Background The interpretation of results from transcriptome profiling experiments via RNA sequencing (RNA-seq) can be a complex task, where the essential information is distributed among different tabular and list formats—normalized expression values, results from differential expression analysis, and results from functional enrichment analyses. A number of tools and databases are widely used for the purpose of identification of relevant functional patterns, yet often their contextualization within the data and results at hand is not straightforward, especially if these analytic components are not combined together efficiently. Results We developed the software package, which serves as a comprehensive toolkit for streamlining the interpretation of functional enrichment analyses, by fully leveraging the information of expression values in a differential expression context. is implemented in R and Shiny, leveraging packages that enable HTML-based interactive visualizations for executing drilldown tasks seamlessly, viewing the data at a level of increased detail. is integrated with the core classes of existing Bioconductor workflows, and can accept the output of many widely used tools for pathway analysis, making this approach applicable to a wide range of use cases. Users can effectively navigate interlinked components (otherwise available as flat text or spreadsheet tables), bookmark features of interest during the exploration sessions, and obtain at the end a tailored HTML report, thus combining the benefits of both interactivity and reproducibility. Conclusion is distributed as an R package in the Bioconductor project (https://bioconductor.org/packages/GeneTonic/) under the MIT license. Offering both bird’s-eye views of the components of transcriptome data analysis and the detailed inspection of single genes, individual signatures, and their relationships, aims at simplifying the process of interpretation of complex and compelling RNA-seq datasets for many researchers with different expertise profiles.
The generation and interpretation of results from transcriptome profiling experiments via RNA sequencing (RNA‐seq) can be a complex task. While raw data quality control, alignment, and quantification can be streamlined via efficient algorithms that can deliver the preprocessed expression matrix, a common bottleneck in the analysis of such large datasets is the subsequent in‐depth, iterative processes of data exploration, statistical testing, visualization, and interpretation. Specific tools for these workflow steps are available but require a level of technical expertise which might be prohibitive for life and clinical scientists, who are left with essential pieces of information distributed among different tabular and list formats. Our protocols are centered on the joint use of our Bioconductor packages (pcaExplorer, ideal, GeneTonic) for interactive and reproducible workflows. All our packages provide an interactive and accessible experience via Shiny web applications, while still documenting the steps performed with RMarkdown as a framework to guarantee the reproducibility of the analyses, reducing the overall time to generate insights from the data at hand. These protocols guide readers through the essential steps of Exploratory Data Analysis, statistical testing, and functional enrichment analyses, followed by integration and contextualization of results. In our packages, the core elements are linked together in interactive widgets that make drill‐down tasks efficient by viewing the data at a level of increased detail. Thanks to their interoperability with essential classes and gold‐standard pipelines implemented in the open‐source Bioconductor project and community, these protocols will permit complex tasks in RNA‐seq data analysis, combining interactivity and reproducibility for following modern best scientific practices and helping to streamline the discovery process for transcriptome data. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Exploratory Data Analysis with pcaExplorer Basic Protocol 2: Differential Expression Analysis with ideal Basic Protocol 3: Interpretation of RNA‐seq results with GeneTonic Support Protocol: Downloading and installing pcaExplorer, ideal, and GeneTonic Alternate Protocol: Using functions from pcaExplorer, ideal, and GeneTonic in custom analyses
Although knowledge on inflammatory signaling pathways driving cancer initiation and progression has been increasing, molecular mechanisms in hepatocarcinogenesis are still far from being completely understood. Hepatocyte-specific deletion of the MAPKKK Tak1 in mice recapitulates important steps of hepatocellular carcinoma (HCC) development, including the occurrence of cell death, steatohepatitis, dysplastic nodules, and HCCs. However, overactivation of Tak1 in mice upon deletion of its deubiquitinase Cyld also results in steatohepatitis and HCC development. To investigate Tak1 and Cyld in human HCCs, we created a tissue microarray to analyze their expression by immunohistochemistry in a large and well-characterized cohort of 871 HCCs of 561 patients. In the human liver and HCC, Tak1 is predominantly present as its isoform Tak1A and predominantly localizes to cell nuclei. Tak1 is upregulated in diethylnitrosamine-induced mouse HCCs as well as in human HCCs independent of etiology and is further induced in distant metastases. A high nuclear Tak1 expression is associated with short survival and vascular invasion. When we overexpressed Tak1A in Huh7 cells, we observed increased tumor cell migration, whereas overexpression of full-length Tak1 had no significant effect. A combined score of low Cyld and high Tak1 expression was an independent prognostic marker in a multivariate Cox regression model.
