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. SplAdder in its default configuration, 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
  • quantify all alternative splicing events on each of the provided alignment files

SplAdder is invoked using the following:

  1. Input (s)
    1. The folder containing the list of alignment files (bam files)
    2. The genome annotation file (gtf file)
  2. Parameters
    1. Alternative Splicing event type (Default is Exon_skip, Intron_retention, alternate_3',alternate_5', multiexon_skip):
      • Exon_skip
      • Intron_retention
      • alternate_3'
      • alternate_5'
      • multiexon_skip
    2. Confidence level (Default is 3)
    3. Primary_alignments_only (Default is no)
    4. Merge Strategy (Default is merge_graphs)
  3. Outpus
    1. An output directory where results files are stored (Default is Output). 

Please work through the documentation and add your comments on the bottom of this page, or email comments to support@cyverse.org. Thank you.

Test Data

 The test data for Spladder-1.0.0 consists of 6 bam files and an annotation files from Arabidopsis thaliana 

All files are located in the Community Data directory of the iPlant Discovery Environment at the following path: Community Data > iplantcollaborative > example_data > spladder (/iplant/home/shared/iplantcollaborative/example_data/spladder/)

Run SplAdder as following:

  1. Input (s)
    1. The folder containing the list of alignment files - /iplant/home/shared/iplantcollaborative/example_data/spladder/
    2. The genome annotation file (/iplant/home/shared/iplantcollaborative/example_data/spladder/TAIR10_GFF3_genes.tiny.gff)
  2. Parameters
    1. Alternative Splicing event type - Exon_skip, Intron_retention, alternate_3',alternate_5', multiexon_skip
    2. Confidence level - 3
    3. Primary_alignments_only - no
    4. Merge Strategy - merge_graphs
  3. Outputs
    1. Output folder containing the following files

merge_graphs_exon_skip_C3.confirmed.txt contains the following output

contig	strand	event_id	gene_name	exon_pre_start	exon_pre_end	exon_start	exon_end	exon_aft_start	exon_aft_end	NMD_DBL1.tiny:exon_pre_cov	NMD_DBL1.tiny:exon_cov	NMD_DBL1.tiny:exon_aft_cov	NMD_DBL1.tiny:intron_pre_conf	NMD_DBL1.tiny:intron_aft_conf	NMD_DBL1.tiny:intron_skip_conf	NMD_DBL1.tiny:psi	NMD_DBL2.tiny:exon_pre_cov	NMD_DBL2.tiny:exon_cov	NMD_DBL2.tiny:exon_aft_cov	NMD_DBL2.tiny:intron_pre_conf	NMD_DBL2.tiny:intron_aft_conf	NMD_DBL2.tiny:intron_skip_conf	NMD_DBL2.tiny:psi	NMD_WT1.tiny:exon_pre_cov	NMD_WT1.tiny:exon_cov	NMD_WT1.tiny:exon_aft_cov	NMD_WT1.tiny:intron_pre_conf	NMD_WT1.tiny:intron_aft_conf	NMD_WT1.tiny:intron_skip_conf	NMD_WT1.tiny:psi	NMD_WT2.tiny:exon_pre_cov	NMD_WT2.tiny:exon_cov	NMD_WT2.tiny:exon_aft_cov	NMD_WT2.tiny:intron_pre_conf	NMD_WT2.tiny:intron_aft_conf	NMD_WT2.tiny:intron_skip_conf	NMD_WT2.tiny:psi
Chr1	+	exon_skip_1	AT1G21690	7616028	7616107	7616267	7616332	7616604	7616726	32.4	10.2	39.3	3	18	24	0.30	63.6	11.1	59.1	7	8	52	0.13	33.9	0.2	60.6	0	0	49	0.00	74.2	0.5	71.0	0	0	72	0.00


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