Run the ORF discovery pipeline

For this tutorial, we use the c-elegans-chrI-example with the c-elegans-test.yaml configuration file.

How to run the pipeline

To run the pipeline, change the paths in the configuration file to point to the location where you extracted the data, e.g. /home/user/data/c-elegans-chrI-example. You can do this using a text editor, or simply by modifying the file in place

sed -i 's|/path/to/your/c-elegans-example|'`pwd`'|g' c-elegans-test.yaml

Important

Default parameters were modified for the example and included in the configuration file. If you use this configuration file as a general template for your data, do not forget to remove everything below the line “REMOVE BELOW THIS LINE IF YOU USE THIS CONFIGURATION FILE AS TEMPLATE FOR YOUR DATA”.

You can now create the genome indices and annotations. For this small example, it is important to downscale the STAR --genomeSAindexNbases parameter as follows

prepare-rpbp-genome c-elegans-test.yaml --star-options "--genomeSAindexNbases 10" --num-cpus 4 --logging-level INFO --log-file rpbp-genome.log

The file rpbp-genome.log contains logging output for the reference preprocessing. You now have a new directory called WBcel235.79.chrI with genome indices and annotations.

Finally, run the ORF discovery pipeline

run-all-rpbp-instances c-elegans-test.yaml --merge-replicates --run-replicates --keep-intermediate-files --num-cpus 4 --logging-level INFO --log-file rpbp-pipeline.log

The file rpbp-pipeline.log contains logging output for the different processing steps. You now have four new directories (with-, without-) including output from Flexbar, Bowtie2, and STAR, and directories with Rp-Bp output: metagene-profiles, orf-profiles, and orf-predictions. The orf-predictions include the output for each sample c-elegans-rep-1 and c-elegans-rep-2 as well as for the merged replicates c-elegans-test.

How to summarize the results and launch the apps

Prepare the summary output for the profile construction dashboard

summarize-rpbp-profile-construction c-elegans-test.yaml --num-cpus 4 --logging-level INFO --log-file rpbp-profile-summary.log

and for the predictions dashboard

summarize-rpbp-predictions c-elegans-test.yaml --no-replicates --circos-bin-width 10000 --circos-show-chroms I --logging-level INFO --log-file rpbp-predictions-summary.log

Due to the size of the data, we reduce the bin width for the Circos plot. We also need to specify which sequences or chromosomes we want to include (by default, only numbered chromosomes and X/x, Y/y are shown). You now have a new directory analysis with profile_construction and rpbp_predictions output.

Launch any of the web applications with

rpbp-profile-construction-dashboard -c c-elegans-test.yaml

or

rpbp-predictions-dashboard -c c-elegans-test.yaml

To navigate the apps is easy, just follow the “hints”. Most items are interactive. Press CTRL+C to quit.

Attention

For the apps only, the configuration file is passed using a (required) named argument -c/--config CONFIG.

Note

Any of the above command will open a browser page with the web application running locally. You can also specify a --host and a --port, e.g. if launching the app from a remote server. In the latter case, you have to open a browser page at the correct address. For example, if you use --host 123.123.123.123, then open a page on http://123.123.123.123:8050/.