Error: gtf_to_fasta returned an error.

[hathiram2]

Hello guys,

I am Hathi, a postdoc in EMBL, Heidelberg.

I am doing alignment of my RNA-Seq data from Arabidopsis with Tophat


I gave the following command

tophat -G genes.gtf -p 5 -o controlone genome
C2AFAACXX_Hr-02_13s004770-1-1_ram_lane313s004770_sequence.fastq,C2AFAACXX_Hr-02_13s004770-2-1_ram_lane513s004770_sequence.fastq

Genes.gtf is gene annotation file, while bowtie2 index files are starting with 'genome' name.

These both the files were downloaded from this link

http://tophat.cbcb.umd.edu/igenomes.shtml

and the out put looks like this

[2013-08-27 15:23:42] Beginning TopHat run (v2.0.9)
-----------------------------------------------
[2013-08-27 15:23:42] Checking for Bowtie
Bowtie version: 2.1.0.0
[2013-08-27 15:23:42] Checking for Samtools
Samtools version: 0.1.19.0
[2013-08-27 15:23:42] Checking for Bowtie index files (genome)..
[2013-08-27 15:23:42] Checking for reference FASTA file
[2013-08-27 15:23:42] Generating SAM header for genome
format: fastq
quality scale: phred33 (default)
[2013-08-27 15:23:42] Reading known junctions from GTF file
[2013-08-27 15:23:45] Preparing reads
left reads: min. length=52, max. length=52, 58033296 kept reads
(3282 discarded)
[2013-08-27 15:36:32] Building transcriptome data files..
[FAILED]
Error: gtf_to_fasta returned an error.




Can anyone please tell me that where is the problem??

Thank you so much and look forward to your posts eagerly....

[sphil]

Did you check that the names of the index and the GTF file are the same? That is the most common problem. (And don't assume they are the same just because you downloaded the files from somewhere...)

[hathiram2]

Thanks sphil,,

Yes,,they have different names..
Let em try with same names..

[hathiram2]

Hi Sphil,

I tried with the same names of index and GTF files, but still I get same error message. Can't understand anything that whys this happening

[dpryan]

You can look in the run log for the exact command that's being run and then run it to get the exact error message that's being thrown.

[hathiram2]

Thanks dpryan,

I am also getting your reply in another thredd
please see the link

http://seqanswers.com/forums/showthread.php?t=31344

[sphil]

hmm that's strange, normally that fixes the problem. No clue whats going on than, myb send an E-Mail to the Mailing list or one of the authors to get help...

[sphil]

hey i checked it, since the index file has different names your reference sequence has also different names. I guess you need to change this as well or, change the tags in your GTF so that the tags in your sequence reference as well as in your index can stay the same!

keep me posted

[varshacp]

Error: gtf_to_fasta returned an error

Hi

I am getting the following error
Error: gtf_to_fasta returned an error

as mentioned in the previous blogs I checked the names in annotation gff file, genome sequence file as well as the bowtie index file, it is same Here is the run log

/opt/tophat/bin/gtf_to_fasta --min-anchor 8 --splice-mismatches 0 --min-report-intron 50 --max-report-intron 500000 --min-isoform-fraction 0.15 --output-dir cp04_thout1/ --max-multihits 20 --max-seg-multihits 40 --segment-length 25 --segment-mismatches 2 --min-closure-exon 100 --min-closure-intron 50 --max-closure-intron 5000 --min-coverage-intron 50 --max-coverage-intron 20000 --min-segment-intron 50 --max-segment-intron 500000 --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --max-insertion-length 3 --max-deletion-length 3 -z gzip -p2 --gtf-annotations ref_ASM33114v1_top_level_edited3.gff3 --gtf-juncs cp04_thout1/tmp/ref_ASM33114v1_top_level_edited3.juncs --no-closure-search --no-coverage-search --no-microexon-search ref_ASM33114v1_top_level_edited3.gff3 caref_all.fa cp04_thout1/tmp/ref_ASM33114v1_top_level_edited3.fa > cp04_thout1/logs/g2f.out

When I copy paste it on terminal
I get -bash: cp04_thout1/logs/g2f.out: No such file or directory

Kindly help

Thankx in advance

[GenoMax]

Hi

When I copy paste it on terminal
I get -bash: cp04_thout1/logs/g2f.out: No such file or directory

Kindly help

Thankx in advance

Is there a directory called "logs" under cp04_thout1? If there is, do you have permissions to write to that directory?

[varshacp]

Hi Genomax

Thank you for your reply. Yes there is a logs directory and I do not have permission to write for it only read and execute.




Thank you

[GenoMax]

Hi Genomax

Thank you for your reply. Yes there is a logs directory and I do not have permission to write for it only read and execute.

Thank you

Varsha: Just replace the "cp04_thout1/logs/" part with a directory name that you can write to and give it a try.

[varshacp]

Error (GFaSeqGet) opening file 'caref_ncbiall.fa'

Hi Am running the tophat with following command

tophat -p 2 -G caref_seq.gff -o cp04_thout5 caref_ncbiall cp04.fastq
and getting the error

Error: gtf_to_fasta returned an error
When I check the run.log file and copy paste the following command

[e09812@unixprdap218 logs]$ /opt/tophat/bin/gtf_to_fasta --min-anchor 8 --splice-mismatches 0 --min-report-intron 50 --max-report-intron 500000 --min-isoform-fraction 0.15 --output-dir cp04_thout5/ --max-multihits 20 --max-seg-multihits 40 --segment-length 25 --segment-mismatches 2 --min-closure-exon 100 --min-closure-intron 50 --max-closure-intron 5000 --min-coverage-intron 50 --max-coverage-intron 20000 --min-segment-intron 50 --max-segment-intron 500000 --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --max-insertion-length 3 --max-deletion-length 3 -z gzip -p2 --gtf-annotations caref_seq.gff --gtf-juncs cp04_thout5/tmp/caref_seq.juncs --no-closure-search --no-coverage-search --no-microexon-search caref_seq.gff caref_ncbiall.fa cp04_thout5/tmp/caref_seq.fa

I get the following message
Error (GFaSeqGet) opening file 'caref_ncbiall.fa'

Kindly help

Thankx in advance

Varsha

[GenoMax]

Varsha: You do not appear to have write permissions to "cp04_thout5" and every directory below it (cp04_thout5/tmp would not work).

Replace that section (cp04_thout5/..) with a directory that you are able to write to.

[varshacp]

Hi Genomax

Thank you for your reply.

I am not sure I can change the permissions or move to some other directory to which I have read and write permissions as the output directory "cp04_thout5" is created while the tophat is executing (tophat -p 2 -G caref_seq.gff -o cp04_thout5 caref_ncbiall cp04.fastq)


I do not get this message with other genome sequence file.

I checked the caref_ncbiall file and could not find any error in it.

Kindly help to solve it


Thank you

Varsha