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RNAseq analysis in R

May 11-12, 2016

9:00 am - 4:30 pm

Instructors: Belinda Phipson, Maria Doyle, Jovana Maksimovic, Harriet Dashnow, Matt Ritchie

Helpers: Jane Hawkey, Sepideh Foroutan, Ramyar Molania, Chenkai Ma, Luke Zappia, Katrina Bell, Anthony Hawkins, Enid Lam, Yih-Chih Chan, Matt Wakefield, Piyush Madhamshettiwar


General Information

In this workshop, you will be learning how to analyse RNA-seq count data, using R. This will include reading the data into R, quality control and performing differential expression analysis and gene set testing, with a focus on the limma-voom analysis workflow. You will learn how to generate common plots for analysis and visualisation of gene expression data, such as boxplots and heatmaps. You will also be learning how alignment and counting of raw RNA-seq data can be performed in R. In addition, you will be introduced to Degust, a user-friendly interactive tool for RNA-seq analysis. This workshop is aimed at biologists interested in learning how to perform differential expression analysis of RNA-seq data when reference genomes are available.

With special guest speakers Alicia Oshlack and Wei Shi.

Who: The course is aimed at graduate students and other researchers. Some basic R knowledge is assumed. This is not an introduction to R course. If you are not familiar with the R statistical programming language we strongly encourage you to work through an introductory R course before you attend this workshop. We recommend the Software Carpentry R for Reproducible Scientific Analysis lessons up to and including vectorisation (topic 9).

Where: LAB-14, 700 Swanston Street, Carlton VICTORIA 3053. Get directions with OpenStreetMap or Google Maps.

Thank you to VLSCI for hosting this workshop.

VLSCI EMBL Australian Bioinformatics Resource

Requirements: Participants must bring a laptop with a Mac, Linux, or Windows operating system (not a tablet, Chromebook, etc.) that they have administrative privileges on. They should have a few specific software packages installed (listed below). If you choose to bring your own RNAseq data, it must be count data (because we don't have the time or computational resources to map large data sets during the workshop). If you're not sure, here is an example of count data format.

Accessibility: We are committed to making this workshop accessible to everybody. The workshop organisers have checked that:

  • The room is wheelchair / scooter accessible.
  • Accessible restrooms are available.

Contact: Please mail for more information.


Note: this is a preliminary schedule. There may be changes to the timing and content.

If you have any trouble installing the software or packages, please arrive at 8:45am on the first day so we can help before the workshop starts.

In the first session of Day 1 we will cover some R skills that are particularly useful for RNAseq analysis, for example data frame manipulation, factors and subsetting using logical statements. If you are a more advanced R user, you may not need to attend this session.

Day 1

09:00 R for RNAseq
11:00 Morning tea
11:30 Introduction to RNA-seq theory
12:00 Differential expression
13:00 Lunch
13:45 Differential expression continued
15:00 Afternoon tea
15:30 Differential expression continued
16:30 Wrap-up

Day 2

09:30 Alignment and feature counting
11:00 Morning tea
11:30 Gene set testing
13:00 Lunch
13:45 Applying RNAseq (or BYO data)
15:00 Afternoon tea
15:30 Applying RNAseq (or BYO data) continued
16:30 Wrap-up

We will use this Etherpad for chatting, taking notes, and sharing URLs and bits of code.


You can find all of the lesson notes for this workshop here.


To participate in this workshop, you will need access to the software described below. In addition, you will need an up-to-date web browser.

Even if you already have R installed, it is important that you have the latest version because some of the packages we will be using will not work with earlier versions of R. The latest version (available through the links below) is 3.2.5 for Windows and 3.2.4 for Mac 10.9 and above. For Mac 10.6-10.8 the latest version is 3.2.1 (available from here) and for that version you will also need to download this gfortran package gfortran package).

Software Carpentry maintains a list of common issues that occur during installation as a reference for instructors that may be useful on the Configuration Problems and Solutions wiki page.


R is a programming language that is especially powerful for data exploration, visualization, and statistical analysis. To interact with R, we use RStudio.


Video Tutorial

Install R by downloading and running this .exe file from CRAN. Also, please install the RStudio IDE.

Mac OS X

Video Tutorial

Install R by downloading and running this .pkg file from CRAN. Also, please install the RStudio IDE.


You can download the binary files for your distribution from CRAN. Or you can use your package manager (e.g. for Debian/Ubuntu run sudo apt-get install r-base and for Fedora run sudo yum install R). Also, please install the RStudio IDE.

R packages

You will also need to install the following R packages: limma, edgeR, gplots,, EDASeq, RColorBrewer, GO.db, BiasedUrn, DESeq2, Glimma, Rsubread.

These can all be obtained from Bioconductor, except for Glimma (see below).

Open RStudio and run the following commands to install packages from Bioconductor:

> source("")

> biocLite("limma")

Repeat this for each package.

> biocLite("edgeR")

> biocLite("RColorBrewer")

We will install Glimma from source (Mac users: Glimma_0.99.5.tgz, Windows: We can help with this during the workshop.


Mac and Linux users can install Rsubread using

> biocLite("Rsubread")

Windows users will need to set up a GVL instance. Instructions to follow.

Common problems:

  • If the source command doesn't work, try https instead of http.
  • Make sure you are directly connected to the internet and not using a proxy.
  • Make sure the package name is in quotes.
If you have any trouble please see the Instructions for installing Bioconductor packages. You can also arrive early on the first day of the workshop for help with set up.