so apparently, I have a problem with running tuxedo protocol.
Now I have run tophat on my data with novel junctions function enabled (as default) as well as supplying reference annotation gtf file as -G. My data is paired end run RNA-seq. Then I have run cufflinks transcriptome assembly sample by sample on each accepted_hits.bam file, also with supplying cufflinks with the same reference annotation gtf file as -g.
Further , I merged all the transcripts from my samples with supplying cuffmerge with the reference annotation one more time as -g.
So at the end I have added it 3 times, in tophat, cufflinks,cuffmerge along the protocol.
However, the output of cuffmerge is not as expected.
I understand that it did repeat some exons shared among different isoforms either assembled by cufflinks or it was available in reference annotation.
1. Why did cuffmerge annotates all exons as their source is " cufflinks" ?! however some exons are surely form the reference annotation?
did I make a mistake supplying the reference annotation too many times in the protocol? should I really modify one of those teps to get what I want i.e list of annotated exons and novel exons with no repetition in one file, with the novel exons identified as novel exons?