Hi List,

I have a user running a Tuxedo pipeline on our local Galaxy but it has been 
fraught with errors.

The first issue was with running cufflinks with an annotation file which had 
duplicate IDs - fixed by me running the following:
awk '($3 == "exon" || $3 == "CDS")' dataset_8640.dat >> newref
Just tossing out the transcript and gene lines seemed to help.

Second issue was Error: sequence lines in a FASTA record must have the same 
length!
Converting to and from tabular fixed that.

Now the issue is with cuffmerge. The user has run Tophat on a pair of fastq 
files and a fasta genome. They then ran cufflinks on the Tophat assembly with 
an annotation file (not the previously mentioned one). This worked. But using 
the gtf file produced by cufflinks in a cuffmerge step results in:
Error running cuffmerge. 
[Sat Mar 22 14:00:10 2014] Beginning transcriptome assembly merge
-------------------------------------------

[Sat Mar 22 14:00:10 2014] Preparing output location cm_output/
[Sat Mar 22 14:02:37 2014] Converting GTF files to SAM
[14:02:38] Loading reference annotation.
Error: duplicate GFF ID 'CDS:GBG_brugia_K07A12.4b' encountered!
        [FAILED]
Error: could not execute gtf_to_sam
I took out the sequence and annotation file in the cuffmerge step with no 
change in result. I ran gffread on the cufflinks output and sure enough, it 
explodes. But why would Cufflinks create an invalid file?

The file itself has these entries:
Bmal_v3_scaffold1   Cufflinks   transcript   280476  280630  1   +   .   
gene_id ""; transcript_id "CDS:GBG_brugia_K07A12.4b"; FPKM "0.0000000000"; frac 
"0.000000"; conf_lo "0.000000"; conf_hi "0.000000"; cov "0.000000";
Bmal_v3_scaffold1   Cufflinks   exon   280476  280630  1   +   .   gene_id ""; 
transcript_id "CDS:GBG_brugia_K07A12.4b"; exon_number "1"; FPKM "0.0000000000"; 
frac "0.000000"; conf_lo "0.000000"; conf_hi "0.000000"; cov "0.000000";

Bmal_v3_scaffold1   Cufflinks   transcript   281149  281207  1   +   .   
gene_id ""; transcript_id "CDS:GBG_brugia_K07A12.4b"; FPKM "0.0000000000"; frac 
"0.000000"; conf_lo "0.000000"; conf_hi "0.000000"; cov "0.000000";
Bmal_v3_scaffold1   Cufflinks   exon   281149  281207  1   +   .    gene_id ""; 
transcript_id "CDS:GBG_brugia_K07A12.4b"; exon_number "1"; FPKM "0.0000000000"; 
frac "0.000000"; conf_lo "0.000000"; conf_hi "0.000000"; cov "0.000000";

# Shortening for brevity:
Bmal_v3_scaffold1   Cufflinks   transcript   281521  281622  1  ...
Bmal_v3_scaffold1   Cufflinks   exon    281521  281622  1  ...

Bmal_v3_scaffold1   Cufflinks   transcript   281743  281863  1  ...
Bmal_v3_scaffold1   Cufflinks   exon   281743  281863  1  ...

Bmal_v3_scaffold1   Cufflinks   transcript   282355  282537  1  ..
Bmal_v3_scaffold1   Cufflinks   exon   282355  282537  1  ...

Bmal_v3_scaffold1   Cufflinks   transcript   283063  283190  1  ...
Bmal_v3_scaffold1   Cufflinks   exon   283063  283190  1  ...

Bmal_v3_scaffold1   Cufflinks   transcript   283879  284035  1  ...
Bmal_v3_scaffold1   Cufflinks   exon   283879  284035  1  ...

Bmal_v3_scaffold1   Cufflinks   transcript   280652  280683  1  ...
Bmal_v3_scaffold1   Cufflinks   exon   280652  280683  1  ...

Here's my setup:
Galaxy changeset: dc067a95261d was my last pull

$ cuffmerge --version
merge_cuff_asms v1.0.0 
$ cufflinks 
cufflinks v2.2.0
linked against Boost version 104700
$ tophat --version
TopHat v1.3.3

Using tools:
Cuffdiff devteam revision: 604fa75232a2
Cufflinks devteam revision: 9aab29e159a7
Cuffmerge devteam revision: 424d49834830
Tophat devteam revision: 1030acbecce6 

Has anyone else seen this? I'm re-running the workflow from scratch but I don't 
really have any leads.

Sincerely,

Carrie Ganote
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