Question: Solution For: Error Running Cuffdiff. Error: Cannot Open Reference Gtf File Condition, Control For Reading
0
Carlos Borroto • 390 wrote:
Hi,
I ran into this error running cuffdiff. I had a hard time debugging
this user error, so I though it would be nice to share the solution.
This was in a local instance, but I don't see why it wouldn't happen
in Galaxy Main under the same circumstances.
Tool execution generated the following error message:
Error running cuffdiff. Error: cannot open reference GTF file
CONDITION,CONTROL for reading
The tool produced the following additional output:
cuffdiff v1.3.0 ()
cuffdiff --no-update-check -q -p 4 -c 1000 --FDR 0.050000 -N -b
--labels CONDITION,CONTROL
/local/db/genomes/illumina/Homo_sapiens/Ensembl/GRCh37/Annotation/Gene
s/genes.gtf
/local/opt/galaxy_central/database/files/001/dataset_1339.dat,/local/o
pt/galaxy_central/database/files/001/dataset_1391.dat
/local/opt/galaxy_central/database/files/001/dataset_1452.dat,/local/o
pt/galaxy_central/database/files/001/dataset_1478.dat
The problem ended being the use of "Perform Bias Correction"(-b) and a
GTF file with no "Database/Build" associated. Looking at cuffdiff
wrapper I found, if a FASTA reference is not selected from the
history, the FASTA reference of the GTF file associated build is used.
If there is not build association, your cuffdiff run will fail with
this not so helpful error.
My feeling is, cuffdiff should check for a non-dashed string after
'-b' and complain if is absents, but this doesn't happen currently.
Kind regards,
Carlos
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modified 6.8 years ago
by
Jeremy Goecks • 2.2k
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written
6.8 years ago by
Carlos Borroto • 390
Hi, I'm getting the same error but don't know how to solve it. My aim is to obtain the per gene & transcript FPKM count matrices to perform analysis using R packages. My analysis steps are: Hisat2 > Cufflinks > Cuffmerge > Cuffquant > Cuffnorm *Cuflinks version 2.2.1
Cufflinks (using UCSC hg38 annotation & sequence)
command ran on each library
cufflinks -o ./cufflinks --GTF genes.gtf --frag-bias-correct genome.fa --multi-read-correct --total-hits-norm --max-bundle-frags 10000000 library1.hisat2aligned.sorted.sam
Cuffmerge
cuffmerge -o ./cuffmerge -g genes.gtf -s genome.fa list_GTF.txt # (GFT list file from Cufflinks outputs)
Cuffquant
command ran on each library
cuffquant -o ./cuffquant merged.gtf --frag-bias-correct genome.fa --multi-read-correct library1.hisat2aligned.sorted.sam
Cuffnorm
cuffnorm -o ./cuffnorm -L SAMPLE_1,SAMPLE_2 merge.gtf ./cuffquant/SAMPLE_1.repl1.cxb,./cuffquant/SAMPLE_1.repl2.cxb \ ./cuffquant/SAMPLE_2.repl1.cxb,./cuffquant/SAMPLE_2.repl2.cxb
Error: cannot open reference GTF file merge.gtf for reading
Found it was a typo in merged.gtf