Cuffdiff - results replicates

[adrian]

Hi:

I have 4 different cell types. A , B, C and D. Each class has 4 replicates.

In Cuffdiff, I gave replicates with comma separated lines

A1.bam, A2.bam, A3.bam,A4.bam B1.bam,b2.bam,b4.bam ... and so on.


From gene_exp.diff, I wanted to get list of differentially expressed genes.
I observe that cuffdiff also compared q1 q2 (which is a1.bam a2.bam).

why cuffdiff comparing between two replicates. Could anyone provide their expert input.

Also is this the only file to look for differentially expressed genes?

Thanks
Adrian

[Bukowski]

q1 and q2 will be the replicate groups, not the sample id's if you've specified your replicate groupings correctly.

Use -L to specify labels instead

genes_exp.diff is the gene level summaries of the differential expression testing between conditions.

The other diff files are summaries of the differential expression at transcript leve, tss level, cds level etc.

[adrian]

Hi :
Thanks for your reply.

A1 A2 A3 B1 B2 B3 C1 C2 C3 D1 D2 D3

If q1, q2, Q3 and Q4 are replicate groups, I should not get anything above q4 since I have only 4 replicate groups. However, I am seeing in columns Sample_1 and Sample_2:

q1 q4

q1 q8

q1 q9

q1 q12

etc.

This lead me to think that cuffdiff is comparing every replicate sample to other samples in other groups and reporting the significant FDR (yes or no calls).

Yes, I did not specify -L option.


Is the following correct syntax for -L option?

cuffdiff combined.gtf -L sample1, sample2, sample3 and sample4 a1.bam,a2.bam,a3.bam b1.bam,b2.bam,b3.bam c1.bam,c2.bam,c3.bam d1.bam,d2.bam,d3.bam


Thanks a lot again.

-Adrian

[DonDolowy]

cuffdiff combined.gtf -L sample1,sample2,sample3,sample4 a1.bam,a2.bam,a3.bam b1.bam,b2.bam,b3.bam c1.bam,c2.bam,c3.bam d1.bam,d2.bam,d3.bam

This should be the right one.

[adrian]

Thanks a lot for your reply.

I have one more question:

Is is okay if I do cuffdiff only on two replicate groups.

Say cuffdiff between a1,a2,a3 and b1,b2,b3 only.

since my cuffmerge gtf assembly file is made from all samples in the experiment, will I loose power by only comparing two replicate groups and not all.

Thanks again.

-Adrian