I'm trying to use cufflinks and related programs (cuffmerge and cuffdiff) to find differentially expressed genes and some splicing events between two conditions (3 replicate for each sample) for a plant. This is my first experience and just based on my reading, I decided to use cufflinks for transcript assembly along with GTF file as a guide (-g option in cufflinks) for each bam file, then merge all assembly files together and with reference annotation (GTF file) to make a single annotation as the input for cuffdiff. Could you please help me on the following issue:
1) is right the above pipeline? 2) The default value of "--overlap-radius" in cufflinks is 50, please kindly tell me to go ahead with default or reduce it (what number suggest)? 3) The experiment was done at 4 time points, so for cuffdiff analysis, I should just "-T" option in the command, is there anything to care about it?
Thanks so much