Tutorial Event Detection

After having generated an augmented splicing graph representation, we can now use SplAdder to detect alternative splicing events.

In default mode of SplAdder this would be done automatically. However, to step through this tutorial, we have set -T no which switched off event detection. So the only alteration we need to make now is to switch event detection back on:

$> python spladder.py -a examples/TAIR10_GFF3_genes.tiny.gff \
                      -b examples/NMD_WT1.tiny.bam,examples/NMD_WT2.tiny.bam,examples/NMD_DBL1.tiny.bam,examples/NMD_DBL2.tiny.bam \
                      -o tutorial \
                      -T y

If we now again check the output directory, many new files have appeared:

ls -1 tutorial/
merge_graphs_alt_3prime_C3.confirmed.pickle
merge_graphs_alt_3prime_C3.counts.hdf5
merge_graphs_alt_3prime_C3.pickle
merge_graphs_alt_5prime_C3.confirmed.pickle
merge_graphs_alt_5prime_C3.counts.hdf5
merge_graphs_alt_5prime_C3.pickle
merge_graphs_exon_skip_C3.confirmed.gff3
merge_graphs_exon_skip_C3.confirmed.pickle
merge_graphs_exon_skip_C3.confirmed.txt
merge_graphs_exon_skip_C3.counts.hdf5
merge_graphs_exon_skip_C3.pickle
merge_graphs_intron_retention_C3.confirmed.pickle
merge_graphs_intron_retention_C3.counts.hdf5
merge_graphs_intron_retention_C3.pickle
merge_graphs_mult_exon_skip_C3.confirmed.pickle
merge_graphs_mult_exon_skip_C3.counts.hdf5
merge_graphs_mult_exon_skip_C3.pickle
spladder

For each event type (per default these are exon skips, intron retentions and alternative 3'/5' splice site usage) a set of result files has been created. Again, the files ending in *.pickle contain internal information and should not concern us any further. All detected events have been quantified their quantification values are provided in HDF5 format. In addition to that you see that some event types have additional files containing the label confirmed. SplAdder uses the provided RNA-Seq data to verify each event in each sample. An event will be labeled as confirmed, if either of its two isoforms was supported by read data in at least one of the given samples. Note, that this does not necessarily mean that both isoforms of the event have been confirmed in the same sample.

For all confirmed events, SplAdder generates three additional output files. Again a *.pickle file which we ignore, as well as an annotation file in GFF3 format that contains the event structure for each event and a tab delimited plain text file that contains the quantification information for each confirmed event in each sample. This information is also provided in the HDF5 file, where field conf_idx identifies the events that have been confirmed. For further details on content and structure of the output files, please refer to the wiki page on file formats.

If you are only interested in one (or a subset) of the event types, you can use the option -t. For example, -t exon_skip,intron_retention would only detect and quantify exon skip and intron retention events.