--- title: "Pathway Analysis" canonical: "https://help.biobam.com/space/OED0324/3525084170/Pathway%20Analysis" format: markdown --- # Introduction The pathway analysis feature in OmicsBox allows identifying pathways from multiple pathway databases for any set of sequences. In combination with differential expression data, the tool allows calculating pathway enrichment. In order to generate the pathway example dataset, there was the need to run a full RNA-seq analysis as well as retrieve the *Mus musculus* annotated genome. The RNA-seq analysis steps performed were [FastQC](https://biobam.atlassian.net/wiki/spaces/OBD/pages/598114595), [Preprocessing](https://biobam.atlassian.net/wiki/spaces/OBD/pages/597917932), [RNA-Seq Alignment](https://biobam.atlassian.net/wiki/spaces/OBD/pages/618856449), [Gene Level Quantification](https://biobam.atlassian.net/wiki/spaces/OBD/pages/598114455) and [Pairwise Differential Expression Analysis](https://biobam.atlassian.net/wiki/spaces/OBD/pages/598081602) with OmicsBox using the [Transcriptomics Module](https://www.biobam.com/transcriptomics/) and the *Mus musculus* annotated genome has been retrieved using the [BioMart](http://manual.omicsbox.biobam.com/user-manual/omicsbox-modules/module-functional-analysis/load-data/#LoadData-LoadAnnotationsfromBioMart) feature available in OmicsBox. ### Dataset description Transcriptome analysis of *Mus musculus *luminal and basal cell subpopulations in the lactating versus pregnant mammary gland. - Organism: *[Mus musculus](https://www.ncbi.nlm.nih.gov/genome/52)* - Type of experiment: RNA-Seq - Experimental design: 12 samples - 2 replicates for each stage (Virgin, Pregnant, Lactating) - 6 for each cell type (Luminal and Basal) - Instrument: Illumina HiSeq 2000 - Layout: Single-end ### Publication [Fu NY, Rios AC, Pal B, Soetanto R, Lun AT, Liu K, Beck T, Best SA, Vaillant F, Bouillet P, Strasser A, Preiss T, Smyth GK, Lindeman GJ, Visvader JE. EGF-mediated induction of Mcl-1 at the switch to lactation is essential for alveolar cell survival. Nat Cell Biol. 2015 Apr;17(4):365-75. doi: 10.1038/ncb3117. Epub 2015 Mar 2. PMID: 25730472.](https://pubmed.ncbi.nlm.nih.gov/25730472/)
Abstract Expansion and remodelling of the mammary epithelium requires a tight balance between cellular proliferation, differentiation and death. To explore cell survival versus cell death decisions in this organ, we deleted the pro-survival gene Mcl-1 in the mammary epithelium. Mcl-1 was found to be essential at multiple developmental stages including morphogenesis in puberty and alveologenesis in pregnancy. Moreover, Mcl-1-deficient basal cells were virtually devoid of repopulating activity, suggesting that this gene is required for stem cell function. Profound upregulation of the Mcl-1 protein was evident in alveolar cells at the switch to lactation, and Mcl-1 deficiency impaired lactation. Interestingly, EGF was identified as one of the most highly upregulated genes on lactogenesis and inhibition of EGF or mTOR signalling markedly impaired lactation, with concomitant decreases in Mcl-1 and phosphorylated ribosomal protein S6. These data demonstrate that Mcl-1 is essential for mammopoiesis and identify EGF as a critical trigger of Mcl-1 translation to ensure survival of milk-producing alveolar cells.
### Original Data Link to the public repositories where the original data can be found: - BioProject: [PRJNA258286](https://www.ncbi.nlm.nih.gov/bioproject/PRJNA258286) - SRA: [SRP045534](https://www.ncbi.nlm.nih.gov/sra?term=SRP045534) - [GEO Datasets](https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE60450) - Reference: [NCBI](https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/001/635/GCF_000001635.27_GRCm39/GCF_000001635.27_GRCm39_genomic.fna.gz) # Bioinformatic Analysis ## Combined Pathway Analysis ### Application [Combined Pathway Analysis](https://biobam.atlassian.net/wiki/spaces/OBD/pages/2394914849) ### Input *Mus musculus* [Genome Annotation project](https://drive.google.com/file/d/1prvusjr-E_CDwJwJMhMXatxLes8oXLSF/view?usp=sharing) Pairwise differential expression [project](https://drive.google.com/file/d/144kbgx0LNaDShKt8MCXJ2VT19emMt0qD/view?usp=sharing) ### Parameters ### Execution Time 55 mins ### Output A [table](https://drive.google.com/file/d/1f5sCy52J6qejv9J2w4cvJdJsyrwj6bxu/view?usp=sharing) containing all the linked pathways to the sequences as well as the enriched pathways with differential expression genes.