DiffSplice: Interpreting DiffSplice Results

 

This page is under active construction.

 

Main result files

1. Splice graph and ASMs

 result/splice_graph.gtf and result/asm_path.gtf โ€” gtf tracks for the splice graph and alternative ASM paths.


2. Differentially expressed/transcribed regions

 result/differential_expression.txt โ€” summary of differential expression level on genes

chromosome chromosome name of the differentially expressed gene
position_start start position of the differentially gene
position_end end position of the differentially gene
stat_diff_expr(|stat_d_expected-stat_d|) statistic of differential gene expression, larger value indicates greater difference (statistical difference)     
fold_change fold change from group1 to group2 (practical difference)
coverage_group1 estimated gene expression of group1, measured by read coverage
coverage_group2 estimated gene expression of group2, measured by read coverage
significant whether the difference is significant under the specified false discovery rate

 result/differential_transcription.txt โ€” summary of differential transcription on ASMs

chromosome chromosome name of the differentially transcribed ASM
position_start start position of the differentially transcribed ASM
position_end end position of the differentially transcribed ASM
category category of the ASM
stat_diff_trans(|stat_d_expected-stat_d|) statistic of differential ASM transcription, larger value indicates greater difference (statistical difference) 
sqrtJSD square root of JSD between group1 and group2 (practical difference), larger value indicates great difference
coverage_group1 estimated ASM expression of group1, measured by read coverage
coverage_group2 estimated ASM expression of group2, measured by read coverage
significant whether the difference is significant under the specified false discovery rate

 

3. False discovery rate table

 FDR_expression_two_group_comparison.txt and FDR_transcription_two_group_comparison.txt

The statistical significance of the differential signals is measured by the difference between the expected and calculated divergence statistic, |stat_d_expected - stat_d|, and a differential signal will be called significant if such difference is larger than some cutoff delta. Therefore, the selection of delta will affect the set of genes/ASMs called significant and will affect the false discovery rate (FDR). To assist your choice of FDR, we shift the value of delta and provide the number of significant genes/ASMs you will have at different levels of delta. You may choose your desired FDR according to this list and give to DiffSplice (need to rerun the differential test step, specified by -x option, to see the updated results).

 

Result tracks on UCSC genome browser

To view result tracks on UCSC genome browser, please take the following steps:

 1. Upload the two gtf tracks generated by DiffSplice (splice_graph.gtf and asm_path.gtf), either through browser or public server. You may also upload the wiggle files or bigwig files of you data if you have generated them from the SAM/BAM files;

 2. Select the region of interest from differential_expression.txt for differentially expressed genes or from differential_transcription.txt for differentially transcribed ASMs;

 3. Paste in the region to genome browser and it will lead you to the place with differential expression/transcription;

 4. If you are checking a place with differential transcription, the location provided in differential_transcription.txt gives the coordinates of the ASM, not including the surrounding exons. You may zoom out to see the surrounding exons and the whole gene.

Below is an example.

                                  

The track given by splice_graph.gtf shows the part of the splice graph that is shared by all transcripts in this gene. The track given by asm_path.gtf then shows the alternative splicing variants in each alternative splicing event. For the example shown above, there are two alternative splicing events detected in this gene: ASM1 indicates 4 alternative transcription start sites and ASM2 indicates an exon-skipping event. You may find more examples and explanation in the DiffSplice article (link listed on our website).
 

 

Other result files

 

1. Splice junctions from RNA-seq alignments

result/stat/splice_all.bed all splice junctions from the input RNA-seq alignments    
result/stat/splice_filtered.bed splice junctions after filtering