Outrigger is a tool to de novo annotate splice sites and exons
Project description
Outrigger
Outrigger is a program which uses junction reads from RNA seq data, and a graph database to create a de novo alternative splicing annotation with a graph database, and quantify percent spliced-in (Psi) of the events.
Free software: BSD license
Features
Finds novel splicing events, including novel exons! (outrigger index) from .bam files
(optional) Validates that exons have correct splice sites, e.g. GT/AG and AT/AC for mammalian systems (outrigger validate)
Calculate “percent spliced-in” (Psi/Ψ) scores for all your samples given the validated events (or the original events if you opted not to validate)
Installation
To install outrigger, we recommend using the Anaconda Python Distribution and creating an environment.
You’ll want to add the `bioconda <https://bioconda.github.io/>`__ channel to make installing `bedtools <bedtools.readthedocs.io>`__ and its Python wrapper, `pybedtools <https://daler.github.io/pybedtools/>`__ easy.
conda config --add channels r conda config --add channels bioconda
Create an environment called outrigger-env. Python 2.7, Python 3.4, and Python 3.5 are supported.
conda create -n outrigger-env pandas pybedtools gffutils biopython bedtools joblib
Now activate that environment using source activate outrigger-env and install outrigger from PyPI, using pip:
source activate outrigger-env pip install outrigger
To check that it installed properly, try the command with the help option (-h), outrigger -h. The output should look like this:
$ outrigger -h usage: outrigger [-h] {index,validate,psi} ... Calculate "percent-spliced in" (Psi) scores of alternative splicing on a *de novo*, custom-built splicing index positional arguments: {index,validate,psi} Sub-commands index Build an index of splicing events using a graph database on your junction reads and an annotation validate Ensure that the splicing events found all have the correct splice sites psi Calculate "percent spliced-in" (Psi) values using the splicing event index built with "outrigger index" optional arguments: -h, --help show this help message and exit
Bleeding edge code from Github (here)
For advanced users, if you have git and Anaconda Python installed, you can:
Clone this repository
Change into that directory
Create an environment with the necessary packages from Anaconda
Activate the environment
Install remaining packages from PyPI (`graphlite <https://github.com/eugene-eeo/graphlite>`__ is only available on PyPI, not as a conda package)
Install this package
These steps are shown in code below.
git clone git@github.com:YeoLab/outrigger cd outrigger conda create --name outrigger --yes --file conda_requirements.txt --channel bioconda source activate outrigger pip install -r requirements.txt pip install .
Quick start
If you just want to know how to run this on your data with the default parameters, start here. Let’s say you performed your alignment in the folder called ~/projects/tasic2016/analysis/tasic2016_v1, and that’s where your SJ.out.tab files from the STAR aligner are (they’re output into the same folder as the .bam files). First you’ll need to change directories to that folder with cd.
cd ~/projects/tasic2016/analysis/tasic2016_v1
Then you need find all alternative splicing events, which you do by running outrigger index on the splice junction files and the gtf. Here is an example command:
outrigger index --sj-out-tab *SJ.out.tab \ --gtf /projects/ps-yeolab/genomes/mm10/gencode/m10/gencode.vM10.annotation.gtf
Next, you’ll want to validate that the splicing events you found follow biological rules, such as being containing GT/AG (mammalian major spliceosome) or AT/AC (mammalian minor splicesome) sequences. To do that, you’ll need to provide the genome name (e.g. mm10) and the genome sequences. An example command is below:
outrigger validate --genome mm10 \ --fasta /projects/ps-yeolab/genomes/mm10/GRCm38.primary_assembly.genome.fa
Finally, you can calculate percent spliced in (Psi) of your splicing events! Thankfully this is very easy:
outrigger psi
It should be noted that ALL of these commands should be performed in the same directory, so no moving.
