confused about cuffdiff sample design

Question

[zizigolu]

Hi,

In my experiment, before high temperature treatment (48 c), Arabidopsis cotyledons were pre-heated for 2, 8 and 24 hours. In the other hand I have cotyledons keeping in room temperature for 2, 8 and 24 hours, I mean without pre-heating these cotyledons directly face the high temperature. I don't have any replication

Something like below

• pre-heated-2h
• pre-heated-8h
• pre-heated-24h
• unpre-heated-2h
• unpre-heated-8h
• unpre-heated-24h

I got totally confused if I should use this syntax in cuffdiff

cuffdiff \
  -o diff_out \
  -b genome.fa \
  -p 8 \
  -u merged_asm/merged.gtf \
  pre-heated-2h.bam, pre-heated-8h.bam, pre-heated-24h.bam, unpre-heated-2h.bam, unpre-heated-8h.bam, unpre-heated-24h.bam

or

cuffdiff \
  -o diff_out \
  -b genome.fa \
  -p 8 \
  -L pre,unpre \
  -u merged_asm/merged.gtf \
  pre-heated-2h.bam, pre-heated-8h.bam, pre-heated-24h.bam, unpre-heated-2h.bam, unpre-heated-8h.bam, unpre-heated-24h.bam

Using the first syntax the cummerbund result was nonsense..and the number of differentially expressed genes was 0, how possible

I am going to know the effect of pre-heating on heat stress tolerance

Please help me to be clear a little bit

Thank you

Answer 1

[Devon Ryan]

Assuming there's no effect due to duration of treatment (or that it's minimal):

cuffdiff -o diff_out -b genome.fa -p 8 -L pre,unpre -u merged_asm/merged.gtf  pre-heated-2h.bam,pre-heated-8h.bam,pre-heated-24h.bam unpre-heated-2h.bam,unpre-heated-8h.bam,unpre-heated-24h.bam

[zizigolu]

thank you,

sorry, no way to consider the effect of time points too?

[Devon Ryan]

Not with cuffdiff, no.

[zizigolu]

thank you dear Devon

Answer 2

[GouthamAtla]

You should at least put some effort and time (maybe 10 min ?) to read the manual and copy paste?

If you look at the manual, it clearly states:

cuffdiff [options]* <transcripts.gtf> \
<sample1_replicate1.sam[,...,sample1_replicateM.sam]> \
<sample2_replicate1.sam[,...,sample2_replicateM.sam]> ... \
[sampleN.sam_replicate1.sam[,...,sample2_replicateM.sam]]

If you apply that exact command to your data:

cuffdiff -o diff_out -b genome.fa -p 8 -L pre,unpre -u merged_asm/merged.gtf \
pre-heated-2h.bam,pre-heated-8h.bam,pre-heated-24h.bam \
unpre-heated-2h.bam,unpre-heated-8h.bam,unpre-heated-24h.bam