~/classdata/Session5/RNAseq-AnalysisDifferential Expression Analysis
An R script template, and a folder of pre-computed FeatureCounts outputs are at:
A set of nine human neuronal differential RNAseq samples have been sequenced, consisting of 6x control samples (3x two different individuals) and 3x a deletion mutant of the 1q21.1 cytogenetic region of the human genome, and 3x of a duplication of this region.
Deletions and alterations to this region has a range of impacts on neuropsychiatric disorders and is under active study. See the omim and the Wikipedia page is remarkably informative! 1q21.1_deletion_syndrome.
Exercises: Differential Gene Analysis
We will be using the script Sartools-template-deseq2.r provided on the server and github pages.
We will use the server Rstudio to do the analysis.
Copy either the FeatureCounts/markdup or rmdup, and targets.txt data to your local directory
Inside of Rstudio, edit the sartools script in the fields highlighted with ” * * * ” for your data
Explore the html report generated and the differentiation of the sample types
Look at the most differentially expressed genes (DEGs) in the outputted tables (excel)
Visualising Expression Patterns
Once an analysis has yielded a list of DEGs, it’s common to visualise them in a heatmap. There is a wonderful R package called ComplexHeatmap, which will do almost anything you can think of to a heatmap.
Exercises: Differential Gene Analysis
We will be using the script Heatmaps.r provided on the server and github pages. We will use the server Rstudio to do the analysis.
Explore the heatmap code provided in the script, and then try customising it.
Extension: Useful Packages for Downstream Analysis
Use the list of ~200 most differentially expressed genes in gProfiler, or other online resources below, to investigate the functional connotations (More on this in the extension session).