I would like to assemble transcripts of several chromosomes. I do have sequences of these chromosomes from related species and would therefore like to do reference-guided transcriptome assembly.
I have two concerns:
1, which programs should I use? I have read about Cufflinks and have very little experience with it but it seems to provide only gtf file instead of sequences of all my isoforms. Since my reference is related species, I do think that differencies will be too big to be expressed just with gtf/bed file.
Is velvet's columbus option? What program would you recommend?
2, if I use as reference sequences from my related species, their gene content will vastly overlap. I suppose that this will make a lot of my reads impossible to align uniquely. Should I then use only one species reference each time?
Thanks a lot for advice.