[GenoMax]

TopHat is picky about the order of options on the command line. Can you try the following:

$ tophat -o cp04_thout5 -p 2 -G caref_seq.gff caref_ncbiall cp04.fastq

Let me also verify that the basename for your genome index files is "caref_ncbiall", that is there are several files (that comprise of the index) that have that prefix?

[varshacp]

Hi

I forgot to mention that the fastq file is also in same directory. The gff file and genome sequences were downloaded from NCBI (NCBI has separate fasta file for each chromosomes and all the unplaced scafolds are in one fasta file. I concatenated these files to make the genome file and renamed it as per the gff file. The same gff file is working with other genome sequence file which does not have unplaced sequences.


Thank you

Varsha

[varshacp]

Hi GenoMAx

All the files are in the same directory. I also made sure that the names are same in gff and genome file.


Thank you

Varsha

[GenoMax]

I did not pay close attention to your original question (i.e. you are getting the error while running tophat itself and not the actual script). My fault.

Let us start over.

Is caref_ncbiall available in the same directory that you are running tophat from? If not, can you provide the full path to that file when you execute tophat. The same should apply to cp04.fastq and caref_seq.gff. Also make certain that your gff file matches the format specification as noted here: http://cufflinks.cbcb.umd.edu/gff.html

[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

[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]

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]

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]

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

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]

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

[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

[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...

[hathiram2]

Thanks dpryan,

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

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

[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.