Weird tophat2 error message

[feralBiologist]

I am trying to run tophat2 on a set of SE reads:

tophat2 ../../../references/genomes/xen-trop/ens71/xen-trop-4-2-71 CO1_CGATGT_L008.fastq --min-intron-length 6 --max-insertion-length 3 --max-deletion-length 3 --b2-seed 73 --solexa1.3-quals --microexon-search --num-threads 32 --library-type fr-unstranded --no-coverage-search --GTF ../../../references/gtf/xen-trop/ens71/Xenopus_tropicalis.JGI_4.2.71.gtf

But I am getting a weird error message:

[2013-08-10 14:34:22] Beginning TopHat run (v2.0.9)
[2013-08-10 14:34:22] Checking for Bowtie
              Bowtie version:        2.1.0.0
[2013-08-10 14:34:22] Checking for Samtools
            Samtools version:        0.1.19.0
[2013-08-10 14:34:22] Checking for Bowtie index files (genome)..
[2013-08-10 14:34:22] Checking for reference FASTA file 
[2013-08-10 14:34:22] Generating SAM header for ../../../references/genomes/xen-trop/ens71/xen-trop-4-2-71
Traceback (most recent call last): File "/galaxy/software/tophat2/2.0.9/tophat", line 4072, in ?
sys.exit(main())
File "/galaxy/software/tophat2/2.0.9/tophat", line 3926, in main
params.read_params = check_reads_format(params, reads_list)
File "/galaxy/software/tophat2/2.0.9/tophat", line 1829, in check_reads_format
zf = ZReader(f_name, params)
File "/galaxy/software/tophat2/2.0.9/tophat", line 1782, in __init__
self.file=open(filename)
IOError: [Errno 2] No such file or directory: '--min-intron-length'

What is going on here? It seems tophat2 is looking for a file/directory when it encounters "--min-intron-length" yet I don't understand why. Can you help?

[GenoMax]

Are you running this command from the directory where the "CO1_CGATGT_L008.fastq" file is located?

Try explicitly specifying the file location as: "./CO1_CGATGT_L008.fastq" (without quotes)

[feralBiologist]

@GenoMax: Yes, I do. I tried "./", too. Interestingly, If I put just:

tophat2 ../../../references/genomes/xen-trop/ens71/xen-trop-4-2-71 CO1_CGATGT_L008.fastq

than it works. Adding the additional parameters leads to the error. Also, if I submit

tophat2 ../../../references/genomes/xen-trop/ens71/xen-trop-4-2-71 CO1_CGATGT_L008_1.fastq CO1_CGATGT_L008_2.fastq --min-intron-length 6 --max-insertion-length 3 --max-deletion-length 3 --b2-seed 73 --solexa1.3-quals --microexon-search --num-threads 32 --library-type fr-unstranded --no-coverage-search --GTF ../../../references/gtf/xen-trop/ens71/Xenopus_tropicalis.JGI_4.2.71.gtf

than it works, too. Somehow only the combination of using a single file and additional parameters is leading to the error. But this is a SE sample, so it is a single file so right now my options are to either submitted it with no additional parameters or not submit it at all. It is really bizarre.

[dpryan]

I just commented on this over at biostars, but I'll do so here as well. In short, try:

tophat2 --min-intron-length 6 --max-insertion-length 3 --max-deletion-length 3 --b2-seed 73 --solexa1.3-quals --microexon-search --num-threads 32 --library-type fr-unstranded --no-coverage-search --GTF ../../../references/gtf/xen-trop/ens71/Xenopus_tropicalis.JGI_4.2.71.gtf ../../../references/genomes/xen-trop/ens71/xen-trop-4-2-71 CO1_CGATGT_L008.fastq

since that's likely to be parsed correctly (write a few programs and you'll start to get a good feeling for why this might occur).

[feralBiologist]

Thank you dpryan - this was, indeed, the problem. I responded with more detail on biostar: http://www.biostars.org/p/78628/#78704

[dpryan]

Cool, glad that was an easy fix. You'd be surprised how many programs are affected by this sort of issue.