Dear all,
I'm trying to detect alternative splicing events from RNA-seq experiments. I have used the NCBI SRA Tools to extract the single-end reads I want to analyse in fastqsanger format and, then, the TopHat (Galaxy version 2.1.0) to map these reads to Arabidopsis thaliana TAIR 10 genome. However, TopHat splice junctions output file has no data produced and, as a result, the software that I run cannot detect any alternative splicing event using the output .bam file. Do I need to change something in the input parameters of TopHat? I would appreciate any help.
The input parameters of TopHat run are:
Use a built in reference genome or own from your history indexed
Select a reference genome Arabidopsis_thaliana_TAIR10
TopHat settings to use full
Max realign edit distance 1000
Max edit distance 2
Library Type FR Unstranded
Final read mismatches 2
Use bowtie -n mode No
Anchor length (at least 3) 4
Maximum number of mismatches that can appear in the anchor region of spliced alignment 0
The minimum intron length 40
The maximum intron length 2000
Allow indel search Yes
Max insertion length. 3
Max deletion length. 3
Maximum number of alignments to be allowed 20
Minimum intron length that may be found during split-segment (default) search 40
Maximum intron length that may be found during split-segment (default) search 2000
Number of mismatches allowed in each segment alignment for reads mapped independently 2
Minimum length of read segments 25
Output unmapped reads False
Do you want to supply your own junction data No
Use coverage-based search for junctions Yes
Minimum intron length that may be found during coverage search 40
Maximum intron length that may be found during coverage search 2000
Use Microexon Search No
Do Fusion Search No
Set Bowtie2 settings No
Specify read group? no
Job Resource Parameters no
Best,
Thanasis
