RNA-seq Bioinformatics

Introduction to bioinformatics for RNA sequence analysis

Tool Installation


First, make sure your environment is set up correctly.

Tools needed for this analysis are: samtools, bam-readcount, HISAT2, stringtie, gffcompare, htseq-count, flexbar, R, ballgown, fastqc and picard-tools. In the following installation example the installs are local and will work whether you have root (i.e. admin) access or not. However, if root is available some binaries can/will be copied to system-wide locations (e.g., /usr/bin/).

Set up tool installation location:

mkdir student_tools
cd student_tools


cd $RNA_HOME/student_tools/
wget https://github.com/samtools/samtools/releases/download/1.9/samtools-1.9.tar.bz2
bunzip2 samtools-1.9.tar.bz2
tar -xvf samtools-1.9.tar
cd samtools-1.9


cd $RNA_HOME/student_tools/
export SAMTOOLS_ROOT=$RNA_HOME/student_tools/samtools-1.9
git clone https://github.com/genome/bam-readcount.git
cd bam-readcount
cmake -Wno-dev $RNA_HOME/student_tools/bam-readcount


cd $RNA_HOME/student_tools/
wget ftp://ftp.ccb.jhu.edu/pub/infphilo/hisat2/downloads/hisat2-2.1.0-Linux_x86_64.zip
unzip hisat2-2.1.0-Linux_x86_64.zip
cd hisat2-2.1.0


cd $RNA_HOME/student_tools/
wget http://ccb.jhu.edu/software/stringtie/dl/stringtie-1.3.4d.Linux_x86_64.tar.gz
tar -xzvf stringtie-1.3.4d.Linux_x86_64.tar.gz
cd stringtie-1.3.4d.Linux_x86_64


cd $RNA_HOME/student_tools/
wget http://ccb.jhu.edu/software/stringtie/dl/gffcompare-0.10.6.Linux_x86_64.tar.gz
tar -xzvf gffcompare-0.10.6.Linux_x86_64.tar.gz
cd gffcompare-0.10.6.Linux_x86_64


cd $RNA_HOME/student_tools/
wget https://github.com/simon-anders/htseq/archive/release_0.11.0.tar.gz
tar -zxvf release_0.11.0.tar.gz
cd htseq-release_0.11.0/
python setup.py install --user
chmod +x scripts/htseq-count


Note, this tool is currently only installed for the gtf_to_fasta tool used in kallisto section

cd $RNA_HOME/student_tools/
wget https://ccb.jhu.edu/software/tophat/downloads/tophat-2.1.1.Linux_x86_64.tar.gz
tar -zxvf tophat-2.1.1.Linux_x86_64.tar.gz
cd tophat-2.1.1.Linux_x86_64/


cd $RNA_HOME/student_tools/
wget https://github.com/pachterlab/kallisto/releases/download/v0.44.0/kallisto_linux-v0.44.0.tar.gz
tar -zxvf kallisto_linux-v0.44.0.tar.gz
cd kallisto_linux-v0.44.0/


In addition to installing on the cloud you should also install FastQC on your own laptop/desktop

cd $RNA_HOME/student_tools/
wget https://www.bioinformatics.babraham.ac.uk/projects/fastqc/fastqc_v0.11.8.zip --no-check-certificate
unzip fastqc_v0.11.8.zip
cd FastQC/
chmod 755 fastqc
./fastqc --help


pip3 install multiqc
multiqc --help


cd $RNA_HOME/student_tools/
wget https://github.com/broadinstitute/picard/releases/download/2.18.15/picard.jar -O picard.jar
java -jar $RNA_HOME/student_tools/picard.jar


cd $RNA_HOME/student_tools/
wget https://github.com/seqan/flexbar/releases/download/v3.4.0/flexbar-3.4.0-linux.tar.gz
tar -xzvf flexbar-3.4.0-linux.tar.gz
cd flexbar-3.4.0-linux/
export LD_LIBRARY_PATH=$RNA_HOME/student_tools/flexbar-3.4.0-linux:$LD_LIBRARY_PATH


cd $RNA_HOME/student_tools/
git clone https://github.com/griffithlab/regtools
cd regtools/
mkdir build
cd build/
cmake ..


