Installation

###Note: First, make sure your environment is set up correctly.

#1-i. Installation Tools needed for this analysis are: samtools, bam-readcount, bowtie, tophat, star, HISAT2, cufflinks, htseq-count, R, cummeRbund, fastqc, picard-tools, and samstat. 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:

cd $RNA_HOME
mkdir tools
cd tools

##SAMtools

cd $RNA_HOME/tools/
wget https://github.com/samtools/samtools/releases/download/1.3.1/samtools-1.3.1.tar.bz2
bunzip2 samtools-1.3.1.tar.bz2
tar -xvf samtools-1.3.1.tar
cd samtools-1.3.1
make
./samtools

##bam-readcount

cd $RNA_HOME/tools/
mkdir git
cd git
git clone --recursive git://github.com/genome/bam-readcount.git
cd $RNA_HOME/tools/
mkdir bam-readcount
cd bam-readcount
export SAMTOOLS_ROOT=$RNA_HOME/tools/samtools-1.3.1
cmake $RNA_HOME/tools/git/bam-readcount
make
./bin/bam-readcount

##Bowtie2

cd $RNA_HOME/tools/
wget http://sourceforge.net/projects/bowtie-bio/files/bowtie2/2.2.9/bowtie2-2.2.9-linux-x86_64.zip
unzip bowtie2-2.2.9-linux-x86_64.zip
cd bowtie2-2.2.9
./bowtie2
./bowtie2-build

##TopHat2

cd $RNA_HOME/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
./tophat2

##STAR

cd $RNA_HOME/tools/
wget https://github.com/alexdobin/STAR/archive/2.5.1b.tar.gz
tar -zxvf 2.5.1b.tar.gz
cd STAR-2.5.1b/source
make STAR
file STAR

##HISAT2

cd $RNA_HOME/tools/
wget ftp://ftp.ccb.jhu.edu/pub/infphilo/hisat2/downloads/hisat2-2.0.4-Linux_x86_64.zip
unzip hisat2-2.0.4-Linux_x86_64.zip
cd hisat2-2.0.4
./hisat2

##Cufflinks2

cd $RNA_HOME/tools/
wget http://cole-trapnell-lab.github.io/cufflinks/assets/downloads/cufflinks-2.2.1.Linux_x86_64.tar.gz
tar -zxvf cufflinks-2.2.1.Linux_x86_64.tar.gz
cd cufflinks-2.2.1.Linux_x86_64
./cufflinks

##htseq-count

cd $RNA_HOME/tools/
wget --no-check-certificate https://pypi.python.org/packages/source/H/HTSeq/HTSeq-0.6.1p1.tar.gz
tar -zxvf HTSeq-0.6.1p1.tar.gz
cd HTSeq-0.6.1p1/
python setup.py install --user
chmod +x scripts/htseq-count
./scripts/htseq-count

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

cd $RNA_HOME/tools/
wget http://www.bioinformatics.babraham.ac.uk/projects/fastqc/fastqc_v0.11.5.zip
unzip fastqc_v0.11.5.zip
cd FastQC/
chmod 755 fastqc
./fastqc --help

##Picard

cd $RNA_HOME/tools/
wget https://github.com/broadinstitute/picard/releases/download/1.140/picard-tools-1.140.zip -O picard-tools-1.140.zip
unzip picard-tools-1.140.zip
java -jar $RNA_HOME/tools/picard-tools-1.140/picard.jar

##SAMStat

export PATH=$RNA_HOME/tools/samtools-1.3.1:$PATH
cd $RNA_HOME/tools/
wget http://downloads.sourceforge.net/project/samstat/samstat-1.5.1.tar.gz
tar -xzvf samstat-1.5.1.tar.gz
cd samstat-1.5.1
./configure
make
./src/samstat

##Flexbar

cd $RNA_HOME/tools/
wget https://github.com/seqan/flexbar/releases/download/v2.4.0/flexbar_v2.4_linux64.tgz
tar -xzvf flexbar_v2.4_linux64.tgz
cd flexbar_v2.4_linux64
export LD_LIBRARY_PATH=$RNA_HOME/tools/flexbar_v2.4_linux64:$LD_LIBRARY_PATH
./flexbar

##R 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!

#cd $RNA_HOME/tools/
#export R_LIBS=
#wget https://cran.r-project.org/src/base/R-3/R-3.2.5.tar.gz
#tar -zxvf R-3.2.5.tar.gz
#cd R-3.2.5
#./configure --prefix=$RNA_HOME/tools/R-3.2.5/ --with-x=no
#make
#make install
#./bin/Rscript

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:

##R Libraries For this tutorial we require:

• gplots
• ggplot2

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.

#install.packages("gplots")
#install.packages("ggplot2")
#quit()

##Bioconductor For this tutorial we require:

• cummeRbund
• edgeR

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.

#source("http://bioconductor.org/biocLite.R")
#biocLite("cummeRbund")
#biocLite("edgeR")
#quit()

---

##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/tools/

• Hint: google "bedtools" to find the source code
• Hint: there is a README file that will give you hints on how to install
• Hint: If your install has worked you should be able to run bedtools as follows:

$RNA_HOME/tools/bedtools2/bin/bedtools

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 RNA_HOME=~/workspace/rnaseq
export PATH=$RNA_HOME/tools/samtools-1.2:$RNA_HOME/tools/bam-readcount/bin:$RNA_HOME/tools/bowtie2-2.2.6:$RNA_HOME/tools/tophat-2.1.0.Linux_x86_64:$RNA_HOME/tools/STAR-STAR_2.5.0a/source:$RNA_HOME/tools/cufflinks-2.2.1.Linux_x86_64:$RNA_HOME/tools/HTSeq-0.6.1p1/scripts:$RNA_HOME/tools/R-3.2.2/bin:$RNA_HOME/tools/FastQC:$RNA_HOME/tools/picard-tools-1.140:$RNA_HOME/tools/samstat-1.5.1/src:/home/ubuntu/bin/bedtools2/bin:$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://github.com/griffithlab/rnaseq_tutorial_v1/wiki/config/.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
tree

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