running cuffdiff for 2 conditions without biological replicates

Question

[hana]

Hi

I want to run cuffdiff for analysis of 2 samples which have different conditions (control , disease) and without biological replicates. I would like to know is it correct that before running cuffdiff , I use cuffmerge and merge the transcripts.gtf files of 2 samples (assembled with cufflinks) with references annotations file?

Thank you

[hana]

l want just to learn how can run cuffdiff correctly. I will have more biological replicates later. For this 2 current samples:

First, I used cuffmerge to merge the gtf files form cufflinks and then run cuffdiff. I got 71 sig genes.

In second run I just use cuffdiff with gtf annotation files and accepted_hits files (without gtf files from cufflinks) and got 412 sig genes.

the command that I used is:

cuffdiff \
  -L A,B \
  -o /home/cuffdiff \
  /home/Homo_sapiens_UCSC_hg19/Homo_sapiens/UCSC/hg19/Annotation/Archives/archive-2014-06-02-13-47-56/Genes/genes.gtf \
  /home/A/accepted_hits.bam /home/B/accepted_hits.bam

I would like to know which one is correct?

Thanks in advance

[Devon Ryan]

Both are correct, but the first result is probably more reliable, due to better matching the actual gene models used.

Answer 1

[Devon Ryan]

Yes, you run cuffmerge and merge the GTF files beforehand (do specify a reference annotation if you have one).

BTW, don't read too much into the results. A 1 vs. 1 comparison will never produce reliable p-values or fold-changes.

[hana]

Thank you Devon for your reply. l want just to learn how can run cuffdiff correctly. I will have more biological replicates later. For this 2 current samples:

First, I used cuffmerge to merge the gtf files form cufflinks and then run cuffdiff. I got 71 sig genes.

In second run I just use cuffdiff with gtf annotation files and accepted_hits files (without gtf files from cufflinks) and got 412 sig genes.

the command that I used is:

cuffdiff \
  -L A,B \
  -o /home/cuffdiff \
  /home/Homo_sapiens_UCSC_hg19/Homo_sapiens/UCSC/hg19/Annotation/Archives/archive-2014-06-02-13-47-56/Genes/genes.gtf \
  /home/A/accepted_hits.bam /home/B/accepted_hits.bam

I would like to know which one is correct?

Thanks in advance