This tutorial assumes use of a Linux computer with an ‘x86_64’ architecture. The rest of the tutorial should be conducted in a linux Terminal session. In other words you must already be logged into the Amazon EC2 instance as described in the previous section.
Before proceeding you must define a global working directory by setting the environment variable: ‘RNA_HOME’ Log into a server and SET THIS BEFORE RUNNING EVERYTHING.
Create a working directory and set the ‘RNA_HOME’ environment variable
mkdir -p ~/workspace/rnaseq/ export RNA_HOME=~/workspace/rnaseq
Make sure whatever the working dir is, that it is set and is valid
You can place the RNA_HOME variable (and other environment variables) in your .bashrc and then logout and login again to avoid having to worry about it. This has been done for you in the pre-configured amazon instance that you will be using.
Environment variables used throughout this tutorial:
export RNA_HOME=~/workspace/rnaseq export RNA_DATA_DIR=$RNA_HOME/data export RNA_DATA_TRIM_DIR=$RNA_DATA_DIR/trimmed export RNA_REFS_DIR=$RNA_HOME/refs export RNA_REF_INDEX=$RNA_REFS_DIR/chr22_with_ERCC92 export RNA_REF_FASTA=$RNA_REF_INDEX.fa export RNA_REF_GTF=$RNA_REF_INDEX.gtf export RNA_ALIGN_DIR=$RNA_HOME/alignments/hisat2
In order to use fastqc on these instances, we need to change a java variable. We can do this by running the following command.
If these variables are not part of your .bashrc, you can type the following. First, you can open your .bashrc file with nano by simply typing:
You can now see the contents of this file. Then, you want to add the above environment variables to the bottom of the file. You can do this by copying and pasting. Once you have the variables in the file, you’ll want to type
o to save the file, then
enter to confirm you want the same filename, then
x to exit nano.
Since all the environment variables we set up for the RNA-seq workshop start with ‘RNA’ we can easily view them all by combined use of the
grep commands as shown below. The
env command shows all environment variables currently defined and the
grep command identifies string matches.
env | grep RNA