I am working on fungi transcriptome. Existing annotation for my species is not reliable. To improve the annotation we generated RNAseq data with different stress conditions to make all transcripts to express in different conditions and using those expressed transcripts in different conditions I am trying to build one comprehensive transcriptome using cufflink protocol.
I ran cufflink and cuffmerge with and without reference in following combinations.
1) cufflink with reference cuffmerge with reference 2)cufflink with reference cuffmerge without reference 3)cufflink without reference cuffmerge with reference 4)cufflink without reference cuffmerge without reference
All of them has it's pros and cons. By doing some manual observation on IGV i decided to go with 4th combination which is cufflink without reference cuffmerge without reference. Still I am not satisfied the way it did annotations
For example consider following scenario (attached image). Though I have clearly 3 transcripts expressed in all my conditions (shown in the green box) none of the above combinations of cufflink and cuffmerge could detect the true transcript structure.
Can anyone tell why this situation ? Also suggest if any better option available than cufflink cuffmerge.