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 Dong
Bauer Lab, MCBD
Simon Hall: 313-317
212 S. Hawthorne Dr.
Bloomington, IN 47405
Lab Phone:812-855-8443
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