Question: Cuffdiff Error- return value of zero and empty output files
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gravatar for Lindsay44
4.4 years ago by
Lindsay4420
Lindsay4420 wrote:

I am trying to run Cuffdiff to conduct a differential expression analysis. I am running the following command (I am using two data sets as a sample, one from each experimental group, until I can get it to run correctly):

cuffdiff  c:/research/Cuffmerge/merged_asm/merged.gtf c:/research/alignment_data/TB1_alignment_sorted.sam c:/research/alignment_data/NT1_alignment_sorted.sam

I keep on receiving empty output files either with or without an error message (depending on small changes I make to the script). I tried troubleshooting quite a bit- I am running the command with different arguments and also made sure that the transcripts were properly labeled with tss_id and p_id.

I broke up the command to try to find the problem:

I first ran it without any labels. The output is a set of empty files and the following command line message:

You are using Cufflinks v2.2.1, which is the most recent release.

Then I ran the script without the paths and got this error message:

You are using Cufflinks v2.2.1, which is the most recent release. 
open: No such file or directory 
Error: cannot open file TB1_alignment_sorted.sam for reading. Unrecognized file type

Some outputs also make it seem like the second file isn't being detected:

cuffdiff requires at least 2 SAM files

The return value on these commands is 0- so I don't know why they aren't being executed properly.

What is going wrong?

rna-seq cuffdiff • 1.7k views
ADD COMMENTlink modified 3.0 years ago by Biostar ♦♦ 20 • written 4.4 years ago by Lindsay4420

Are you running this on cygwin?

You want to run this as (label 1 and label 2 are the two groups you are comparing, sample1/2 are for label1 and sample3/4 for label2, note the comma separation within labels and then space separation between labels for the file names)

cuffdiff (options) -L label1,label2 -u file.gtf sample1.bam,sample2.bam sample3.bam,sample4.bam

something like

cuffdiff  -L TB,NT -u c:/research/Cuffmerge/merged_asm/merged.gtf c:/research/alignment_data/TB1_alignment_sorted.sam c:/research/alignment_data/NT1_alignment_sorted.sam
ADD REPLYlink modified 4.4 years ago • written 4.4 years ago by GenoMax94k

I am using cygwin.

I ran the following command and recieved the same empty output files:

cuffdiff  -L TB,NT -u c:/research/Cuffmerge/merged_asm/merged.gtf c:/research/alignment_data/TB1_alignment_sorted.sam c:/research/alignment_data/NT1_alignment_sorted.sam

What could be going wrong?

ADD REPLYlink modified 4.4 years ago by _r_am32k • written 4.4 years ago by Lindsay4420

Add --verbose option when you run that command to get additional log information from cuffdiff. That may give an idea of what is going wrong.

ADD REPLYlink written 4.4 years ago by GenoMax94k

I tried that already. This is the only command line output, and the files are all still empty:

You are using Cufflinks v2.2.1, which is the most recent release.

ADD REPLYlink written 4.4 years ago by Lindsay4420
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