Tophat Error

[JasonServiss]

Hello!

I have repeatedly experienced the same error when running tophat and despite trying to solve the problem have had no luck... The command line I use is located below:

./tophat --keep-tmp -G Homo_sapiens.GRCh37.66.gtf -p 8 -M -o /Users/jasonserviss/Desktop/Tophat\ results e66humanWP NG-6038_iPSD199_lib10333_1_sequence.fastq NG-6038_iPSD199_lib10333_2_sequence.fastq

And the error is listed here:

[2012-10-30 02:06:44] Building Bowtie index from Homo_sapiens.GRCh37.66.fa
[2012-10-30 02:37:17] Mapping left_kept_reads to transcriptome Homo_sapiens.GRCh37.66 with Bowtie
[2012-10-30 03:57:37] Mapping right_kept_reads to transcriptome Homo_sapiens.GRCh37.66 with Bowtie
[2012-10-30 05:20:01] Resuming TopHat pipeline with unmapped reads
Traceback (most recent call last):
File "./tophat", line 4035, in <module>
sys.exit(main())
File "./tophat", line 4002, in main
user_supplied_deletions)
File "./tophat", line 3423, in spliced_alignment
if not nonzeroFile(initial_reads[0]) and \
File "./tophat", line 1119, in nonzeroFile
samtools_view = subprocess.Popen(samtools_view_cmd, stdout=subprocess.PIPE)
File "/System/Library/Frameworks/Python.framework/Versions/2.7/lib/python2.7/subprocess.py", line 672, in __init__
errread, errwrite)
File "/System/Library/Frameworks/Python.framework/Versions/2.7/lib/python2.7/subprocess.py", line 1202, in _execute_child
raise child_exception
OSError: [Errno 2] No such file or directory

I have looked through the forum and found 2 suggestions, one stating that the subprocess.py file is missing and that is causing the error. So I checked and it seems as though the file is present in the directory. As well, another person suggested the this error occurs when Bowtie is not in the PATH but this doesn't seem to be the case since the mapping with bowtie completes successfully and bowtie, tophat, and samtools are all in the same location on my computer...

I would be really thankful for any advice that you could offer to help me solve this issue!!

Kind Regards,
Jason

[Richard Barker]

I'm not sure whats wrong but you could try a different approach... I personally use the iPlant collaborative cloud infrastructure http://www.iplantcollaborative.org/...

Simple go to their website setup an account, this will create a essentially limitless online data store where you can upload your data for safe keeping (fastq files i assume?). You can align your RNA fragments using a GUI they built which will complete relatively fast as all the processing occurs on their super computers. It works so you won't get the error message and they have a really responsive team of experts to help.

Alternatively you can also use the iPlant infrastructure to create a virtual machine, they have a long list to choose from, one of which already has NGS tools instralled and ready for use...

[JasonServiss]

Thanks for your reply Richard! I'd like to try to work through the problem in my current system first and if I don't have any luck I may try your solution...

Maybe I should have mentioned in my first post...
I am running mac OSx, tophat 2.0.5, bowtie 0.12.8, and samtools 0.1.18

[JasonServiss]

Update

I can also add that I have now tried updating to Bowtie2 and Tophat 2.0.6 and get the exact same error. I also tried running with the "simple test data", provided at the Tophat website to no avail.


