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  • Error: gtf_to_fasta returned an error.

    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




    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....

  • #2
    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...)

    Comment


    • #3
      Thanks sphil,,

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

      Comment


      • #4
        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

        Comment


        • #5
          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.

          Comment


          • #6
            Thanks dpryan,

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

            Discussion of next-gen sequencing related bioinformatics: resources, algorithms, open source efforts, etc

            Comment


            • #7
              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...

              Comment


              • #8
                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

                Comment


                • #9
                  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

                  Comment


                  • #10
                    Originally posted by varshacp View Post
                    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?

                    Comment


                    • #11
                      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

                      Comment


                      • #12
                        Originally posted by varshacp View Post
                        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.

                        Comment


                        • #13
                          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

                          Comment


                          • #14
                            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.

                            Comment


                            • #15
                              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

                              Comment

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