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README.md Update README.md Nov 15, 2016
diff-expr.R Differential expression template Oct 17, 2016
sample_description.csv
yaml_example.yaml Add files via upload Oct 10, 2016

README.md

Laboratory of Systems Pharmacology - RNAseq pipeline

Converting FASTQ files to count matrices

  1. If this is your first time using a linux command shell, make sure you type the commands in exactly as is. Things like spaces and quotes matter. The easiest way to do this is to copy and paste the commands from this README into your command shell.

  2. Login to Orchestra using the instructions available at https://wiki.med.harvard.edu/Orchestra/NewUserGuide

  3. Familiarize yourself with nano, a text editor in linux. Specifically, take a look at keyboard shortcuts like CTRL+O to save, CTRL+X to exit

  4. Use nano ~/.bashrc to open your .bashrc file and add the following lines to have the path to bcbio in your orchestra setup:

    # Environment variables for running bcbio
    export PATH=/opt/bcbio/centos/bin:$PATH
    unset PYTHONHOME
    unset PYTHONPATH
    export PYTHONNOUSERSITE=1
    module load dev/java/jdk1.7
    

    Save and exit (CTRL+O and CTRL+X in nano).
    This only needs to be done once.

  5. Change into the project directory: cd /groups/<groupname>/<yourname>/<projectname>/ (For example, this could be /groups/springer/sarah/Isog/)

  6. Move your fastq files to this directory. (Using mv command on Orchestra.)

  7. Move your yaml file to this directory as well. An example .yaml file is available in this GitHub repository as yaml_example.yaml:

    • OPTION 1: Open a new file with a text editor. Copy and paste the content of yaml_example.yaml to that file. Save and exit.
    • OPTION 2: Download the file directly from GitHub using the following command: wget https://raw.githubusercontent.com/sorgerlab/rnaseq/master/yaml_example.yaml
  8. Make a .csv file describing your samples. Use sample_description.csv in this GitHub repository as a template. As before, you can either create a new file and copy-and-paste OR using wget https://raw.githubusercontent.com/sorgerlab/rnaseq/master/sample_description.csv

  9. Run the following line of code:

    bcbio_nextgen.py -w template yaml_example.yaml sample_description.csv *.fq 
    
    • NOTE: Use *.fq or *.fastq depending on how the files are named
    • NOTE: fastq filenames CANNOT end in _1 or _anynumber.fastq or it will cause FATAL ERROR
  10. Running this setup creates a project directory and the following subdirectories inside of that: work, final, config

    • NOTE: The project directory will be named sample_description. You can control that by substituting a different name in all filenames and commands above.
  11. Descend into the work subdirectory of your project (by default, this is done using cd sample_description/work). Using a text editor (e.g., nano submit_bcbio.lsf) create the following submission file:

    #!/bin/sh
    
    #BSUB -q priority
    #BSUB -J bcbio_project
    #BSUB -n 1
    #BSUB -W 100:0
    #BSUB -R "rusage[mem=10000]"
    #BSUB -e project.err
    #BSUB -o project.out
    
    bcbio_nextgen.py ../config/sample_description.yaml -n 32 -t ipython -s lsf -q parallel -r mincores=2 -r minconcores=2 '-rW=72:00' --retries 3 --timeout 380
    
    • NOTE: If you changed the name of your project from sample_description to something else, use that new name when specifying ../config/sample_description.yaml above. The path has to point to an actual file in the config subdirectory.
  12. Submit the job to Orchestra using the following command on the newly-created submission file:

    bsub < submit_bcbio.lsf
    
    • The job will sometimes time out or have memory issue. In this case, one can just re-submit the job with different settings. The job will look at where the previous run left off and start from there.
    • Use the following alternate submission settings: bcbio_nextgen.py ../config/yaml_example.yaml -n 32 -t ipython -s lsf -q parallel -r mincores=2 -r minconcores=2 '-rW=72:00' --retries 3 --timeout 380
  13. Note: The instructions assume hg19 - genome build 37. Additional reading:

Running differential expression on the count matrices

To run the next part of the analysis, you will need a few R packages. To install it on Orchestra, start up R, by typing the following commands in your shell:

module load stats/R/3.3.1
R

This gets you into an R. You can now install the packages by running the following command in R:

install.package( c("ggplot2", "reshape", "gplots", "edgeR", "CHBUtils", "pheatmap",
              "DESeq2", "tximport", "DT", "DEGreport", "dplyr", "rmarkdown") )

Go get a cup of coffee!

Once you obtained the count matrices and finish installing Rmarkdown, the basic differential expression pipeline can be run via the following command:

bcbio-rnaseq summarize path-to-project-summary-yaml -f "~celltype"

where path-to-project-summary-yaml refers to a file that you should be able to find in your final subdirectory (which is on the same level as the config and work subdirectories covered above). If you didn't change the name of sample_description.csv, the summary yaml file will live at /groups/<groupname>/<yourname>/<projectname>/sample_description/final/<date>_sample_description/project-summary.yaml.

The bcbio-rnaseq command above will run the DESeq2 differential expression pipeline and produce a report in the R markdown format (http://rmarkdown.rstudio.com/). The formula that you provide after the -f flag must be in the appropriate format (see https://bioconductor.org/packages/release/bioc/vignettes/DESeq2/inst/doc/DESeq2.pdf) and reference the column names in your sample_description.csv file. In the example above, we provide "~celltype", which implies that we are interested in performing a differential expression comparison along the third column of our sample_description.csv.

Note that if you omit the formula, the script will perform basic quality control instead:

bcbio-rnaseq summarize path-to-project-summary-yaml