Cuffdiff Error- return value of zero and empty output files

Question

[Lindsay44]

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?

[GenoMax]

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

[Lindsay44]

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?

[GenoMax]

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.

[Lindsay44]

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.