Cannot find formatdb

[Heather E]

Hi all.  I am new to qiime and am trying to run the script parallel_identify_chimeric_seqs.py.  However I am getting the following error:

Traceback (most recent call last):

  File "/macqiime/anaconda/bin/parallel_identify_chimeric_seqs.py", line 173, in <module>

    main()

  File "/macqiime/anaconda/bin/parallel_identify_chimeric_seqs.py", line 169, in main

    suppress_submit_jobs=opts.suppress_submit_jobs)

  File "/macqiime/anaconda/lib/python2.7/site-packages/qiime/parallel/util.py", line 133, in __call__

    self._precommand_initiation(input_fp, output_dir, working_dir, params)

  File "/macqiime/anaconda/lib/python2.7/site-packages/qiime/parallel/identify_chimeric_seqs.py", line 70, in _precommand_initiation

    build_blast_db_from_fasta_path(reference_seqs_fp)

  File "/macqiime/anaconda/lib/python2.7/site-packages/bfillings/formatdb.py", line 116, in build_blast_db_from_fasta_path

    fdb = FormatDb(WorkingDir=output_dir, HALT_EXEC=HALT_EXEC)

  File "/macqiime/anaconda/lib/python2.7/site-packages/burrito/util.py", line 201, in __init__

    self._error_on_missing_application(params)

  File "/macqiime/anaconda/lib/python2.7/site-packages/burrito/util.py", line 468, in _error_on_missing_application

    "Is it in your path?" % command)

burrito.util.ApplicationNotFoundError: Cannot find formatdb. Is it installed? Is it in your path?

I understand that this has something to do with blast not being in my path?

When I type which blastall and then $echo PATH, I get the following:

/macqiime/QIIME/bin:/macqiime/bin:/macqiime/rtax-0.984:/macqiime/rtax-0.984/scripts:/macqiime/anaconda/bin:/macqiime/microbiomeutil_2010-04-29/ChimeraSlayer:/macqiime/microbiomeutil_2010-04-29/NAST-Ier:/macqiime/microbiomeutil_2010-04-29/WigeoN:/macqiime/blat:/Library/Frameworks/Python.framework/Versions/2.7/bin:/usr/bin:/bin:/usr/sbin:/sbin:/usr/local/bin:/opt/X11/bin

But I do not know how go get blast into my path.  I have downloaded ncbi-blast-2.6.0+ and it is currently installed in my home directory.  I tried modifying my PATH file by adding the following line:

export PATH=/ncbi-blast-2.6.0+/bin:${PATH} 

but that didn't work.  How do I get the blast files into my path correctly?

Thank you!

[Colin Brislawn]

Hello Heather,

Thanks for getting in touch. You are on the right track with downloading blast and adding it to your path. Qiime needs a specific version of legacy blast (not blast+). The link to download for OSX and Linux is on this page:

http://qiime.org/install/alternative.html#installing-qiime-natively-with-a-full-install 

Try setting that up, and let me know what you find.

Colin

[Heather E]

Thanks, Colin.  The link  you sent went to a page that said "The webpage at ftp://ftp.ncbi.nlm.nih.gov/blast/executables/release/2.2.22/blast-2.2.22-universal-macosx.tar.gz might be temporarily down or it may have moved permanently to a new web address."

However, I was able to download from here:

http://mirrors.vbi.vt.edu/mirrors/ftp.ncbi.nih.gov/blast/executables/blast%2B/2.2.22/

I downloaded the file (macosx.tar.gz) and installed according to the directions on the Werner lab page (http://www.wernerlab.org/software/macqiime/macqiime-installation/installing-blast-in-os-x) but I am still getting the same error and my path still looks the same when I type which blastall then echo $PATH.  If I type those commands in a regular terminal (not with a macqiime terminal) there is shortened output:

DENR-MNS-49F9VN:~ genlab$ which blastall

DENR-MNS-49F9VN:~ genlab$ echo $PATH

/Library/Frameworks/Python.framework/Versions/2.7/bin:/usr/bin:/bin:/usr/sbin:/sbin:/usr/local/bin:/opt/X11/bin

I don't know that that info helps at all, just trying to put everything out there!

Also, my file is named ncbi-blast-2.2.22+, which is slightly different than the one mentioned (blast-2.2.22).  Is this still the right program?

[Colin Brislawn]

Hello Heather,

Thanks for alerting us to this broken link... I'll see if can fix the one on the website. Try this one: 

ftp://ftp.ncbi.nlm.nih.gov/blast/executables/legacy/2.2.22/ 

Also, my file is named ncbi-blast-2.2.22+, which is slightly different than the one mentioned (blast-2.2.22).  Is this still the right program?

I'm not sure. If you see the program 'formatdb' along with blast, it might be right. That + makes me think it may be blast+, instead of the needed version of legacy-blast.

Sorry about that link. Let me know if this works better,

Colin

[Heather E]

Ok, I was able to install the correct version of blast and my command ran without error.  However, the file was empty.  Which seems odd. 

This is my command:

parallel_identify_chimeric_seqs.py -i ./Documents/Evans/18S_chimera/rep_set_aligned.fasta -a ./Documents/Evans/18S_chimera/99_otus_aligned.fasta -o ./Documents/Evans/18S_chimera/chimeric_seqs.txt -m ChimeraSlayer -O 2

I am using SILVA128 as my reference database for 18S sequence.

My print_qiime_config.py is below:

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


Did I really have no chimeras or was it comparing against the wrong database?

[Colin Brislawn]

Hello Heather,

I'm glad you got it installed and running. I'm not very familiar with chimera slayer, so I'm not sure why it may not have found results... 

Perhaps you could try another database, or maybe a different algorithm. The chimera slayer devs now recommend using uchime, so that may be worth a shot.

Colin