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Old 09-08-2013, 08:26 AM   #1
gwilymh
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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 \
/gpfs/group1/f/flyinv/Outputs_TopHat/transcriptiomeSequence_exonsAndGeneAnnotationData/SC_JR32_Larvae/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:
http://seqanswers.com/forums/showthr...rence+GTF+file

http://seqanswers.com/forums/showthr...rence+GTF+file


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?
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Old 10-16-2013, 02:44 PM   #2
hpimentel
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Location: Berkeley

Join Date: Nov 2010
Posts: 6
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Hello there,

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

Code:
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.)

Code:
 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.


HTH
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Old 11-19-2013, 02:06 PM   #3
liweixie
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Location: Ann Arbor

Join Date: Oct 2013
Posts: 21
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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
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