perform well in benchmarks, but having the major limitation of long runtimes on large datasets. Here, we present an update to the InParanoid algorithm that can use the faster tool DIAMOND instead of BLAST for the homolog search step. We show that it reduces the runtime by 94%, while still obtaining similar performance in the Quest for Orthologs benchmark.

Accurate inference of gene regulatory networks (GRN) is an essential component of systems biology, and there is a constant development of new inference methods. The most common approach to assess accuracy for publications is to benchmark the new method against a selection of existing algorithms. This often leads to a very limited comparison, potentially biasing the results, which may stem from tuning the benchmark's properties or incorrect application of other methods. These issues can be avoided by a web server with a broad range of data properties and inference algorithms, that makes it easy to perform comprehensive benchmarking of new methods, and provides a more objective assessment. Here we present https://GRNbenchmark.org/ - a new web server for benchmarking GRN inference methods, which provides the user with a set of benchmarks with several datasets, each spanning a range of properties including multiple noise levels. As soon as the web server has performed the benchmarking, the accuracy results are made privately available to the user via interactive summary plots and underlying curves. The user can then download these results for any purpose, and decide whether or not to make them public to share with the community.

Functional analysis of gene sets derived from experiments is typically done by pathway annotation. Although many algorithms exist for analyzing the association between a gene set and a pathway, an issue which is generally ignored is that gene sets often represent multiple pathways. In such cases an association to a pathway is weakened by the presence of genes associated with other pathways. A way to counteract this is to cluster the gene set into more homogenous parts before performing pathway analysis on each module. We explored whether network-based pre-clustering of a query gene set can improve pathway analysis. The methods MCL, Infomap, and MGclus were used to cluster the gene set projected onto the FunCoup network. We characterized how well these methods are able to detect individual pathways in multi-pathway gene sets, and applied each of the clustering methods in combination with four pathway analysis methods: Gene Enrichment Analysis, BinoX, NEAT, and ANUBIX. Using benchmarks constructed from the KEGG pathway database we found that clustering can be beneficial by increasing the sensitivity of pathway analysis methods and by providing deeper insights of biological mechanisms related to the phenotype under study. However, keeping a high specificity is a challenge. For ANUBIX, clustering caused a minor loss of specificity, while for BinoX and NEAT it caused an unacceptable loss of specificity. GEA had very low sensitivity both before and after clustering. The choice of clustering method only had a minor effect on the results. We show examples of this approach and conclude that clustering can improve overall pathway annotation performance, but should only be used if the used enrichment method has a low false positive rate.

Motivation: Pathway annotation tools are indispensable for the interpretation of a wide range of experiments in life sciences. Network-based algorithms have recently been developed which are more sensitive than traditional overlap-based algorithms, but there is still a lack of good online tools for network-based pathway analysis. Results: We present PathwAX II-a pathway analysis web tool based on network crosstalk analysis using the BinoX algorithm. It offers several new features compared with the first version, including interactive graphical network visualization of the crosstalk between a query gene set and an enriched pathway, and the addition of Reactome pathways.

Motivation: Inferring an accurate gene regulatory network (GRN) has long been a key goal in the field of systems biology. To do this, it is important to find a suitable balance between the maximum number of true positive and the minimum number of false-positive interactions. Another key feature is that the inference method can handle the large size of modern experimental data, meaning the method needs to be both fast and accurate. The Least Squares Cut-Off (LSCO) method can fulfill both these criteria, however as it is based on least squares it is vulnerable to known issues of amplifying extreme values, small or large. In GRN this manifests itself with genes that are erroneously hyper-connected to a large fraction of all genes due to extremely low value fold changes. Results: We developed a GRN inference method called Least Squares Cut-Off with Normalization (LSCON) that tackles this problem. LSCON extends the LSCO algorithm by regularization to avoid hyper-connected genes and thereby reduce false positives. The regularization used is based on normalization, which removes effects of extreme values on the fit. We benchmarked LSCON and compared it to Genie3, LASSO, LSCO and Ridge regression, in terms of accuracy, speed and tendency to predict hyper-connected genes. The results show that LSCON achieves better or equal accuracy compared to LASSO, the best existing method, especially for data with extreme values. Thanks to the speed of least squares regression, LSCON does this an order of magnitude faster than LASSO.

