my experimental design for cuffdiff

Question

[zizigolu]

Hi,

I have a paied-end RNA-seq study like below

• Arabidopsis-heat for 2 hours
• Arabidopsis-control for 2 hours
• Arabidopsis-heat for 8 hours
• Arabidopsis-control for 8 hours
• Arabidopsis-heat for 24 hours
• Arabidopsis-control for 24 hours

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

[Devon Ryan]

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.

Answer 1

[andrew.j.skelton73]

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.

[Devon Ryan]

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.

[zizigolu]

thank you both, I don;t have any biological replication only these six samples. then you mean I should better use something like deseq2?

[Devon Ryan]

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.

[zizigolu]

thank you for your explanations

[andrew.j.skelton73]

Ah damnit, I was on my phone and didn't check the manual, thanks for correcting me!