Quick start summary
Here is a summary the commands in the order you would use them for outrigger!
cd ~/projects/tasic2016/analysis/tasic2016_v1 outrigger index --sj-out-tab *SJ.out.tab \ --gtf /projects/ps-yeolab/genomes/mm10/gencode/m10/gencode.vM10.annotation.gtf outrigger validate --genome mm10 \ --fasta /projects/ps-yeolab/genomes/mm10/GRCm38.primary_assembly.genome.fa outrigger psi
This will create a folder called outrigger_output, which at the end should look like this:
$ tree outrigger_output outrigger_output ├── index │ ├── gtf │ │ ├── gencode.vM10.annotation.gtf │ │ ├── gencode.vM10.annotation.gtf.db │ │ └── novel_exons.gtf │ ├── junction_exon_direction_triples.csv │ ├── mxe │ │ ├── events.csv │ │ ├── exon1.bed │ │ ├── exon2.bed │ │ ├── exon3.bed │ │ ├── exon4.bed │ │ ├── splice_sites.csv │ │ └── validated │ │ └── events.csv │ └── se │ ├── events.csv │ ├── exon1.bed │ ├── exon2.bed │ ├── exon3.bed │ ├── splice_sites.csv │ └── validated │ └── events.csv ├── junctions │ ├── metadata.csv │ └── reads.csv └── psi ├── mxe │ └── psi.csv ├── outrigger_psi.csv └── se └── psi.csv 10 directories, 22 files
For Developers
How to run with the Python debugger
How to run the code with the Python debugger. To run the command line functions such that when they break, you jump into the pdb (Python debugger), here is the code:
python -m pdb outrigger/commandline.py index \ --sj-out-tab outrigger/test_data/tasic2016/unprocessed/sj_out_tab/* \ --gtf outrigger/test_data/tasic2016/unprocessed/gtf/gencode.vM10.annotation.snap25.myl6.gtf
Notice that you replace outrigger with python -m pdb outrigger/commandline.py, which is relative to this github directory.
How to run the tests
If you want to run the tests without calculating what percentage of lines are covered in the test suite, run
make test
If you want to run the tests and see which lines are covered by tests and get an overall percentage of test coverage, run
make coverage
If you want to run an example with ENSEMBL GTF files, do:
make arabdopsis
By default, Travis-CI does all three:
script: - make coverage - make lint - make arabdopsis
History
v0.2.9 (November 11th, 2016)
This is a non-breaking release with many speed improvements, and upgrade is recommended.
v0.2.9 New features
Add bam alignment files as input option
Miscellaneous
Parallelized Psi calculation, the exact number of processors can be specified with --n-jobs, and by default, --n-jobs is -1, which means use as many processors as are available.
v0.2.8 (October 23rd, 2016)
Updated README/HISTORY files
v0.2.7 (October 23rd, 2016)
v0.2.7 New features
Added outrigger validate command to check for canonical splice sites by default: GT/AG (U1, major spliceosome) and AT/AC (U12, minor spliceosome). Both of these are user-adjustable as they are only the standard for mammalian genomes.
v0.2.7 API changes
Added --resume and --force options to outrigger index to prevent the overwriting of interrupted indexing operations, or to force overwriting. By default, outrigger complains and cowardly exits.
v0.2.7 Bug fixes
Support ENSEMBL gtf files which specify chromsome names with a number, e.g. 4 instead of chr4. Thank you to lcscs12345 for pointing this out!
v0.2.7 Miscellaneous
Added version info with outrigger --version
Sped up gffutils queries and event finding by running ANALYZE on SQLite databases.
v0.2.6 (September 15th, 2016)
This is a non-breaking patch release
v0.2.6 Bug fixes
Wasn’t concatenating exons properly after parallelizing
v0.2.6 Miscellaneous
Clarified .gtf file example for directory output
v0.2.5 (September 14th, 2016)
v0.2.5 Bug fixes
Added joblib to requirements
v0.2.4 (September 14th, 2016)
This is a non-breaking patch release of outrigger.
v0.2.4 New features
Actually parallelized exon finding for novel exons. Before had written the code and tested the non-parallelized version but now using actually parallelized version!
v0.2.4 Bug fixes
Don’t need to turn on --debug command for outrigger to even run
v0.2.3 (September 13th, 2016)
This is a patch release of outrigger, with non-breaking changes from the previous one.
Bug fixes
Subfolders get copied when installing
Add test for checking that outrigger -h command works
v0.2.2 (September 12th, 2016)
This is a point release which includes the index submodule in the __all__ statement.
v0.2.1 (September 12th, 2016)
This is a point release which actually includes the requirements.txt file that specifies which packages outrigger depends on.
v0.2.0 (September 9th, 2016)
This is the second release of outrigger!
New features
Parallelized exon finding for novel exons
Added outrigger validate command to check that your new exons have proper splice sites (e.g. GT/AG and AT/AC)
Added more test data for other event types (even though we don’t detect them yet)
v0.1.0 (May 25, 2016)
This is the initial release of outrigger
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