Cufflinks (Cuffdiff) v0.9.3 multiple samples

[keebs42]

Hi all,

I'm running through the cufflinks-cuffcompare-cuffdiff pipeline using three samples, but for some reason cuffdiff is only producing one of each kind of differential expression file (*_exp.diff) rather than one for each pair of samples. In the *.fpkm_tracking files, all 3 samples are showing up (q1,q2,q3) but I'm only getting expression files comparing q1 and q2. Any ideas?? Here's the basics of the cuffdiff command I'm using:

cuffdiff -q -r ref.fa cuffcompare_6hr.4/stdout.combined.gtf tophat_out_1/sample1.bam tophat_out_2/sample2.bam tophat_out_3/sample3.bam

Anyone else running cuffdiff on >2 samples using the latest version?

Thanks!

[roryk]

Hi all,

I'm running through the cufflinks-cuffcompare-cuffdiff pipeline using three samples, but for some reason cuffdiff is only producing one of each kind of differential expression file (*_exp.diff) rather than one for each pair of samples. In the *.fpkm_tracking files, all 3 samples are showing up (q1,q2,q3) but I'm only getting expression files comparing q1 and q2. Any ideas?? Here's the basics of the cuffdiff command I'm using:

cuffdiff -q -r ref.fa cuffcompare_6hr.4/stdout.combined.gtf tophat_out_1/sample1.bam tophat_out_2/sample2.bam tophat_out_3/sample3.bam

Anyone else running cuffdiff on >2 samples using the latest version?

Thanks!

I think if you look carefully at the .diff files, you will see that they are doing all pairwise comparisons for each sample (q1 vs q2, q2 vs q3, etc).

[keebs42]

Yep.. you're right. Just didn't check down within the file. Thanks for the tip!

[jp.]

Hi
I got problem with cuffdiff. I am working of Linuxe server and when give commands:
cuffdiff -o diff_out -b genome.fa -p 8 –L 1,2 -u merged_asm/merged.gtf \
./1_R1_thout/accepted_hits.bam,./1_R2_thout/accepted_hits.bam,./1_R3_thout/accepted_hits.bam \
./2_R1_thout/accepted_hits.bam,./2_R_thout/accepted_hits.bam,./2_R3_thout/accepted_hits.bam
Error: cannot open alignment file

When I changed (removed \ and ./):
cuffdiff -o diff_out -b genome.fa -p 8 –L 1,2 -u merged_asm/merged.gtf /1_R1_thout/accepted_hits.bam /2_R1_thout/accepted_hits.bam
It goes well.
I can not add replicates. any idea ?
Thank you

Yep.. you're right. Just didn't check down within the file. Thanks for the tip!

[moredd]

If you have more than one replicate for a sample, supply the SAM files for the sample as a single comma-separated list. It is not necessary to have the same number of replicates for each sample, says the manual @ http://cufflinks.cbcb.umd.edu/manual.html#cuffdiff