Summary and Schedule

This course was designed for students who have a background in molecular biology and are interested in learning how to analyze RNA sequencing (RNA-Seq) data. RNA-Seq is an analytical method which measures the amount of RNA that has been transcribed from each gene. It involves some sophisticated molecular biology, expensive instruments, and generates large volumes of data which are beyond what standard office software and laptops can handle. This means that you will need to use both specialized software and remote high- performance computing systems. In order to work with these systems, you will need to learn some basic programming skills. This course is designed to help you learn these skills and to apply them to RNA-Seq analysis.

The actual schedule may vary slightly depending on the topics and exercises chosen by the instructor.

Preparations

This is a hands-on lesson and students must have R and RStudio installed on their computers. They also need to be able to install a number of R packages, create directories, and download files.

To avoid troubleshooting during the lesson, students should follow the instruction below to download and install everything beforehand. If they are using their own computers this should be no problem, but if the computer is managed by their organization’s IT department they might need help from an IT administrator.

Install R and RStudio

R and RStudio are two separate pieces of software:

  • R is a programming language that is especially powerful for data exploration, visualization, and statistical analysis.
  • RStudio is an integrated development environment (IDE) that makes using R easier. In this course we use RStudio to interact with R.

Installation Instructions

If you don’t already have R and RStudio installed, follow the instructions for your operating system below. You have to install R before you install RStudio.

  • Download R from the CRAN website.
  • Run the .exe file that was just downloaded
  • Go to the RStudio download page
  • Under All Installers, download the RStudio Installer for Windows.
  • Double click the file to install it
  • Once it’s installed, open RStudio to make sure it works and you don’t get any error messages.
  • Download R from the CRAN website.
  • Select the .pkg file for the latest R version
  • Double click on the downloaded file to install R
  • It is also a good idea to install XQuartz (needed by some packages)
  • Go to the RStudio download page
  • Under All Installers, download the RStudio Installer for MacOS.
  • Double click the file to install RStudio
  • Once it’s installed, open RStudio to make sure it works and you don’t get any error messages.
  • Follow the instructions for your distribution from CRAN, they provide information to get the most recent version of R for common distributions. For most distributions, you could use your package manager (e.g., for Debian/Ubuntu run sudo apt-get install r-base, and for Fedora sudo yum install R), but we don’t recommend this approach as the versions provided by this are usually out of date. In any case, make sure you have at least R 3.3.1.
  • Go to the RStudio download page
  • Under All Installers, select the version that matches your distribution and install it with your preferred method (e.g., with Debian/Ubuntu sudo dpkg -i rstudio-YYYY.MM.X-ZZZ-amd64.deb at the terminal).
  • Once it’s installed, open RStudio to make sure it works and you don’t get any error messages.

Update R and RStudio

If you already have R and RStudio installed, first check if your R version is up to date:

  • When you open RStudio your R version will be printed in the console on the bottom left. Alternatively, you can type sessionInfo() into the console. If your R version is 4.3.1 or later, you don’t need to update R for this lesson. If your version of R is older than that, download and install the latest version of R from the R project website for Windows, for MacOS, or for Linux
  • It is not necessary to remove old versions of R from your system, but if you wish to do so you can check How do I uninstall R?
  • Note: The changes introduced by new R versions are usually backwards-compatible. That is, your old code should still work after updating your R version. However, if breaking changes happen, it is useful to know that you can have multiple versions of R installed in parallel and that you can switch between them in RStudio by going to Tools > Global Options > General > Basic.
  • After installing a new version of R, you will have to reinstall all your packages with the new version. For Windows, there is a package called installr that can help you with upgrading your R version and migrate your package library.

To update RStudio to the latest version, open RStudio and click on Help > Check for Updates. If a new version is available follow the instruction on screen. By default, RStudio will also automatically notify you of new versions every once in a while.

Install required R packages

During the course we will need a number of R packages. Packages contain useful R code written by other people. We will use the packages tidyverse, hexbin, patchwork, and RSQLite.

To try to install these packages, open RStudio and copy and paste the following command into the console window (look for a blinking cursor on the bottom left), then press the Enter (Windows and Linux) or Return (MacOS) to execute the command.

R 

install.packages(c("tidyverse", "BiocManager"))

Then load the BiocManager package and use it to install bioinformatics packages.

R 

library(BiocManager)
BiocManager::install(c('AnnotationHub', 'DESeq2', 'GenomicRanges'))

Alternatively, you can install the packages using RStudio’s graphical user interface by going to Tools > Install Packages and typing the names of the packages separated by a comma.

R tries to download and install the packages on your machine. When the installation has finished, you can try to load the packages by pasting the following code into the console:

R 

library(tidyverse)
library(DESeq2)

If you do not see an error like there is no package called ‘...' you are good to go!

Updating R packages

Generally, it is recommended to keep your R version and all packages up to date, because new versions bring improvements and important bugfixes. To update the packages that you have installed, click Update in the Packages tab in the bottom right panel of RStudio, or go to Tools > Check for Package Updates....

Sometimes, package updates introduce changes that break your old code, which can be very frustrating. To avoid this problem, you can use a package called renv. It locks the package versions you have used for a given project and makes it straightforward to reinstall those exact package version in a new environment, for example after updating your R version or on another computer. However, the details are outside of the scope of this lesson.

Download the data

We will download the data directly from R during the lessons. However, if you are expecting problems with the network, it may be better to download the data beforehand and store it on your machine.

The data files for the lesson can be downloaded manually here: TBD