outrigger 1.1.1

Outrigger detects *de novo* exons and quantifies their percent spliced-in

Large-scale detection and calculation of alternative splicing with 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

• Documentation is available here: http://yeolab.github.io/outrigger/

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) via outrigger psi

Installation

To install outrigger, we recommend using the Anaconda Python Distribution and creating an environment.

You’ll want to add the bioconda channel to make installing bedtools and its Python wrapper, pybedtools easy (these programs are necessary for both outrigger index and outrigger validate).

conda config --add channels r
conda config --add channels bioconda

Create an environment called outrigger-env. Python 2.7, Python 3.4, Python 3.5, and Python 3.6 are supported.

conda create --name outrigger-env outrigger

Now activate that environment:

source activate outrigger-env

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] [--version] {index,validate,psi} ...

outrigger (1.0.0dev). Calculate "percent-spliced in" (Psi) scores of
alternative splicing on a *de novo*, custom-built splicing index -- just for
you!

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
  --version             show program's version number and exit

Bleeding edge code from Github (here)

For advanced users, if you have git and Anaconda Python installed, you can:

1. Clone this repository

2. Change into that directory

3. Create an environment named outrigger-env with the necessary packages from Anaconda and the Python Package Index (PyPI).

4. Activate the environment

These steps are shown in code below.

git clone https://github.com/YeoLab/outrigger.git
cd outrigger
conda env create --file environment.yml
source activate outrigger-env

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:

Input: .SJ.out.tab files

outrigger index --sj-out-tab *SJ.out.tab \
    --gtf /projects/ps-yeolab/genomes/mm10/gencode/m10/gencode.vM10.annotation.gtf

Input: .bam files

If you’re using .bam files instead of SJ.out.tab files, never despair! Below is an example command. Keep in mind that for this program to work, the events must be sorted and indexed.

outrigger index --bam *sorted.bam \
    --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 the one below. Each file and folder is annotated with which command produced it.

$ tree outrigger_output
outrigger_output..........................................................index
├── index.................................................................index
│   ├── gtf...............................................................index
│   │   ├── gencode.vM10.annotation.gtf...................................index
│   │   ├── gencode.vM10.annotation.gtf.db................................index
│   │   └── novel_exons.gtf...............................................index
│   ├── exon_direction_junction_triples.csv...............................index
│   ├── mxe...............................................................index
│   │   ├── event.bed.....................................................index
│   │   ├── events.csv....................................................index
│   │   ├── exon1.bed.....................................................index
│   │   ├── exon2.bed.....................................................index
│   │   ├── exon3.bed.....................................................index
│   │   ├── exon4.bed.....................................................index
│   │   ├── intron.bed....................................................index
│   │   ├── splice_sites.csv...........................................validate
│   │   └── validated..................................................validate
│   │       └── events.csv.............................................validate
│   └── se................................................................index
│       ├── event.bed.....................................................index
│       ├── events.csv....................................................index
│       ├── exon1.bed.....................................................index
│       ├── exon2.bed.....................................................index
│       ├── exon3.bed.....................................................index
│       ├── intron.bed....................................................index
│       ├── splice_sites.csv...........................................validate
│       └── validated..................................................validate
│           └── events.csv.............................................validate
├── junctions.............................................................index
│   ├── metadata.csv......................................................index
│   └── reads.csv.........................................................index
└── psi.....................................................................psi
    ├── mxe.................................................................psi
    |   ├── psi.csv.........................................................psi
    │   └── summary.csv.....................................................psi
    ├── outrigger_psi.csv...................................................psi
    └── se..................................................................psi
        ├── psi.csv.........................................................psi
        └── summary.csv.....................................................psi

10 directories, 26 files

History

v1.1.0 (June 28th, 2017)

This is a minor release to `outrigger.

Bug fixes

• Fixed UNIQUE ID error that happened somewhat stochastically when adding new exons to the database

Miscellaneous

• Explicitly added Python 3.6 compatibility

• Change logo location to logo/ instead of logo/v1 since there’s only one version anyway…

v1.0.0 (April 3rd, 2017)

This is the first major release of outrigger!!!

v1.0.0 New features

• Parallelized event across chromosomes

• Added --low-memory flag for index, validate, and psi commands to use a smaller memory footprint when reading CSV files.

• Added --splice-types option to specify only one kind of splicing you’d like to find

• So the user can double-check the Psi calculation, create a summary.csv file indicating the number of reads found at each junction, for all samples - This also shows which “Case” corresponds to each event in each sample, so you can see whether there were sufficient or insufficient reads on the junctions of each event, and how outrigger judged it.

• Added functions to extract constitutive and alternative exons separately

v1.0.0 Bug fixes

• Fixed a bug that stalled on .bam files while counting the junctions

v1.0.0 Miscellaneous

• Added GC/AG to valid splice sites

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