pip3 install RSeQC


cd $RNA_HOME/student_tools/
mkdir bedops_linux_x86_64-v2.4.35
cd bedops_linux_x86_64-v2.4.35
wget -c https://github.com/bedops/bedops/releases/download/v2.4.35/bedops_linux_x86_64-v2.4.35.tar.bz2
tar -jxvf bedops_linux_x86_64-v2.4.35.tar.bz2


cd $RNA_HOME/student_tools/
mkdir gtfToGenePred
cd gtfToGenePred
wget -c http://hgdownload.cse.ucsc.edu/admin/exe/linux.x86_64/gtfToGenePred
chmod a+x gtfToGenePred


cd $RNA_HOME/student_tools/
mkdir genePredToBed
cd genePredToBed
wget -c http://hgdownload.cse.ucsc.edu/admin/exe/linux.x86_64/genePredToBed
chmod a+x genePredToBed


This install takes a while so check if you have R installed already by typing which R. It is already installed on the Cloud, but for completeness, here is how it was done. Please skip all R installation!

#sudo apt-get install r-base-dev
#export R_LIBS=
#cd $RNA_HOME/student_tools/
#wget https://stat.ethz.ch/R/daily/R-patched.tar.gz
#tar -xzvf R-patched.tar.gz
#cd R-patched
#./configure --prefix=$RNA_HOME/student_tools/R-patched/ --with-x=no
#make install

Note, if X11 libraries are not available you may need to use --with-x=no during config, on a regular linux system you would not use this option. Also, linking the R-patched bin directory into your PATH may cause weird things to happen, such as man pages or git log to not display. This can be circumvented by directly linking the R* executables (R, RScript, RCmd, etc.) into a PATH directory.

R Libraries

For this tutorial we require:

launch R (enter R at linux command prompt) and type the following at an R command prompt. NOTE: This has been pre-installed for you, so these commands can be skipped.



For this tutorial we require:

launch R (enter R at linux command prompt) and type the following at an R command prompt. If prompted, type “a” to update all old packages. NOTE: This has been pre-installed for you, so these commands can be skipped.




PRACTICAL EXERCISE 1 - Software Installation

Assignment: Install bedtools on your own. Make sure you install it in your tools folder. Download, unpack, compile, and test the bedtools software.

cd $RNA_HOME/student_tools/


Solution: When you are ready you can check your approach against the Solutions

Add locally installed tools to your PATH [OPTIONAL]

To use the locally installed version of each tool without having to specify complete paths, you could add the install directory of each tool to your ‘$PATH’ variable


export LD_LIBRARY_PATH=$RNA_HOME/student_tools/flexbar-3.4.0-linux:$LD_LIBRARY_PATH

echo $PATH

You can make these changes permanent by adding the above lines to your .bashrc file use a text editor to open your bashrc file. For example:

vi ~/.bashrc

Vi instructions

  1. Using your cursor, navigate down to the “export PATH” commands at the end of the file.
  2. Delete the line starting with PATH using the vi command “dd”.
  3. Press the “i” key to enter insert mode. Go to an empty line with you cursor and copy paste the new RNA_HOME and PATH commands into the file
  4. Press the “esc” key to exit insert mode.
  5. Press the “:” key to enter command mode.
  6. Type “wq” to save and quit vi

If you would like to learn more about how to use vi, try this tutorial/game: VIM Adventures

NOTE: If you are worried your .bashrc is messed up you can redownload as follows:

cd ~
wget -N https://raw.githubusercontent.com/griffithlab/rnaseq_tutorial/master/setup/.bashrc
source ~/.bashrc

Installing tools from official ubuntu packages [OPTIONAL]

Some useful tools are available as official ubuntu packages. These can be installed using the linux package management system apt. Most bioinformatic tools (especially the latest versions) are not available as official packages. Nevertheless, here is how you would update your apt library, upgrade existing packages, and install an Ubuntu tool called tree.

#sudo apt-get update
#sudo apt-get upgrade
#sudo apt-get install tree