Bowtie...Error: could not execute Bowtie

[Brajbio]

Hi,

I am trying to run tophat with the sample data reads_1.fq and reads_2.fq.

I built the bowtie index successfully and moved the generated index files to the Bowtie indexes subdirectory. I run the command

tophat -r 20 test_ref reads_1.fq reads_2.fq

and the output i got is an error showing on console showing:
Error: could not execute Bowtie

[Wed Jun 2 13:46:29 2010] Beginning TopHat run (v1.0.7)
-----------------------------------------------
[Wed Jun 2 13:46:29 2010] Preparing output location ./tophat_out/
[Wed Jun 2 13:46:29 2010] Checking for Bowtie index files
[Wed Jun 2 13:46:29 2010] Checking for reference FASTA file
[Wed Jun 2 13:46:29 2010] Checking for Bowtie
Bowtie version: 0.12.3.0
[Wed Jun 2 13:46:29 2010] Checking reads
seed length: 75bp
format: fastq
quality scale: phred
Splitting reads into 3 segments
[Wed Jun 2 13:46:29 2010] Mapping reads against test_ref with Bowtie [FAILED]
Error: could not execute Bowtie
Traceback (most recent call last):
File "/usr/bin/tophat", line 1476, in ?
sys.exit(main())
File "/usr/bin/tophat", line 1450, in main
user_supplied_juncs)
File "/usr/bin/tophat", line 1297, in spliced_alignment
unmapped_seg)
File "/usr/bin/tophat", line 612, in bowtie
exit(1)
TypeError: 'str' object is not callable


I am really struggling hard to resolve this....and need someones help urgently

Thanks

Brajendra

[raela]

Is bowtie in your PATH? You can either copy the bowtie executable to the same dir as tophat, or `export PATH=$PATH:/location/of/bowtie`. Exporting the path would need to be done every time you log on, or it could be added to the rc file of the shell you use (~/.bashrc for bash, ~/.zshrc for zsh, etc). You could also try a symbolic link - in the tophat directory, `ln -s /path/to/bowtie bowtie`

[Brajbio]

To raela,

I had placed all the following requisite executables in root '/usr/bin'

1. bowtie
2. bowtie-inspect
3. bowtie-build
4. tophat

My input files : reads_1.fq, reads_2.fq
are in Path: /home/user1/NGS/TopHat_Test_data/test_data)
whereas
My Bowtie index files are in Path: /home/user2/bowtie_install/bowtie-0.12.3/indexes/

Note: user1 doesn't have permission to directly access the user2 directory where the Bowtie 'indexes' sub directory is except through login as the admin.

I am running the command

tophat -r 20 test_ref reads_1.fq reads_2.fq

from the directory: /home/user1/NGS/TopHat_Test_data/test_data

I also tried running the same command as

tophat -r 20 /home/user2/bowtie_install/bowtie-0.12.3/indexes/test_ref reads_1.fq reads_2.fq

then i am getting the error as

-bash-3.1$ tophat -r 20 /home/user2/bowtie_install/bowtie-0.12.3/indexes/test_ref reads_1.fq reads_2.fq

[Wed Jun 2 17:24:16 2010] Beginning TopHat run (v1.0.7)
-----------------------------------------------
[Wed Jun 2 17:24:16 2010] Preparing output location ./tophat_out/
[Wed Jun 2 17:24:16 2010] Checking for Bowtie index files
Error: Could not find Bowtie index files /home/user2/bowtie_install/bowtie-0.12.3/indexes/test_ref.*
Traceback (most recent call last):
File "/usr/bin/tophat", line 1476, in ?
sys.exit(main())
File "/usr/bin/tophat", line 1411, in main
(ref_fasta, idx_bfa) = check_index(bwt_idx_prefix)
File "/usr/bin/tophat", line 378, in check_index
check_bowtie_index(idx_prefix)
File "/usr/bin/tophat", line 295, in check_bowtie_index
exit(1)
TypeError: 'str' object is not callable

[raela]

It looks like tophat really wants the indexes and reads to be in the same directory. You should try copying either the index files or the reads to the other directory, or both to the same, and run again. Maybe copy both to /tmp if you have the disk space, then transfer the resulting output to your users' /home?

[john_mu]

When you try to execute Bowtie directly, does it work?

If you type "Bowtie" does this successfully print out the Bowtie help?

[Brajbio]

I apologize for the delay

To John:

Yeah when i type 'bowtie' on terminal, its displaying the help, so it is confirmed that bowtie is installed successfully.

To reaela :

Actually, it was the permission problem.
When i run the command being as root login it run successfully. The mistake i did was, though the bowtie executables were placed in 'bin' folder so that all user can access but the bowtie index sub directory was left in user 2 work space.
So when i was running the tophat from my user login the index directory was not getting accessed.

[Ajayi Oyeyemi]

Hi,
I was running the alignment using tophat:

[2013-02-12 16:32:41] Beginning TopHat run (v2.0.7)
-----------------------------------------------
[2013-02-12 16:32:41] Checking for Bowtie
Bowtie version: 2.0.6.0
[2013-02-12 16:32:42] Checking for Samtools
Samtools version: 0.1.18.0
[2013-02-12 16:32:42] Checking for Bowtie index files
[2013-02-12 16:32:42] Checking for reference FASTA file
Warning: Could not find FASTA file /workdir/ooa4/index/bovine.fa
[2013-02-12 16:32:42] Reconstituting reference FASTA file from Bowtie index
Executing: /programs/bin/bowtie2/bowtie2-inspect /workdir/ooa4/index/bovine > tophat_output/tmp/bovine.fa
[2013-02-12 16:35:54] Generating SAM header for /workdir/ooa4/index/bovine
format: fastq
quality scale: phred33 (default)
[2013-02-12 16:36:56] Reading known junctions from GTF file
[2013-02-12 16:36:59] Preparing reads
left reads: min. length=72, max. length=72, 24813301 kept reads (29188 discarded)
[2013-02-12 16:47:07] Creating transcriptome data files..
[2013-02-12 16:48:19] Building Bowtie index from genes.fa
[FAILED]
Error: Couldn't build bowtie index with err = 1.

I went through the log file in my tophat output and it gave:
bowtie_inspect_recons.log g2f.err g2f.out gtf_juncs.log prep_reads.log run.log tophat.log

I used the code: tophat -o tophat_output -p 8 -G Bos_taurus/NCBI/Btau_4.6.1/Annotation/Archives/archive-2012-03-08-21-54-00/Genes/genes.gtf /workdir/ooa4/index/bovine SRR594497.fastq

All my .bt2 files are in the folder called index e.g bovine.bt1 etc...



I don't know whats happening and I need your help. I've been struggling real hard on this. Please help me out.

Yemi.