[2012-11-08 19:51:26] Beginning TopHat run (v2.0.5)
-----------------------------------------------
[2012-11-08 19:51:26] Checking for Bowtie
Bowtie version: 2.0.0.7
[2012-11-08 19:51:26] Checking for Samtools
Samtools version: 0.1.18.0
[2012-11-08 19:51:26] Checking for Bowtie index files
[2012-11-08 19:51:26] Checking for reference FASTA file
[2012-11-08 19:51:26] Generating SAM header for test_ref
format: fastq
quality scale: phred33 (default)
[2012-11-08 19:51:26] Preparing reads
left reads: min. length=75, max. length=75, 100 kept reads (0 discarded)
right reads: min. length=75, max. length=75, 100 kept reads (0 discarded)
Traceback (most recent call last):
File "./tophat", line 4035, in <module>
sys.exit(main())
File "./tophat", line 4002, in main
user_supplied_deletions)
File "./tophat", line 3470, in spliced_alignment
if reads == None or not nonzeroFile(reads):
File "./tophat", line 1119, in nonzeroFile
samtools_view = subprocess.Popen(samtools_view_cmd, stdout=subprocess.PIPE)
File "/System/Library/Frameworks/Python.framework/Versions/2.6/lib/python2.6/subprocess.py", line 623, in __init__
errread, errwrite)
File "/System/Library/Frameworks/Python.framework/Versions/2.6/lib/python2.6/subprocess.py", line 1141, in _execute_child
raise child_exception
OSError: [Errno 2] No such file or directory

Any help with figuring out what this error is indicating would be very appreciated!!!!

[GenoMax]

What version of OS X are you using? Did you get pre-compiled binaries or compiled the program yourself?

It also appears that you switched from Python v.2.7 to v.2.6 in the latest example.

[JasonServiss]

GenoMax: No, I haven't updated it is just that I have tried to run both on my computer at work and my personal computer... thinking that maybe one or the other is missing something essential.

I believe the work computer is mac 10.6.8 whereas my personal computer is 10.7.5

With the latest tophat version (2.0.6) I used the pre-complied binary from the tophat website available at the link titled "Mac OS X x86_64 binary" and previously with tophat 2.0.5 I also used the pre-complied binary.

[EGrassi]

Traceback (most recent call last):
File "./tophat", line 4035, in <module>
sys.exit(main())
File "./tophat", line 4002, in main
user_supplied_deletions)
File "./tophat", line 3470, in spliced_alignment
if reads == None or not nonzeroFile(reads):
File "./tophat", line 1119, in nonzeroFile
samtools_view = subprocess.Popen(samtools_view_cmd, stdout=subprocess.PIPE)
File "/System/Library/Frameworks/Python.framework/Versions/2.6/lib/python2.6/subprocess.py", line 623, in __init__
errread, errwrite)
File "/System/Library/Frameworks/Python.framework/Versions/2.6/lib/python2.6/subprocess.py", line 1141, in _execute_child
raise child_exception
OSError: [Errno 2] No such file or directory[/B]

Any help with figuring out what this error is indicating would be very appreciated!!!!

This traceback seems to suggest a samtools related problem. I would suggest you to try to understand how samtools is invoked by tophat and try that by hand and check if all the related files are created by tophat before that step. It seems a 'samtools view' that fails for a missing file...

[GenoMax]

GenoMax: No, I haven't updated it is just that I have tried to run both on my computer at work and my personal computer... thinking that maybe one or the other is missing something essential.

I believe the work computer is mac 10.6.8 whereas my personal computer is 10.7.5

With the latest tophat version (2.0.6) I used the pre-complied binary from the tophat website available at the link titled "Mac OS X x86_64 binary" and previously with tophat 2.0.5 I also used the pre-complied binary.

I tested tophat (precompiled binary albeit with just the test data) on a Mac that is running 10.7.5 and encountered no problems.

As egrassi said in the post above this one can you verify that samtools executable is present in a directory that is in your PATH (or append the path variable so that it is e.g. PATH=$PATH:/path_to_samtools).

Also as indicated in the install instructions for tophat you will need to make sure that "libbam.a" and the "*.h" files for samtools are in the right directories (steps 6-8 in "installing SAM tools" section on tophat tutorial page http://tophat.cbcb.umd.edu/tutorial.html)

[JasonServiss]

Success...?

So I think I found the problem.... When going back through the tophat installation process I noticed that Boost default installs to usr/local which, for some reason, I don't have on my mac (which probably means it never installed correctly and I didn't notice) I re-installed to an alternate location and ran the tophat test file and it ran ok so I will try the "real" run tonight and see how it goes...

Thank you very much for taking the time to comment and give me a hand with this!!!!