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Old 09-08-2013, 08:26 AM   #1
Location: Milwaukee

Join Date: Dec 2011
Posts: 72
Unhappy cuffDiff "cannot open reference GTF file"

I am running a series of Tuxedo pipelines and have run into the following error message a number of times in CuffDiff "cannot open reference GTF file".

The GTF file was generated in CuffMerge and looks completely normal. Here are some lines to illustrate this:
Ch2 Cufflinks exon 2735 6095 . + . gene_id "XLOC_000001"; transcript_id "TCONS_00000001"; exon_number "1"; oId "CUFF.3.1"; class_code "u"; tss_id "TSS1";
Ch2 Cufflinks exon 6172 6607 . + . gene_id "XLOC_000001"; transcript_id "TCONS_00000001"; exon_number "2"; oId "CUFF.3.1"; class_code "u"; tss_id "TSS1";
Ch2 Cufflinks exon 7279 7433 . + . gene_id "XLOC_000001"; transcript_id "TCONS_00000001"; exon_number "3"; oId "CUFF.3.1"; class_code "u"; tss_id "TSS1";
Ch2 Cufflinks exon 7494 10061 . + . gene_id "XLOC_000001"; transcript_id "TCONS_00000001"; exon_number "4"; oId "CUFF.3.1"; class_code "u"; tss_id "TSS1";

I can find no error in my input command either:
cuffdiff \
-o "/gpfs/group1/f/flyinv/Outputs_CuffDiff/ExonsGenes/SCJR32_a" \
-L SC_JR32_Male,SC_JR32_Female,SC_JR32_Larvae \
--total-hits-norm \
--frag-bias-correct /gpfs/group1/f/flyinv/working_index/Dpse3_0_1.fa \
--multi-read-correct \
--library-norm-method classic-fpkm \
/gpfs/group1/f/flyinv/Outputs_CuffMerge/exonGene/SCR32_a/merged.gtf \
/gpfs/group1/f/flyinv/Outputs_TopHat/transcriptiomeSequence_exonsAndGeneAnnotationData/SC_JR32_Male/accepted_hits.bam \
/gpfs/group1/f/flyinv/Outputs_TopHat/transcriptiomeSequence_exonsAndGeneAnnotationData/SC_JR32_Female/accepted_hits.bam \

I have found other threads where the same problem has been encountered, but there seems to be no real explanation or solution:

One possible cause of this problem could be the GFF3 and GTF files used to guide the upstream TopHat and Cufflinks and CuffMerge analyses. TopHat and CuffLinks were run using a GFF3 file that contained the known gene and exon annotations for the target species. A GFF file generated by TopHat from the same data was used to guide the CuffMerge analyses. I don't see what difference this would make, but could this have somehow caused the error?
gwilymh is offline   Reply With Quote
Old 10-16-2013, 02:44 PM   #2
Junior Member
Location: Berkeley

Join Date: Nov 2010
Posts: 6

Hello there,

Hope this doesn't sound patronizing, but do you have permission to read the GTF file?

ls -lh /gpfs/group1/f/flyinv/Outputs_CuffMerge/exonGene/SCR32_a/merged.gtf
will show you permissions. Alternatively, you can just check if you can read the file by using a program to read the file (e.g. head, less, more, tail, etc.)

 head ls -lh /gpfs/group1/f/flyinv/Outputs_CuffMerge/exonGene/SCR32_a/merged.gtf
If you don't have permission, you should change them using the 'chmod' tool.

hpimentel is offline   Reply With Quote
Old 11-19-2013, 02:06 PM   #3
Location: Ann Arbor

Join Date: Oct 2013
Posts: 21

I think you miss a . before / for your bam or gtf file. It happens on me once and later I figure out it is the wrong directory I put into the cuffdiff command. That is why cuffdiff can not read gtf file
liweixie is offline   Reply With Quote

cuffdiff, cufflink, cuffmerge, rnaseq, top hat

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