White Rose Mechanistic Biology - Analytics 2 (Analysis of RNA-seq data in R)



We will assume you have a basic familiarity with the R language and Rstudio and are reasonably confident in performing the following tasks:-

  • Creating new RStudio projects and markdown files
  • Importing spreadsheets into R
  • Filtering, arranging and selecting with dplyr
  • Plotting using ggplot2

You should also be familiar with the overall workflow of RNA-seq data.


This module is aimed at biology students with little or no knowledge of programming and statistics. It has the following objectives:

  • making students aware of effects of experimental design in the subsequent data analysis;
  • having a good understanding of technologies and methods for Bioinformatics;
  • Introduce basic coding in R and exercise use of workflow and pipelines on real case study.


email: m.j.dunning@sheffield.ac.uk

Installation and setup

Please follow these 5 steps at your earliest convenience. Contact Mark Dunning if you have any problem

1) Download the latest version of R and RStudio for your operating system

Download the pre-compiled binary for your OS from https://cloud.r-project.org/ and install. More specifically:

For Windows

Click “Download R for Windows”, then “base”, then “Download R 4.0.0 for Windows”. This will download an .exe file; once downloaded, open to start the installation. You can accept all the defaults.

For Mac

Click “Download R for (Mac) OS X”, then “R-4.0.0.pkg” to download the installer. Run the installer to complete installation. You can accept all the defaults.

For Linux

Click “Download R for Linux”. Instructions on installing are given for Debian, Redhat, Suse and Ubuntu distributions. Where there is a choice, install both r-base and r-base-dev.

Installing RStudio

Download and install the version for your OS from: https://rstudio.com/products/rstudio/download/#download. You can accept all the defaults.

2) Please download and un-zip this file containing the data for the course


3) Create a New RStudio project from the directory containing the un-zipped files

4) Install the packages required for the course

Run the code in the R script linked below

Package installation code

5) Check your installation.

You can check everything is installed by copying and pasting this into the R console


5) Watch these short introductory videos

A gentle introduction to RNA-seq - 18 minutes RNA-seq count normalisation explained - 10 minutes

Session 1 - Importing RNA-seq counts into R and quality assessment

Learning outcomes

  • Exploring count data and importing these data into R
  • Normalisation strategies for RNA-seq counts
  • Quality Assessment of counts
  • Identifying outliers, batch effects and sample mix-ups


Session 2 - Differential Expression for RNA-seq

Learning outcomes

  • Which statistical tests are appropriate for RNA-seq data
  • Using the DESeq2 package to detect differential expression
  • Using a venn diagram to compare gene lists


Session 3 - Visualisation methods for RNA-seq data

Learning outcomes

  • Using annotation databases to map between gene identifers
  • Construction and interpretation of common visualisations
    • scatter plots
    • volcano plots
    • MA-plots
    • heatmaps
  • Customisation of plots


Session 4 - Pathways and further downstream analysis

Learning outcomes

  • Introduction to assessment
  • Using annotation packages to query pathways
  • Methodology behind gene set testing and enrichment analysis