Metascape Gene List Analysis Report

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Bar Graph Summary

Gene Lists

User-provided gene identifiers are first converted into their corresponding H. sapiens Entrez gene IDs using the latest version of the database (last updated on 2023-09-01). If multiple identifiers correspond to the same Entrez gene ID, they will be considered as a single Entrez gene ID in downstream analyses. The gene lists are summarized in Table 1.

Gene Annotation

The following are the list of annotations retrieved from the latest version of the database (last updated on 2023-09-01) (Table 2).

Pathway and Process Enrichment Analysis

For each given gene list, pathway and process enrichment analysis have been carried out with the following ontology sources: KEGG Pathway, GO Biological Processes, Reactome Gene Sets, Canonical Pathways, CORUM, WikiPathways, and PANTHER Pathway. All genes in the genome have been used as the enrichment background. Terms with a p-value < 0.01, a minimum count of 3, and an enrichment factor > 1.5 (the enrichment factor is the ratio between the observed counts and the counts expected by chance) are collected and grouped into clusters based on their membership similarities. More specifically, p-values are calculated based on the cumulative hypergeometric distribution², and q-values are calculated using the Benjamini-Hochberg procedure to account for multiple testings³. Kappa scores⁴ are used as the similarity metric when performing hierarchical clustering on the enriched terms, and sub-trees with a similarity of > 0.3 are considered a cluster. The most statistically significant term within a cluster is chosen to represent the cluster.

To further capture the relationships between the terms, a subset of enriched terms has been selected and rendered as a network plot, where terms with a similarity > 0.3 are connected by edges. We select the terms with the best p-values from each of the 20 clusters, with the constraint that there are no more than 15 terms per cluster and no more than 250 terms in total. The network is visualized using Cytoscape⁵, where each node represents an enriched term and is colored first by its cluster ID (Figure 2.a) and then by its p-value (Figure 2.b). These networks can be interactively viewed in Cytoscape through the .cys files (contained in the Zip package, which also contains a publication-quality version as a PDF) or within a browser by clicking on the web icon. For clarity, term labels are only shown for one term per cluster, so it is recommended to use Cytoscape or a browser to visualize the network in order to inspect all node labels. We can also export the network into a PDF file within Cytoscape, and then edit the labels using Adobe Illustrator for publication purposes. To switch off all labels, delete the "Label" mapping under the "Style" tab within Cytoscape, and then export the network view.

Protein-protein Interaction Enrichment Analysis

For each given gene list, protein-protein interaction enrichment analysis has been carried out with the following databases: STRING⁶, BioGrid⁷, OmniPath⁸, InWeb_IM⁹.Only physical interactions in STRING (physical score > 0.132) and BioGrid are used (details). The resultant network contains the subset of proteins that form physical interactions with at least one other member in the list. If the network contains between 3 and 500 proteins, the Molecular Complex Detection (MCODE) algorithm¹⁰ has been applied to identify densely connected network components. The MCODE networks identified for individual gene lists have been gathered and are shown in Figure 3.

Pathway and process enrichment analysis has been applied to each MCODE component independently, and the three best-scoring terms by p-value have been retained as the functional description of the corresponding components, shown in the tables underneath corresponding network plots within Figure 3.

Quality Control and Association Analysis

Gene list enrichments are identified in the following ontology categories: COVID, Cell_Type_Signatures, DisGeNET, PaGenBase, TRRUST, Transcription_Factor_Targets. All genes in the genome have been used as the enrichment background. Terms with a p-value < 0.01, a minimum count of 3, and an enrichment factor > 1.5 (the enrichment factor is the ratio between the observed counts and the counts expected by chance) are collected and grouped into clusters based on their membership similarities. The top few enriched clusters (one term per cluster) are shown in the Figure 4-9. The algorithm used here is the same as that is used for pathway and process enrichment analysis.

Reference

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6. Szklarczyk D. et al. STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic Acids Res. (2019) 47:D607-613.
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10. Bader, G.D. et al. An automated method for finding molecular complexes in large protein interaction networks. BMC bioinformatics (2003) 4:2.
11. https://metascape.org/COVID.
12. Subramanian A, et al. Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A 102, 15545-15550 (2005).
13. Pinero J, et al. DisGeNET: a comprehensive platform integrating information on human disease-associated genes and variants. Nucleic acids research 45, D833-D839 (2017).
14. Pan JB, et al. PaGenBase: a pattern gene database for the global and dynamic understanding of gene function. PLoS One 8, e80747 (2013).