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[*Back to home*](Home)
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*Previous: [Secondary analysis](analysis/secondary_analysis)*
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## Tertiary analysis
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The tertiary analysis is about making sence of the lists of gene differentially expressed between conditions found. It is by far the most difficult and time consuming part of an analysis. The pipeline gives you insights on your data by using some of the numerous tools that exist.
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The R package **ClusterProfiler** enables to perform over-representation (ORA) and enrichment (GSEA) analysis. The R script located at "SCRIPTS/DE/annot.R" uses this packages to annotate the list of genes found in the [secondary analysis](analysis/secondary_analysis). It writes multiple files:
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- "annotGo.txt", "annotKegg.txt" : over-representation analysis of Gene Ontology and Kegg Pathways respectively. This is text table listing all ontologies and pathways with their adjusted p-values.
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- "dotplotGO.png", "dotplotKEGG.png" : graphical representation of the ORA results above.
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- "gseGo.txt", "gseKegg.txt" : Gene Set Enrichment Analysis results for Gene Ontology and Kegg Pathways. You can find all about GSEA analysis on the [Broad Institut GSEA page](https://www.gsea-msigdb.org/gsea/index.jsp).
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- "stringFunctionalEnrichment.tsv" : ORA analysis from the [STRING-DB](https://string-db.org/) database.
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![Tertiary Analysis](images/terAnalysis.png)
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The ORA need at least 10 genes to be performed. If there are enough genes found, the images will appear in the final report giving you hints on the most significative ontologies and pathways.
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The GSEA needs genes that can be ranked according to their adjusted p-values. If most genes in the analysis are near 1 in term of p-value, the GSEA will not work properly.
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String-DB will display the interactions between proteins coded by the differentially expressed genes found. If there are more than 50 genes, the graph is hard to explore and will therefore not appear in the report. You can always display the graph on the [String-DB](https://string-db.org/) page.
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<div align="right">
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<i>Next: <a href="analysis/report">The report</a></i>
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</div> |
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