Pathway Analysis

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 to calculate 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, Preprocessing, RNA-Seq Alignment, Gene Level Quantification and Pairwise Differential Expression Analysis with OmicsBox using the Transcriptomics Module and the Mus musculus annotated genome has been retrieved using the BioMart 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

  • 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.

 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:

Bioinformatic Analysis

Combined Pathway Analysis

Application

Combined Pathway Analysis

Input

Mus musculus Genome Annotation project

Pairwise differential expression project

Parameters

Input

Configuration Reactome

  • Run Reactome Pathway Analysis: true

  • Run Blast to link via Protein IDs: true

  • Link with GeneOntology Terms: true

  • Keep Most Specific Pathways: true

  • Give Priority to Taxon: true

  • Top Priority Taxon: Mus musculus

  • Blast Expectation Value: 1.0E-3

  • Include Categories: All

Configuration KEGG

  • Run KEGG Pathway Analysis: true

  • Link KEGG Orthologs via EggNog: true

  • Link via Enzyme Codes: true

  • Include Categories: All

Execution Time

55 mins

Output

A table containing all the linked pathways to the sequences as well as the enriched pathways with differential expression genes.