In the following, we provide some general information on how the setup of SplAdder works in principle and mention several useful things to keep in mind when using the software.
The SplAdder directory setup¶
The general purpose of SplAdder is to build and quantify augmented splicing graphs from RNA-Sequencing data and to utilize them for the detection for alternative splicing events. To achieve this, SplAdder operates on a single project at a time, where a project is characterized by a single shared output (or project) directory. All data of subsequent steps will be written to that directory and a certain substructure will directly be created by SplAdder.
SplAdder has different run modes that reflect the different steps of a typical analysis pipeline:
- for constructing splicing graphs from RNA-Seq data and extracting alternative events
- for the differential analysis between samples
- for the visualization of splicing graphs and alternative events
All of these modes will operate on the same output directory. Please note, that the
always has to precede the
viz modes, as this creates the splicing graph
structures the latter modes operate on.
Please have a look at the SplAdder run modes page for further information.
SplAdder is a heuristic¶
We would like to reiterate here that SplAdder is a heuristic approach, that employs a system of empirical filter rules on RNA-Seq data to extend a splicing graph pre-defined by a given annotation. Due to this heuristic nature, there is a list of things one should keep in mind when working with the software:
- Although the algorithm is deterministic, it is sensitive to the order of input data. Especially when you are working with many input samples and integrate their information to generate a single splicing graph, the order of input files might influence the outcome. However, in most cases the generated splicing graphs are robust against changes of the order of the input.
- Depending on the annotation file that you are using, some annotated gene regions might be subject to filtering. SplAdder will automatically ignore all introns in the input data that could be assigned to more than one gene in the annotation. (This decision is strand-specific, that is both genes and the intron need to be on the same strand.)
- As SplAdder will sample all possible events from the graph, there can be a certain redundancy in the events (although all events are unique as a whole). For instance, if there are 3 possible donors paired with an acceptor in a gene on the positive strand, SplAdder will output all three possible pairs of alternative 3 prime splice site events.
For most of the settings available in SplAdder default parameters are assumed. In a basic call in
spladder build), SplAdder requires at least three parameters: the annotation file
-a), a comma-separated list of alignment files (via
-b) and an output
directory where results files are stored (via
spladder build -o output_directory -b bam_file -a annotation_file
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
Working with large datasets¶
SplAdder can be scaled to larger cohorts and has been run successfully on studies including as many as 10000 samples. Working on such large datasets still needs some consideration and planning and might require to call individual steps differently than how it is done for smaller sample sets.
We provide more detailed information in the section describing how to handle Large cohorts.