Question: Solution For: Error Running Cuffdiff. Error: Cannot Open Reference Gtf File Condition, Control For Reading
gravatar for Carlos Borroto
6.8 years ago by
Washington Metropolitan Area
Carlos Borroto390 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
galaxy • 3.1k views
ADD COMMENTlink modified 6.8 years ago by Jeremy Goecks2.2k • written 6.8 years ago by Carlos Borroto390

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 -o ./cuffmerge -g genes.gtf -s genome.fa list_GTF.txt # (GFT list file from Cufflinks outputs)


command ran on each library

cuffquant -o ./cuffquant merged.gtf --frag-bias-correct genome.fa --multi-read-correct library1.hisat2aligned.sorted.sam


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

ADD REPLYlink written 14 months ago by ugaldemx0

Found it was a typo in merged.gtf

ADD REPLYlink written 14 months ago by ugaldemx0
gravatar for Jeremy Goecks
6.8 years ago by
Jeremy Goecks2.2k
Jeremy Goecks2.2k wrote:
Agreed. I implemented the spirit of this functionality via argument checking in galaxy-central changeset 71031bf3105c Best, J.
ADD COMMENTlink written 6.8 years ago by Jeremy Goecks2.2k
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