Dear team,
I have a few questions regarding RNA-seq data analysis. I want to compare transcriptome profile of a bacterium between 5 time points, and for each time point I have three biological replicates. Right now I have each of the replicates' sequences aligned with Bowtie and assembled transcripts generates using Cufflinks. I understand that if I am just comparing two time points (if each of time point has only one sample, no replicate) I just need to cuffmerge the two time points and cuffdiff the merged file.
My question is, if I need to compare between 5 time points, should I do comparison pairwise?
And also, how should I include three biological replicates for each time point? I can only find 'include replicates' in Cuffdiff. Could you check is the following methodology correct?
let's say I have samples in time 0 , 1hour, 2hours, 3 hours and 4 hours. I will name each of the replicates as 0hour-1, 0hour-2, 0hour-3,etc.
I will first compare expression between 0hour and 1hour.
I will use cuffmerge to merge 0hour-1, 0hour-2, 0hour-3, 1hour-1,1hour-2.1hour-3 to generate one cuffmerge file. Then I will run cuffdiff using the merged file, include two groups, group 1 is 0 hour (add 0hour 1-3 in group 1) and group 2 is 1hour (add 1hour1-3 in group 2).
Does this sound reasonable?
Thank you very much,
Qian