Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • .gtf file issue - Error at parsing .tlst line (invalid strand):

    I'm trying to align my reads to a genome + transcriptome, which is in the form of a .gtf file. It gets half way through and then runs this error:

    Code:
    user@it053392:~$ export PATH=/home/user/Downloads/bowtie2:$PATH
    user@it053392:~$ export PATH=/home/user/Downloads/tophat2:$PATH
    user@it053392:~$ echo $PATH
    /home/user/Downloads/tophat2:/home/user/Downloads/bowtie2:/usr/local/sbin:/usr/local/bin:/usr/sbin:/usr/bin:/sbin:/bin:/usr/games:/usr/local/games
    user@it053392:~$ /home/user/Downloads/tophat2/tophat -G /home/user/Desktop/_sam/RNAseq_beta_data/Transcriptome/merged.remDup.gtf -o /home/user/Desktop/Tophat_out /home/user/Downloads/bowtie2/example/index/DinoAnt /home/user/Desktop/_sam/RNAseq_beta_data/trimmed/23Y70_trimmed.fastq
    
    [2015-11-04 11:52:45] Beginning TopHat run (v2.1.0)
    -----------------------------------------------
    [2015-11-04 11:52:45] Checking for Bowtie
    		  Bowtie version:	 2.2.6.0
    [2015-11-04 11:52:45] Checking for Bowtie index files (genome)..
    [2015-11-04 11:52:45] Checking for reference FASTA file
    	Warning: Could not find FASTA file /home/user/Downloads/bowtie2/example/index/DinoAnt.fa
    [2015-11-04 11:52:45] Reconstituting reference FASTA file from Bowtie index
      Executing: /home/user/Downloads/bowtie2/bowtie2-inspect /home/user/Downloads/bowtie2/example/index/DinoAnt > /home/user/Desktop/Tophat_out/tmp/DinoAnt.fa
    [2015-11-04 11:52:59] Generating SAM header for /home/user/Downloads/bowtie2/example/index/DinoAnt
    [2015-11-04 11:53:01] Reading known junctions from GTF file
    [2015-11-04 11:53:04] Preparing reads
    	 left reads: min. length=85, max. length=85, 23624572 kept reads (13384 discarded)
    [2015-11-04 12:00:30] Building transcriptome data files /home/user/Desktop/Tophat_out/tmp/merged.remDup
    [2015-11-04 12:00:51] Building Bowtie index from merged.remDup.fa
    [2015-11-04 13:22:45] Mapping left_kept_reads to transcriptome merged.remDup with Bowtie2 
    	[FAILED]
    Error running:
    /home/user/Downloads/tophat2/bam2fastx --all /home/user/Desktop/Tophat_out/tmp/left_kept_reads.bam|/home/user/Downloads/bowtie2/bowtie2 -k 60 -D 15 -R 2 -N 0 -L 20 -i S,1,1.25 --gbar 4 --mp 6,2 --np 1 --rdg 5,3 --rfg 5,3 --score-min C,-14,0 -p 1 --sam-no-hd -x /home/user/Desktop/Tophat_out/tmp/merged.remDup -|/home/user/Downloads/tophat2/fix_map_ordering --bowtie2-min-score 15 --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --sam-header /home/user/Desktop/Tophat_out/tmp/merged.remDup.bwt.samheader.sam - - /home/user/Desktop/Tophat_out/tmp/left_kept_reads.m2g_um.bam | /home/user/Downloads/tophat2/map2gtf --sam-header /home/user/Desktop/Tophat_out/tmp/DinoAnt_genome.bwt.samheader.sam /home/user/Desktop/Tophat_out/tmp/merged.remDup.fa.tlst - /home/user/Desktop/Tophat_out/tmp/left_kept_reads.m2g.bam > /home/user/Desktop/Tophat_out/logs/m2g_left_kept_reads.out
    user@it053392:~$
    Running that error tells me this:

    Code:
    user@it053392:~$ /home/user/Downloads/tophat2/bam2fastx --all /home/user/Desktop/Tophat_out/tmp/left_kept_reads.bam|/home/user/Downloads/bowtie2/bowtie2 -k 60 -D 15 -R 2 -N 0 -L 20 -i S,1,1.25 --gbar 4 --mp 6,2 --np 1 --rdg 5,3 --rfg 5,3 --score-min C,-14,0 -p 1 --sam-no-hd -x /home/user/Desktop/Tophat_out/tmp/merged.remDup -|/home/user/Downloads/tophat2/fix_map_ordering --bowtie2-min-score 15 --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --sam-header /home/user/Desktop/Tophat_out/tmp/merged.remDup.bwt.samheader.sam - - /home/user/Desktop/Tophat_out/tmp/left_kept_reads.m2g_um.bam | /home/user/Downloads/tophat2/map2gtf --sam-header /home/user/Desktop/Tophat_out/tmp/DinoAnt_genome.bwt.samheader.sam /home/user/Desktop/Tophat_out/tmp/merged.remDup.fa.tlst - /home/user/Desktop/Tophat_out/tmp/left_kept_reads.m2g.bam > /home/user/Desktop/Tophat_out/logs/m2g_left_kept_reads.out
    
    Error at parsing .tlst line (invalid strand):
    	31958 TCONS_00032473 scaffold40. 5-1634
    (ERR): bowtie2-align exited with value 141
    Which suggests that there is something wrong with the .gtf file at point 00032473. Some googling revealed its something to do with missing strand info, but nobody seemed to provide any solutions. Can anyone help me modify the file or something to make it work? Thanks

  • #2
    For reference, posted also at: https://www.biostars.org/p/164703/

    Comment

    Latest Articles

    Collapse

    • seqadmin
      Strategies for Sequencing Challenging Samples
      by seqadmin


      Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
      03-22-2024, 06:39 AM
    • seqadmin
      Techniques and Challenges in Conservation Genomics
      by seqadmin



      The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

      Avian Conservation
      Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
      03-08-2024, 10:41 AM

    ad_right_rmr

    Collapse

    News

    Collapse

    Topics Statistics Last Post
    Started by seqadmin, 03-27-2024, 06:37 PM
    0 responses
    12 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 03-27-2024, 06:07 PM
    0 responses
    11 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 03-22-2024, 10:03 AM
    0 responses
    53 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 03-21-2024, 07:32 AM
    0 responses
    69 views
    0 likes
    Last Post seqadmin  
    Working...
    X