Difficulties in getting blast to work in assigning taxonomy.

[drcjb...@gmail.com]

Hi there,

Sorry to be a bother, but I am having difficulties in getting blast to work when assigning taxonomies. I get the following error message:

MacQIIME zn10508:~ $ assign_taxonomy.py -i /Users/christopherbarnes/Desktop/Postdoc_lab_results/Thapsia_project/ITSx_rep_set.fasta.ITS2.fasta -r /Users/christopherbarnes/Desktop/Sequencing\ resources/its_12_11_otus/rep_set/97_otus.fasta -t /Users/christopherbarnes/Desktop/Sequencing\ resources/its_12_11_otus/taxonomy/97_otu_taxonomy.txt -o assigned_taxonomy_old_unite_blast -m blast

Traceback (most recent call last):

  File "/macqiime/anaconda/bin/assign_taxonomy.py", line 417, in <module>

    main()

  File "/macqiime/anaconda/bin/assign_taxonomy.py", line 394, in main

    log_path=log_path)

  File "/macqiime/anaconda/lib/python2.7/site-packages/qiime/assign_taxonomy.py", line 500, in __call__

    abspath(reference_seqs_path), output_dir=blast_db_dir)

  File "/macqiime/anaconda/lib/python2.7/site-packages/bfillings/formatdb.py", line 121, in build_blast_db_from_fasta_path

    app_result = fdb(fasta_path)

  File "/macqiime/anaconda/lib/python2.7/site-packages/burrito/util.py", line 285, in __call__

    'StdErr:\n%s\n' % open(errfile).read())

burrito.util.ApplicationError: Unacceptable application exit status: 3

Command:

cd "/tmp/bltax-kTF6Ry/"; formatdb -l "97_otus.fasta.log" -o T -n "97_otus.fasta" -i "/Users/christopherbarnes/Desktop/Sequencing resources/its_12_11_otus/rep_set/97_otus.fasta" -p F > "/dev/null" 2> "/dev/null"

StdOut:

StdErr:

I have blast installed so that the output seems fine:

which blastall

/Users/christopherbarnes/blast-2.2.22/bin/blastall

which formatdb

/Users/christopherbarnes/blast-2.2.22/bin/formatdb

And finally the output from print_qiime_config.py is:

System information

==================

         Platform: darwin

   Python version: 2.7.10 |Anaconda 2.2.0 (x86_64)| (default, May 28 2015, 17:04:42)  [GCC 4.2.1 (Apple Inc. build 5577)]

Python executable: /macqiime/anaconda/bin/python

QIIME default reference information

===================================

For details on what files are used as QIIME's default references, see here:

 https://github.com/biocore/qiime-default-reference/releases/tag/0.1.2

Dependency versions

===================

          QIIME library version: 1.9.1

           QIIME script version: 1.9.1

qiime-default-reference version: 0.1.2

                  NumPy version: 1.9.2

                  SciPy version: 0.15.1

                 pandas version: 0.16.1

             matplotlib version: 1.4.3

            biom-format version: 2.1.4

                   h5py version: 2.4.0 (HDF5 version: 1.8.14)

                   qcli version: 0.1.1

                   pyqi version: 0.3.2

             scikit-bio version: 0.2.3

                 PyNAST version: 1.2.2

                Emperor version: 0.9.51

                burrito version: 0.9.1

       burrito-fillings version: 0.1.1

              sortmerna version: SortMeRNA version 2.0, 29/11/2014

              sumaclust version: SUMACLUST Version 1.0.00

                  swarm version: Swarm 1.2.19 [Jun  2 2015 14:40:16]

                          gdata: Installed.

QIIME config values

===================

For definitions of these settings and to learn how to configure QIIME, see here:

 http://qiime.org/install/qiime_config.html

 http://qiime.org/tutorials/parallel_qiime.html

                     blastmat_dir: None

      pick_otus_reference_seqs_fp: /macqiime/anaconda/lib/python2.7/site-packages/qiime_default_reference/gg_13_8_otus/rep_set/97_otus.fasta

                         sc_queue: all.q

      topiaryexplorer_project_dir: None

     pynast_template_alignment_fp: /macqiime/anaconda/lib/python2.7/site-packages/qiime_default_reference/gg_13_8_otus/rep_set_aligned/85_otus.pynast.fasta

                  cluster_jobs_fp: start_parallel_jobs.py

pynast_template_alignment_blastdb: None

assign_taxonomy_reference_seqs_fp: /macqiime/anaconda/lib/python2.7/site-packages/qiime_default_reference/gg_13_8_otus/rep_set/97_otus.fasta

                     torque_queue: friendlyq

                    jobs_to_start: 1

                       slurm_time: None

            denoiser_min_per_core: 50

assign_taxonomy_id_to_taxonomy_fp: /macqiime/anaconda/lib/python2.7/site-packages/qiime_default_reference/gg_13_8_otus/taxonomy/97_otu_taxonomy.txt

                         temp_dir: /tmp/

                     slurm_memory: None

                      slurm_queue: None

                      blastall_fp: blastall

                 seconds_to_sleep: 60

Any help would be appreciated as I'm completely stumped!

Thanks in advance for any help you can offer.

Chris

[Jenya Kopylov]

Hi Chris,

Could you check whether Blast taxonomy assignment tests are passing on your system?

ex.

$ python /macqiime/QIIME/tests/test_assign_taxonomy.py BlastTaxonAssignerTests
----------------------------------------------------------------------
Ran 11 tests in 2.653s
OK

Also, it seems you're using a 16S database to assign taxonomy to ITS sequences, am I interpreting your command line correctly?

Thanks,

Jenya

[drcjb...@gmail.com]

Hi Jenya,

Thanks for your response. 

Ah, so my command is definitely using fungal databases (UNITE databases), however the defaults from my print_qiime_configs.py are the greengenes database. Does this have an effect if I always specify databases I wish to assign to? I never noticed that before.

Regarding blast output:

MacQIIME zn10508:~ $ python /macqiime/QIIME/tests/test_assign_taxonomy.py BlastTaxonAssignerTests

...........

----------------------------------------------------------------------

Ran 11 tests in 0.634s

OK

All tests of BLAST seem ok, could it be to do with the databases? Either the UNITE databases or something to do with these defaults within QIIIME? 

If it's of relevance, this database works fine using the default UCLUST taxonomic assignments.

Many thanks for your time,

Chris

[Jenya Kopylov]

Hi Chris,

It's good to see the tests passing. One possible issue could be spaces between your folder name (ex. Sequencing resources), could you replace all spaces (ex. with '_') and retry your assign_taxonomy command?

From your command you are indeed using ITS so that shouldn't be an issue.

Jenya

[drcjb...@gmail.com]

Hi Jenya,

It worked, wow I feel a bit silly, totally didn't think about that. I know it's good form not to leave spaces, but I guess this really drums in the message!

Thanks so much for your time, hugely appreciated!

Best,

Chris