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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