RNA-Seq workshop - University of Amsterdam

March 7, 2019 /// 13:00 - 17:00

Instructors: Ernest Aliche, Tijs Bliek, Marc Galland

Helpers: Max van Hooren, Fred White

General Information

This workshop will give a first introduction to a typical messenger RNA-Seq analysis by combining a short lecture with an extensive practical hands-on session.
Upon completion of this workshop, you'll be able to:

Participants will be encouraged to help one another and to apply what they have learned to their own research problems.

Who: The course is aimed researchers in the Life Sciences that have to deal with mRNA-Seq experiments and datasets. You don't need to have any previous knowledge of the tools that will be presented at the workshop.

Where: The workshop will be located in room G3.10 (Room G3.10, Science Park 904, 1098XH Amsterdam, The Netherlands). Find the exact locations on OpenStreetMap or Google Maps.

Requirements: Participants must bring a laptop with a Linux, Mac 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). They are also required to abide by the following Code of Conduct.

Registration: All free for Green Life Sciences students and staff from the University of Amsterdam. If not all seats are claimed by the Green Life Sciences cluster, then the remaining seats will be open for other UvA students and staff. Please get your ticket using the Eventbrite widget (see above).

Organisers: This workshop is organized by the Science Park Study Group members as part of its recurring activities. Please visit our website for other events that take place in Amsterdam (usually Science Park). If there are any questions get in touch and send us an email (see contact)

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

Contact: Please email m.galland@uva.nl or bliek@uva.nl for more information.

Afternoon schedule

13.00 - 13.10 Welcome Workshop presentation Marc
13.10 - 13.20 Introduction 01. Experimental design and Next-Generation Sequencing for RNA-Seq Marc
13.30 - 14.00 Introduction 02. RNA-Seq post-sequencing steps Ernest
14.00 - 14.30 Introduction 03. Set-up: fastq files, genome sequence, annotation, softwares Tijs
14.30 - 15.00 Practical 04. QC and trimming of raw reads using fastQC and Trimmomatic Tijs
15:00 - 15.15 Coffee break
15.15 - 16.00 Practical 05. Read alignment with hisat2 and counting with featureCounts Tijs
16.00 - 16.15 Practical 06. File preparation for use in the ideal online application Marc
16:15 - 17.00 Practical 07. Data exploration using the ideal Shiny app Marc


The Unix Shell

  • Files and directories
  • History and tab completion
  • Pipes and redirection
  • Looping over files
  • Creating and running shell scripts
  • Finding things
  • Reference...


Required Files

You can download all files (fastq sequencing files, genome sequence in fasta format, genome annotation, etc.) here. Unzip/extract the files to create a rnaseq folder.


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

We maintain 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.

The Bash Shell

Bash is a commonly-used shell that gives you the power to do simple tasks more quickly.


Video Tutorial
  1. Download the Git for Windows installer.
  2. Run the installer and follow the steps bellow:
    1. Click on "Next" four times (two times if you've previously installed Git). You don't need to change anything in the Information, location, components, and start menu screens.
    2. Select “Use the nano editor by default” and click on “Next”.
    3. Keep "Use Git from the Windows Command Prompt" selected and click on "Next". If you forgot to do this programs that you need for the workshop will not work properly. If this happens rerun the installer and select the appropriate option.
    4. Click on "Next".
    5. Keep "Checkout Windows-style, commit Unix-style line endings" selected and click on "Next".
    6. Select "Use Windows' default console window" and click on "Next".
    7. Click on "Install".
    8. Click on "Finish".
  3. If your "HOME" environment variable is not set (or you don't know what this is):
    1. Open command prompt (Open Start Menu then type cmd and press [Enter])
    2. Type the following line into the command prompt window exactly as shown:

      setx HOME "%USERPROFILE%"

    3. Press [Enter], you should see SUCCESS: Specified value was saved.
    4. Quit command prompt by typing exit then pressing [Enter]

This will provide you with both Git and Bash in the Git Bash program.


The default shell in all versions of macOS is Bash, so no need to install anything. You access Bash from the Terminal (found in /Applications/Utilities). See the Git installation video tutorial for an example on how to open the Terminal. You may want to keep Terminal in your dock for this workshop.


The default shell is usually Bash, but if your machine is set up differently you can run it by opening a terminal and typing bash. There is no need to install anything.

Text Editor

When you're writing code, it's nice to have a text editor that is optimized for writing code, with features like automatic color-coding of key words. The default text editor on macOS and Linux is usually set to Vim, which is not famous for being intuitive. If you accidentally find yourself stuck in it, try typing the escape key, followed by :q! (colon, lower-case 'q', exclamation mark), then hitting Return to return to the shell.


nano is a basic editor and the default that instructors use in the workshop. It is installed along with Git.

Others editors that you can use are Notepad++ or Sublime Text. Be aware that you must add its installation directory to your system path. Please ask your instructor to help you do this.


nano is a basic editor and the default that instructors use in the workshop. See the Git installation video tutorial for an example on how to open nano. It should be pre-installed.

Others editors that you can use are Text Wrangler or Sublime Text.


nano is a basic editor and the default that instructors use in the workshop. It should be pre-installed.

Others editors that you can use are Gedit, Kate or Sublime Text.


This material was built with heavy inspiration from the Data Carpentry Genomics workshop developped under the supervision of the Carpentries Foundation. We also borrowed material from a course developed by the Harvard Chan Bioinformatics Core.