The regulatory relationships between genes and proteins in a cell form a gene regulatory network (GRN) that controls the cellular response to changes in the environment. A number of inference methods to reverse engineer the original GRN from large-scale expression data have recently been developed. However, the absence of ground-truth GRNs when evaluating the performance makes realistic simulations of GRNs necessary. One aspect of this is that local network motif analysis of real GRNs indicates that the feed-forward loop (FFL) is significantly enriched. To simulate this properly, we developed a novel motif-based preferential attachment algorithm, FFLatt, which outperformed the popular GeneNetWeaver network generation tool in reproducing the FFL motif occurrence observed in literature-based biological GRNs. It also preserves important topological properties such as scale-free topology, sparsity, and average in/out-degree per node. We conclude that FFLatt is well-suited as a network generation module for a benchmarking framework with the aim to provide fair and robust performance evaluation of GRN inference methods.

A vast scenario of potential disease mechanisms and remedies is yet to be discovered. The field of Network Medicine has grown thanks to the massive amount of high-throughput data and the emerging evidence that disease-related proteins form ‘disease modules’. Relying on prior disease knowledge, network-based disease module detection algorithms aim at connecting the list of known disease associated genes by exploiting interaction networks. Most existing methods extend disease modules by iteratively adding connector genes in a bottom-up fashion, while top-down approaches remain largely unexplored. We have created TOPAS, an iterative approach that aims at connecting the largest number of seed nodes in a top-down fashion through connectors that guarantee the highest flow of a Random Walk with Restart in a network of functional associations. We used a corpus of 382 manually selected functional gene sets to benchmark our algorithm against SCA, DIAMOnD, MaxLink and ROBUST across four interactomes. We demonstrate that TOPAS outperforms competing methods in terms of Seed Recovery Rate, Seed to Connector Ratio and consistency during module detection. We also show that TOPAS achieves competitive performance in terms of biological relevance of detected modules and scalability.

Motivation: Accurate inference of gene regulatory interactions is of importance for understanding the mechanisms of underlying biological processes. For gene expression data gathered from targeted perturbations, gene regulatory network (GRN) inference methods that use the perturbation design are the top performing methods. However, the connection between the perturbation design and gene expression can be obfuscated due to problems, such as experimental noise or off-target effects, limiting the methods' ability to reconstruct the true GRN. Results: In this study, we propose an algorithm, IDEMAX, to infer the effective perturbation design from gene expression data in order to eliminate the potential risk of fitting a disconnected perturbation design to gene expression. We applied IDEMAX to synthetic data from two different data generation tools, GeneNetWeaver and GeneSPIDER, and assessed its effect on the experiment design matrix as well as the accuracy of the GRN inference, followed by application to a real dataset. The results show that our approach consistently improves the accuracy of GRN inference compared to using the intended perturbation design when much of the signal is hidden by noise, which is often the case for real data.

Motivation: Pathway annotation is a vital tool for interpreting and giving meaning to experimental data in life sciences. Numerous tools exist for this task, where the most recent generation of pathway enrichment analysis tools, network-based methods, utilize biological networks to gain a richer source of information as a basis of the analysis than merely the gene content. Network-based methods use the network crosstalk between the query gene set and the genes in known pathways, and compare this to a null model of random expectation. Results: We developed PathBIX, a novel web application for network-based pathway analysis, based on the recently published ANUBIX algorithm which has been shown to be more accurate than previous network-based methods. The PathBIX website performs pathway annotation for 21 species, and utilizes prefetched and preprocessed network data from FunCoup 5.0 networks and pathway data from three databases: KEGG, Reactome, and WikiPathways.

The Pfam database is a widely used resource for classifying protein sequences into families and domains. Since Pfam was last described in this journal, over 350 new families have been added in Pfam 33.1 and numerous improvements have been made to existing entries. To facilitate research on COVID-19, we have revised the Pfam entries that cover the SARS-CoV-2 proteome, and built new entries for regions that were not covered by Pfam. We have reintroduced Pfam-B which provides an automatically generated supplement to Pfam and contains 136 730 novel clusters of sequences that are not yet matched by a Pfam family. The new Pfam-B is based on a clustering by the MMseqs2 software. We have compared all of the regions in the RepeatsDB to those in Pfam and have started to use the results to build and refine Pfam repeat families. Pfam is freely available for browsing and download at http://pfam.xfam.org/.