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This README describes the software SplAdder, short for Splicing Adder, a toolbox for alternative splicing analysis based on RNA-Seq alignment data. Briefly, the software takes a given annotation and RNA-Seq read alignments in standardized formats, transforms the annotation into a splicing graph representation, augments the splicing graph with additional information extracted from the read data, extracts alternative splicing events from the graph and quantifies the events based on the alignment data, The quantified events can then be used for differential analysis.

Currently, there are two implementations available, which produce the same results on the same input data. The development of SplAdder was started in Matlab and has been moved to Python in the past two years, mostly for the reason of having fewer dependencies. Both implementations are available in this repository und the respective directories.

Note, that the current Matlab implementation makes extensive use of the HDF5 storage format and uses low level access to the files. Unfortunately, this functionality is not available in Octave, therefor requiring a working Matlab installation. In case the compatibility issues between Matlab and Octave regarding low level HDF5 should get resolved in the near future, SplAdder might be able to run also under Octave.

However, future development is planned to focus on the Python branch. The Python version of the code has been developed in python 2.7 and requires the following packages:

• scipy (version >= 0.12 tested)
• pysam (version >= 0.7 required)
• h5py (version >= 2.2.0 tested)
• matplotlib (for the visualization code only; version >= 1.4.0 tested)

Please see the respective INSTALL files in the matlab or python directories for details on how to install SplAdder on your system.

Information on how to contact the authors can be found in the AUTHORS file.

All licensing information can be found in the COPYRIGHT file.

This README provides a quick walk-through of a basic SplAdder run. For further reading, please consider the file DOCUMENTATION in the matlab or python directory. The following description is generic for both implementations.

Invoking SplAdder without any parameters will print a description of the command line interface to the screen.

In a basic call, SplAdder is invoked with three parameters: the annotation file (via -a), a comma separated list of alignment files (via -b) and an output directory where results files are stored (via -o). This will run SplAdder in its default configuration, which consists of the following steps:

• transform annotation into splicing graph representation
• generate an augmented splicing graph for each alignment file by inferring and adding the following elements:
  • insert intron retentions
  • insert cassette exons
  • insert new intron edges
• merge the augmented splicing graphs into a common splicing graph
• extract the following alternative splicing events:
  • exon skip
  • intron retention
  • alternative 3'/5' splice site
  • multiple exon skip
  • mutually exclusive exons
• quantify all alternative splicing events on each of the provided alignment files