Installing the workshop software

We are going to install the software needed for this workshop

  1. FLASH2 to overlap reads (https://github.com/dstreett/FLASH2)
  2. RDP to classify reads into taxon (https://github.com/rdpstaff/RDPTools)
  3. dbcAmplicons pipeline for processing amplicon sequences to abundance tables (https://github.com/msettles/dbcAmplicons)

optional

  1. bowtie2 (https://github.com/BenLangmead/bowtie2)

System Requirements

1. First, create a directory for the workshop in your home directory:

cd /share/workshop/$USER/

Look at your prompt and make sure your in the right placed

mkdir /share/workshop/$USER/mca_example

and two other directors

mkdir /share/workshop/$USER/mca_example/src
mkdir /share/workshop/$USER/mca_example/bin

2. Now lets add the new bin directory to our PATH in a dbcA_profile file

echo 'export PATH=/share/workshop/$USER/mca_example/bin:$PATH' > /share/workshop/$USER/mca_example/src/dbcA_profile

Then on the command line, execute the commands in the dbcA_profile file using source.

source /share/workshop/$USER/mca_example/src/dbcA_profile

3. Install FLASH2 into src and link the executable into bin

cd /share/workshop/$USER/mca_example/src
git clone https://github.com/dstreett/FLASH2.git
cd FLASH2
make
ln -s /share/workshop/$USER/mca_example/src/FLASH2/flash2 /share/workshop/$USER/mca_example/bin/.
# test installation, should see help documentation
flash2 -h

4.a Install apache ant, need for RDP

cd /share/workshop/$USER/mca_example/src
curl http://mirrors.ibiblio.org/apache/ant/binaries/apache-ant-1.10.5-bin.tar.gz > apache-ant-1.10.5-bin.tar.gz
tar xzvf apache-ant-1.10.5-bin.tar.gz
ln -s /share/workshop/$USER/mca_example/src/apache-ant-1.10.5/bin/ant /share/workshop/$USER/mca_example/bin/.

4.b Install the Ribosomal Database Project (RDP) into src

module load java/jdk1.8 # Need the java jdk1.8 loaded

cd /share/workshop/$USER/mca_example/src
git clone https://github.com/rdpstaff/RDPTools.git
cd RDPTools/
git submodule init
git submodule update
make
# might have problems retrieving data.tgz file ... if so, try again ...
# during 9-5 EST(!) ...
# test installation, should see help documentation for classify
java -jar classifier.jar classify
# this should give you a "Command Error" because you didn\'t specify output
# ... but it should give you a list of options
# feel free to move on if the 'make' fails due to data.tgz file, we can fix this later

4.c Add the location of classifier.jar as a variable in our dbcA_profile file,

echo 'module load java/jdk1.8' >> /share/workshop/$USER/mca_example/src/dbcA_profile
echo 'export RDP_PATH=/share/workshop/$USER/mca_example/src/RDPTools' >> /share/workshop/$USER/mca_example/src/dbcA_profile

Then on the command line, execute the commands in the dbcA_profile file using source.

source /share/workshop/$USER/mca_example/src/dbcA_profile

5.a Setup a python virtual environment for dbcAmplicons, in the src directory. You may need to first install virtualenv and pip, pip can be installed following instructions from here pip and then:

module load anaconda2
#cd /share/workshop/$USER/mca_example/src
#pip install virtualenv

If you already have pip, or having installed it using above, then

cd /share/workshop/$USER/mca_example/src
virtualenv dbcA_virtualenv

5.b This lets you set the virtual environment to activate on login by adding it to our dbcA_profile file.

echo 'module load anaconda2' >> /share/workshop/$USER/mca_example/src/dbcA_profile
echo 'source /share/workshop/$USER/mca_example/src/dbcA_virtualenv/bin/activate' >> /share/workshop/$USER/mca_example/src/dbcA_profile
echo 'export PYTHON_EGG_CACHE=/share/workshop/$USER/mca_example/src' >> /share/workshop/$USER/mca_example/src/dbcA_profile

Then on the command line, execute the commands in the dbcA_profile file using source.

source /share/workshop/$USER/mca_example/src/dbcA_profile

You should now see the text “(dbcA_virtualenv)” at the beginning of your prompt.

6. Install dbcAmplicons

cd /share/workshop/$USER/mca_example/src
easy_install pip
pip install biom_format
git clone https://github.com/msettles/dbcAmplicons.git
cd /share/workshop/$USER/mca_example/src/dbcAmplicons/
python setup.py install
# test installation, should see help documentation
dbcAmplicons -h  # should show options / usage message

Optional. Test dbcAmplicons

You could also test the dbcAmplicons installation by running the script, test_dbAmplicons.sh, under the tests folder (in dbcAmplicions).

cd /share/workshop/$USER/mca_example/src/dbcAmplicons/tests/
./test_dbAmplicons.sh  # could show some ERRORs / WARNINGs, but otherwise give stats after a few minutes

Lets Review

We created a directory for the workshop, in that directory we created two folders src and bin. We’ve installed FLASH2, RDP and dbcAmplicons. We’ve placed the executable for flash in a bin folder and added the folder to our PATH. We created an environment variable for the RDP classifier. We’ve created a python virtual environment and then installed the python package biom-format using pip and the dbcAmplions package using setup.py.

  1. You should have verified all the software works by viewing their help docs
  2. Verify that the flash executable is indeed in the bin folder
  3. We’ve modified your PATH and added 1 new environment variable, RDP_PATH, verify the PATH and the new env variable.
  4. We added a multiple lines to your .bash_profile. How many lines?

Now log out, log back in and verify that each application still works. Ex.

flash2 -h

To verify RDP and dbcAmplicons use

java -jar $RDP_PATH/classifier.jar classify
dbcAmplicons -h

You can check the current version of everything with

dbcVersionReport.sh

We usually save the output of this in the project file to remind ourselves which versions of software were run for that project.

If for some reason installation failed let me know and we’ll get things fixed

These lines should be in your dbcA_profile

export PATH=/share/workshop/$USER/mca_example/bin:$PATH
module load java/jdk1.8
export RDP_PATH=/share/workshop/$USER/mca_example/src/RDPTools
module load anaconda2
source /share/workshop/$USER/mca_example/src/dbcA_virtualenv/bin/activate
export PYTHON_EGG_CACHE=/share/workshop/$USER/mca_example/src

At any time you can initiate the environment for dbcAmplicons using.

source /share/workshop/$USER/mca_example/src/dbcA_profile

7. Last lets copy the workshop data into our home directory.

cd
cd /share/workshop/$USER/mca_example
wget https://bioshare.bioinformatics.ucdavis.edu/bioshare/download/puznld0hwbxpb3c/Slashpile_only_R1.fastq.gz wget https://bioshare.bioinformatics.ucdavis.edu/bioshare/download/puznld0hwbxpb3c/Slashpile_only_R2.fastq.gz wget https://bioshare.bioinformatics.ucdavis.edu/bioshare/download/puznld0hwbxpb3c/Slashpile_only_R3.fastq.gz wget https://bioshare.bioinformatics.ucdavis.edu/bioshare/download/puznld0hwbxpb3c/Slashpile_only_R4.fastq.gz

Take a look at the files … what is inside the Illumina_Reads folder?