Cuffdiff for differential gene expression

Question

[mathanyes]

Hi

I wanted to do differential gene expression testing using RNA-seq data.I have 4 groups. 1 control and 3 diseased.

In each group I have 3 or 4 samples.

G1-4 samples (Control)

G2- 4 Samples (Disease 1)

G3- 3 Samples (Disease 2)

G4- 4 Samples (Disease 3)

In cuffdiff, I have uploaded the combined transcript file from cuffcompare.

Then for the conditions, I have 15 BAM files for 15 samples. In each condition I uploaded each sample (so total no. of conditions would be 15).

I am getting the following error.

Fatal error: Exit code 134 ()
[03:39:08] Loading reference annotation and sequence.
Warning: No conditions are replicated, switching to 'blind' dispersion method
[03:39:31] Inspecting maps and determining fragment length distributions.
[04:02:57] Modeling fragment count overdispersion.

Actually I wanted to download the gene FPKM tracking file from cuffdiff and do the differential gene expression analysis using BRB array tools.

Please help how to solve this.

[HaroonPakistan]

Hi, was your query solved?

Answer 1

[Prakash]

I think you should go like this:

For each condition you generate transcript.gtf

cufflinks -o output_file -G reference_GTF_file bam_file

Then merge all the transcript.gtf of condition for which you want differential gene expression.

cuffmerge -o output_file -g reference_GTF_file transcript.gtf_list

transcript.gtf_list should contain list of all the transcript.gtf to be merged.

then use cuffdiff for differential gene expression.

cuffdiff -o output file merged.gtf bam1 bam2 bam3 .....bamN

bam1, bam2, bam3 are the files of all the condition you want to compare.

Here you will get your gene.fpkm_tracking file.

I hope this would help.