Hi,
I have a paied-end RNA-seq study like below
I am running cuffdiff like below
cuffdiff -o diff_out -b genome.fa -p 8 -u merged_asm/merged.gtf 204.bam, 205.bam, 206.bam, 207.bam, 208.bam, 209.bam
at first telling
Warning: No conditions are replicated, switching to 'blind' dispersion method
then would heat treatment in time points be compared considering controls?
Thank you
If I remember right, it's commas between replicates, spaces between phenotypes. So your command should be:
cuffdiff -o diff_out -b genome.fa -p 8 -u merged_asm/merged.gtf 204.bam,205.bam,206.bam 207.bam,208.bam,209.bam
If 204/5/6 are phenotype A, and 207/8/9 are phenotype B
edit: Devon was right, lesson learnt, always check the manual first.
•
written
by
andrew.j.skelton73
Spaces between groups, commas and no spaces between replicates :) So:
cuffdiff -o diff_out -b genome.fa -p 8 -u merged_asm/merged.gtf 204.bam,205.bam,206.bam 207.bam,208.bam,209.bam
That makes parsing it a bit simpler.
Without replicates you'll get questionable results regardless of what you use (I don't know if DESeq2 will even allow this...I suspect not). If you happen to want to use a model like ~treatment+time where time is a numeric value then you could still use DESeq2 or something similar, though you'd need to recreate the count values. If that's not the case (likely), then DESeq2 won't do anything for you.
Ah damnit, I was on my phone and didn't check the manual, thanks for correcting me!
•
written
by
andrew.j.skelton73
Log in to answer this question.
cuffdiff doesn't really support very complicated designs. In your case it'll do pairwise comparisons between the samples (you don't have biological replicates?). If you need something more complicated then you'll need to use a different tool.