Tophat 2 alignment error

Question

[cjvegame]

Hello everyone, I am trying to aligned a paired end sequences using tophat2. Everything works fine with the practice data I am using, but when I used it with my real data it gives me the error below. Has anybody encountered this problem before?

Code:

tophat2 \
  -p 12 \
  -r 60 \
  --library-type fr-firststrand \
  --rg-id=PQ_3_day_1_Abdomen \
  --rg-sample=BSTRES_02-3d15_PQ_3_day_1_Abdomen_CGATGT_L001 \
  -o /media/ubuntu/"TOSHIBA EXT"/Rna_Seq_2_attempts/trimmed/PQ_3_day_1_Abdomen \
  -G /media/ubuntu/"TOSHIBA EXT"/Rna_Seq_2_attempts/refs/Apis/fasta/gene_Apis.gtf \
  --transcriptome-index /media/ubuntu/"TOSHIBA EXT"/Rna_Seq_2_attempts/tophat/index \
  /media/ubuntu/"TOSHIBA EXT"/Rna_Seq_2_attempts/bwt/Apis/Apis \
  /media/ubuntu/"TOSHIBA EXT"/Rna_Seq_2_attempts/trimmed/BSTRES_02-3d15_PQ_3_day_1_Abdomen_CGATGT_L001__1.fastq.gz \
  /media/ubuntu/"TOSHIBA EXT"/Rna_Seq_2_attempts/trimmed/BSTRES_02-3d15_PQ_3_day_1_Abdomen_CGATGT_L001__2.fastq.gz

Input

[2015-12-28 10:15:53] Beginning TopHat run (v2.1.0)
-----------------------------------------------
[2015-12-28 10:15:53] Checking for Bowtie
          Bowtie version:     2.2.6.0
[2015-12-28 10:15:53] Checking for Bowtie index files (transcriptome)..
[2015-12-28 10:15:53] Checking for Bowtie index files (genome)..
[2015-12-28 10:15:53] Checking for reference FASTA file
[2015-12-28 10:15:53] Generating SAM header for /media/ubuntu/TOSHIBA EXT/Rna_Seq_2_attempts/bwt/Apis/Apis
[2015-12-28 10:15:53] Reading known junctions from GTF file
[2015-12-28 10:15:54] Preparing reads
     left reads: min. length=25, max. length=112, 14848766 kept reads (26376 discarded)
    right reads: min. length=25, max. length=112, 14871677 kept reads (3465 discarded)
[2015-12-28 10:25:39] Using pre-built transcriptome data..
[2015-12-28 10:25:39] Mapping left_kept_reads to transcriptome gene_Apis with Bowtie2
    [FAILED]
Error running bowtie:
Warning: Output file '-' was specified without -S.  This will not work in future Bowtie 2 versions.  Please use -S instead.
Warning: Could not open read file "EXT/Rna_Seq_2_attempts/tophat/index/gene_Apis" for reading; skipping...
Error: No input read files were valid
(ERR): bowtie2-align exited with value 1

This is also my first post in this forum so not sure if I gave as much information as possible. So please tell me if you need more.

Answer 1

[vivekbhr]

Hi

Most probably it's because you have a space in the file name "TOSHIBA EXT"..Remove it and try again ..

Answer 2

[Antonio R. Franco]

Try the using of the TAB key to introduce the PATH at the time of defining the files and archives, and you will avoid many problems

Answer 3

[cjvegame]

Hahaha.. I knew it was something that trivial. I appreciate the input from both of you. This problem is fixed.