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confused about cuffdiff sample design

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

rna-seq next-gen
• 2,093 views • link
updated by Ram Ram • written by zizigolu zizigolu

2 answers

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
• 1 views • link
updated by Ram Ram • written by Devon Ryan Devon Ryan

thank you,

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

• 0 views • link
written by zizigolu zizigolu

Not with cuffdiff, no.

• 0 views • link
written by Devon Ryan Devon Ryan

thank you dear Devon

• 0 views • link
written by zizigolu zizigolu

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
• 0 views • link
updated by Ram Ram • written by GouthamAtla GouthamAtla

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