RNAseq Data Processing & Analysing Course


Pre-course tasks


Install Cardiff Virtual Private Network (VPN) - Global Protect

  • We will need to have the Global Protect VPN installed on our PC’s in order to connect to HAWK off campus.

For MAC

  • Use this link and log in to the Software Downloads website with your university log in.
  • Then click on Global Protect, then click the MAC OS link. Click on the GlobalProtect-6.2.4.pkg download button to initiate the download to your PC.
  • Once the file has downloaded, navigate to it and double-click on it to initiate installation onto your PC.
  • Navigate through the installer by clicking Continue.
  • Check boxes for GlobalProtect and GlobalProtect System Extensions and click Continue.
  • Click Install.
  • Enter your admin password when prompted and click Install Software.
  • Click Close once the installation has completed.
  • When prompted, choose to Allow the system extension in Security and Privacy preferences.
  • Click on the system notification to Allow notifications from GlobalProtect.

For Windows

  • Use this link and log in to the Software Downloads website with your university log in.
  • Then click on Global Protect, then click the Windows link, then click on the 32- or 64-bit client link. To find out what bit your system is, go to Control Panel > System and Security > System. In the System area, the System Type shows if the system is 32 bit or 64 bit.
  • Now click on the GlobalProtect64-6.2.4.msi download button to initiate the download to your PC.
  • Click Run to start installation. Accept any browser security messages if they pop up.
  • Click Next in the setup wizard and accept the default installation folder on your PC.
  • Click Next to install the Agent in the default location in Program Files.
  • Click Next to continue with the installation.
  • You will be notified once installation has completed, click Close to finish.

For Linux

  • Use this link and log in to the Software Downloads website with your university log in.
  • Then click on Global Protect, then click the Linux link.
  • Follow the instructions from there.


Connecting to the VPN:

  • The VPN runs automatically after installation.
  • Enter the portal address as ras.cf.ac.uk
  • Enter your university username and password.
  • Click OK when prompted to allow GlobalProtect access to your Desktop, Documents, and Downloads folders (three prompts).
  • To disconnect, click on the GlobalProtect icon and click ‘Disconnect’.
  • To reconnect, click on the GlobalProtect icon and click ‘Connect’.


Install Visual Studio Code (VSCode)

  • For this course, we will be using VSCode for everything.
  • VSCode is an Integrated Development Environment (IDE) for code development. It is essentially a customisable notebook for coders.
  • The beauty of this tool is that we can install extensions onto the app which means we can do everything in one window.

To install VSCode

  • First, head on over to the download page.

  • Select the version for your operating system, download and install the software.

  • Windows Users: navigate to the location where you downloaded the file, right click on it, and click ‘Run as administator’. This should avoid any problems with your antivirus software.

  • Note: I am not sure how well this app works for Linux users.

  • Once the app has downloaded, it should automatically open.
  • Exit the startup page by clicking on the ‘X’ on the tab at the top of the window.


Install tools and packages in VSCode

  • Now that we have VSCode installed, we can now install the extensions needed for the whole course.

Remote - SSH

  • This extension allows us to connect remotely to HAWK through a terminal window within the software.

  • To install, click on the ‘Extensions’ button.
  • Search for ‘remote ssh’.
  • Click on the ‘Remote - SSH’ extension and then ‘Install’ at the top.

install Remote-SSH

  • Then, to setup the extension to login to your HAWK, click on ‘Remote Explorer’.
  • Then click on the ‘+’ symbol to add our SSH target.
  • You will see a drop down box appear at the top of the screen asking for your login details.
  • Enter the following (add your username) and hit enter:
ssh c.c1234567@hawklogin.cf.ac.uk
  • You will then be asked to select a SSH configuration file to update.
  • Select the top one.

Setting up HAWK

  • You will now see the host has been added to the ‘Remote Explorer’ window.


Optional: Excel Viewer

  • These are not required, but can massively help when editing CSV files.
  • Excel Viewer allows us to open CSV files as a table, rather than a text file.
  • Click ‘Extensions’ button.
  • Search for ‘excel viewer’, then ‘Install’ at the top.
  • To open CSV files with this extension, right click on the file and click ‘Open With…’. This opens a drop down menu at the top of the window.
  • Click on the second option: ‘CSV Editor’.

Installing Excel Viewer


Connecting to HAWK

  • To connect to HAWK, click on the ‘Remote Explorer’ button, then click ‘Connect to Host in New Window’ button. This opens a new VSCode window with the remote host.
  • You will be prompted (top box) to enter the host operating platform.
  • Click Linux.
  • Then you will be prompted to continue, click Yes.
  • Then you will be asked for your password. Do this and hit enter.
  • You are now connected to HAWK.

Connecting to HAWK


Download Gene Set Enrichment Analysis (GSEA) Application

  • GSEA application is needed at the end of the course to analyse our DEGs.
  • Go to the following webpage and download GSEA version 4.3.2 (latest version may not work for everyone so we are using this one).
  • Make sure you click on the download relevant to your operating system: GSEA_MacApp for Mac, GSEA_Win for Windows, GSEA_Linux for Linux. Make sure you download the installer (.exe for Windows)
  • Unzip the file by double clicking or right-clicking and selecting unzip. Windows users: you may need to run as administrator if you are getting any problems with your antivirus software.
  • Now you should have, in the same location, your GSEA_4.3.2.app.
  • You can move this app to anywhere you want and it will still work when you open it.
  • Double-click the app to ensure that it is working correctly.

GSEA window


Learning Materials

  • We won’t be covering how RNAseq technologies work or the specific details of the nf-core pipelines.
  • Please see the following links to learning materials to cover them:

Bulk RNA Sequencing

nf-core Pipelines

  • Details of the fetchngs pipeline, including what tools/packages etc are used can be found here.
  • Details of the rnaseq pipeline, including what tools/packages etc are used can be found here. Also a YouTube video briefly explaining the pipeline.
  • Details of the differentialabundance pipeline, including what tools/packages etc are used can be found here.

GSEA

g:Profiler

EnrichR

Rummagene

RummaGEO

Ingenuity Pathway Analysis (IPA)