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:
Running that error tells me this:
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
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:~$
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
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