Intro to RNA-Seq Data Analysis Course

Course schedule

• This is based on a 2 day Edinburgh Genomics course of the same name, with the following changes:

- "Introduction to Linux" moduel excluded

- "Sequencer technology overview" module excluded.

- No laboratory visit

- 50% of that course was theoretical, this will be reduced to 30%

• Each section begins with a "Talk", and then a practical runthrough.
• If necessary, some talk slides may be skipped, as the main idea is getting through the practicals.
• Having said that if major theoretical points arise during a practical, they will be discussed.
• Course website: http://stab.st-andrews.ac.uk/i2rda/

- this has all the presentations and practicals

Connecting to a remote Machine

Presenting this before introduction as some people might experience delays logging in.

• We shall use a remote machine not the machine you are logged into locally
• The program we shall use is PuTTY.
• Please try to locate PuTTY in the applications section or on AppsAnywhere

Configuring PuTTY for connection

• Server: marvin.st-andrews.ac.uk
• Terminal | keyboard | check VT100+
• Window | Selection | Control use of Mouse | set xterm
• Connect | Data | enter username
• Connection | ssh | X11 Forwarding | Check yes
• Back to PuTTY main screen | select Default setting |click save

You should now be able to "open" a session

• Be aware: typing in your password is done blindly. I.e. it does not appear on the screen.

Note: If you don't have your password, please ask to have it reset for you.

Computing resources

• RNA-Seq like other Next Generation Sequencing technologies, is characterised by;

- heavy computational workloads

- many different software programs, sometimes doing the same thing, which can be arranged into a pipeline.

- long-running tasks.

These have three implications:

- The marvin cluster is an 11-machine shared computing resource, not a personal computer ... others are using it.

- We need to load the special software before using it

- We want to be able to have a process run unattended.

• For these three aspects, we have:

- A queue system to use, we shall request an interactive session (qrsh) from the queue.

- Use the module system to load, list and unload software programs

- We shall use the GNU Screen utility so we can do other things while waiting.

Aspect of using Windows terminals to connect to Linux

• You can run the Windows web-browser (Chrome preferred) and copy text selecting and ctrl+c
• This can then be pasted inside the putty command-line by clicking the middle mouse button.
• In many ways, copy-pasting is not great for learning.

- although some of the commands are too long to type out, even with history and tab-completion.

- try to also use tab-completion, and the history (up/down arrows and Ctrl+r)

Weakness to watch out for:

• The marvin cluster (more precisely, the network it's attached to) doesn't carry graphics so well.
• We shall be using several graphical programs, and they are all likely to run slowly.

- and sometimes even stall

- we'll cross that bridge when we come to it.

GNU Screen 1

A program which allows several command-line sessions open, similar to the idea of open tabs in a web browser. Let's try it out.

• To enter a new session, type screen
• This will open with quite a bare screen except with a indicator line at the bottom.
• Screen works on the activator key concept, you need to use Ctrl+l (while Ctrl-key iis held down briefly, l-key is pressed) to activate any of its functions.
• After pressing Ctrl+l and releasing you then have a series of single key strokes that will do various useful things.
• There will be two command-line windows open when you start it.
• Let's learn how to get out of it first

- type exit, you should see you have one command-line session less.

- type exit again and you will be told you have exited screen

- you are now back in the ordinary command-line.

GNU Screen 2

• Go back into screen
• Switch back and forth between the two open sessions: use Ctrl+l,n (n for next) or Ctrl+l,p (p for previous)
• Don't see anything different when you do this? Look again at the bottom line, the asterisk has changed position.

- the asterisk defines the active session

- you can move to a numbered session with ctrl+l,1 or ctrl+l,2 for session no.1 and no.2 respectively.

- you open a new session with ctrl+l,c which creates a new session.

Getting a Queue slot

We're going to use one of the screen sessions to get a slot from the queue.

• Assuming you've launched screen, type ctrl+l,0 to confirm you are in the first screen session.
• Type qrsh which requests a queue slot ... it will take a little time to give you one.

- we shall not use this slot for the graphical programs, only the processing ones.

Overview of RNA-Seq

• For gene expression analyses, seen as a more powerful replacememnt to microarrays