Background Despite a recent increase in the number of RNA-seq datasets investigating heart failure (HF), accessibility and usability remain critical issues for medical researchers. We address the need for an intuitive and interactive web application to explore the transcriptional signatures of heart failure with this work. Methods We reanalysed the Myocardial Applied Genomics Network RNA-seq dataset, one of the largest publicly available datasets of left ventricular RNA-seq samples from patients with dilated (DCM) or hypertrophic (HCM) cardiomyopathy, as well as unmatched non-failing hearts (NFD) from organ donors and patient characteristics that allowed us to model confounding factors. We analyse differential gene expression, associated pathway signatures and reconstruct signaling networks based on inferred transcription factor activities through integer linear programming. We additionally focus, for the first time, on differential RNA transcript isoform usage (DTU) changes and predict RNA-binding protein (RBP) to target transcript interactions using a Global test approach. We report results for all pairwise comparisons (DCM, HCM, NFD). Results Focusing on the DCM versus HCM contrast (DCMvsHCM), we identified 201 differentially expressed genes, some of which can be clearly associated with changes in ERK1 and ERK2 signaling. Interestingly, the signs of the predicted activity for these two kinases have been inferred to be opposite to each other: In the DCMvsHCM contrast, we predict ERK1 to be consistently less activated in DCM while ERK2 was more activated in DCM. In the DCMvsHCM contrast, we identified 149 differently used transcripts. One of the top candidates is the O-linked N-acetylglucosamine (GlcNAc) transferase (OGT), which catalyzes a common post-translational modification known for its role in heart arrhythmias and heart hypertrophy. Moreover, we reconstruct RBP – target interaction networks and showcase the examples of CPEB1, which is differentially expressed in the DCMvsHCM contrast. Conclusion Magnetique (https://shiny.dieterichlab.org/app/magnetique) is the first online application to provide an interactive view of the HF transcriptome at the RNA isoform level and to include transcription factor signaling and RBP:RNA interaction networks. The source code for both the analyses (https://github.com/dieterich-lab/magnetiqueCode2022) and the web application (https://github.com/AnnekathrinSilvia/magnetique) is available to the public. We hope that our application will help users to uncover the molecular basis of heart failure.
Background: The interpretation of results from transcriptome profiling experiments via RNA sequencing (RNA-seq) can be a complex task, where the essential information is distributed among different tabular and list formats - normalized expression values, results from differential expression analysis, and results from functional enrichment analyses. A number of tools and databases are widely used for the purpose of identification of relevant functional patterns, yet often their contextualization within the data and results at hand is not straightforward, especially if these analytic components are not combined together efficiently. Results: We developed the GeneTonic software package, which serves as a comprehensive toolkit for streamlining the interpretation of functional enrichment analyses, by fully leveraging the information of expression values in a differential expression context. GeneTonic is implemented in R and Shiny, leveraging packages that enable HTML-based interactive visualizations for executing drilldown tasks seamlessly, viewing the data at a level of increased detail. GeneTonic is integrated with the core classes of existing Bioconductor workflows, and can accept the output of many widely used tools for pathway analysis, making this approach applicable to a wide range of use cases. Users can effectively navigate interlinked components (otherwise available as flat text or spreadsheet tables), bookmark features of interest during the exploration sessions, and obtain at the end a tailored HTML report, thus combining the benefits of both interactivity and reproducibility. Conclusion: GeneTonic is distributed as an R package in the Bioconductor project (https://bioconductor.org/packages/GeneTonic/) under the MIT license. Offering both bird's-eye views of the components of transcriptome data analysis and the detailed inspection of single genes, individual signatures, and their relationships, GeneTonic aims at simplifying the process of interpretation of complex and compelling RNA-seq datasets for many researchers with different expertise profiles.
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