We are using the Linux command line to run most of the tools we use today. If you are new to Linux please complete the Intro to Command Line Workshop.
We have several servers that you can login to. For the purpose of this practical we will use bifx-core2. No matter where you login, you will have access to the same files and programs. bifx-core3 is also available, both bifx-core2 and bifx-core3 require you to use a VPN.
There are several options to login to our machines. You can use the Terminal app on a Mac or equivalant Command Prompt or MobaXTerm on Windows. Login via X2G0 if you want a graphical interface.
To login via command line: ssh USER@bifx-core2.bio.ed.ac.uk
Login with your user credentials (the same as EASE)
If you are using MobaXTerm, an alternative way of logging in to the server is shown in the MobaXTerm demo.
Once you have typed in your password, you should see some welcome text and a prompt that looks something like this:
[USERNAME]@bifx-core2:~$
In order to view files created on the server, we need to create a public_html directory.
After logging in you should be in your $HOME directory, check with;
pwdThis should show the PATH of your present working directory, which should now be your home directory as you have just logged in. You can return to this place at any time using the change directory command.
cdYou have permissions to create files and directories under your home folder. Lets create some now which we will use later on.
mkdir ~/public_html
mkdir ~/public_html/TMPHere we have used the absolute path name for each directory using ~/ as a shortcut for your $HOME directory. Nested directories are separated by the forward slash ‘/’ sign.
As you have created ~/public_html, contents of this directory are available online with any web browser
To see it enter the following URL, changing yourUserName to what ever your username is.
http://bifx-core3.bio.ed.ac.uk/~yourUserName
For new users this will be; https://bifx-core3.bio.ed.ac.uk/Public/yourUserName
The datasets used in this exercise are derived from a single end ChIP-seq experiment (actually ChIP-exo) in S.cerevisiae. There are 2 biological replicates (though we recommend using 3 or more!) Reb1_R1 and Reb1_R2 as well as their corresponding input controls Input_R1 and Input_R2. For this experiment immunoprecipitation was performed with antibodies against Reb1. Reb1 recognizes a specific sequence (TTACCCG) and is involved in many aspects of transcriptional regulation by all three yeast RNA polymerases and promotes formation of nucleosome-free regions (NFRs). You can find the original publication here. For the purpose of this workshop we have randomly subsampled and filtered out poor quality reads to speed up runtime.
| Dataset | Description |
|---|---|
| Reb1_R1 | ChIP experiment, replicate 1 |
| Reb1_R2 | ChIP experiment, replicate 2 |
| Input_R1 | Input DNA, replicate 1 |
| Input_R2 | Input DNA, replicate 2 |
First, make a new directory for this tutorial and move into that directory. Then link the directory to your public html folder as we are going to make everything public in this tutorial.
cd
mkdir ChIP-seq_workshop
cd ChIP-seq_workshop
ln -s $PWD ~/public_html/Next, create a subfolder called fastq for all of our sequence files and link the raw datasets to this folder:
mkdir fastq
cp /homes/library/training/ChIP-seq_workshop/data/*fq.gz fastq/.When you receive data from a sequencing centre the file should also be provided with an alphanumeric string known as an md5 checksum. We can think of this as a files passport or fingerprint and use it to verify our data and ensure it wasn’t corrupted or truncated during download. The md5 checksums for these files are below. Lets check that now using the md5sum command:
| md5 checksum | filename |
|---|---|
| 914b4dda687a76b0d50e545e3ce705d6 | Input_R1.fq.gz |
| f421ed18b71a801b236612cdde49dbaf | Input_R2.fq.gz |
| dd363301ad237ecb6c81c59ae97995a2 | Reb1_R1.fq.gz |
| 06623f9e556876dd6c4d1dfdc4348698 | Reb1_R2.fq.gz |
cd fastq #Move into the fastq directory
md5sum *.fq.gz > md5
cat md5 #prints out the contents of md5
#To check the files and md5 sums match at any time
md5sum -c md5