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Old 11-05-2015, 01:28 AM   #1
4galaxy7
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Location: UK

Join Date: Nov 2015
Posts: 19
Default .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:
[email protected]:~$ export PATH=/home/user/Downloads/bowtie2:$PATH
[email protected]:~$ export PATH=/home/user/Downloads/tophat2:$PATH
[email protected]:~$ echo $PATH
/home/user/Downloads/tophat2:/home/user/Downloads/bowtie2:/usr/local/sbin:/usr/local/bin:/usr/sbin:/usr/bin:/sbin:/bin:/usr/games:/usr/local/games
[email protected]:~$ /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
[email protected]:~$
Running that error tells me this:

Code:
[email protected]:~$ /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
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Old 11-05-2015, 03:42 AM   #2
GenoMax
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Location: East Coast USA

Join Date: Feb 2008
Posts: 6,494
Default

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