ERROR RAISED DURING STEP: Pick OTUs

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estefania...@gmail.com

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Aug 24, 2016, 2:20:14 PM8/24/16
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Hi,
I've been having problems my data.
I've launched the qiime instance in Amazon EC2 and when I run the command pick_de_novo_otus.py, its says theres and error:
Traceback (most recent call last)
  File "/usr/local/bin/pick_de_novo_otus.py", line 180, in <module>
    main()
  File "/usr/local/bin/pick_de_novo_otus.py", line 177, in main
    status_update_callback=status_update_callback)
  File "/usr/local/lib/python2.7/dist-packages/qiime/workflow/upstream.py", line 306, in run_pick_de_novo_otus
    close_logger_on_success=close_logger_on_success)
  File "/usr/local/lib/python2.7/dist-packages/qiime/workflow/util.py", line 122, in call_commands_serially
    raise WorkflowError(msg)
qiime.workflow.util.WorkflowError:

*** ERROR RAISED DURING STEP: Pick OTUs
Command run was:
 pick_otus.py -i combined_seqs.fna -o denovo/uclust_otus//uclust_picked_otus
Command returned exit status: 1
Stdout:

Stderr
Traceback (most recent call last):
  File "/usr/local/bin/pick_otus.py", line 1004, in <module>
    main()
  File "/usr/local/bin/pick_otus.py", line 792, in main
    result_path=result_path, log_path=log_path, HALT_EXEC=False)
  File "/usr/local/lib/python2.7/dist-packages/qiime/pick_otus.py", line 1286, in __call__
    HALT_EXEC=HALT_EXEC)
  File "/usr/local/lib/python2.7/dist-packages/bfillings/uclust.py", line 585, in get_clusters_from_fasta_filepath
    raise ApplicationError('Error running uclust. Possible causes are '
burrito.util.ApplicationError: Error running uclust. Possible causes are unsupported version (current supported version is v1.2.22) is installed or improperly formatted input file was provided.

  • My data weight 2.1 GB and has 21 samples with 200,00
 to 300,000 sequence per sample.
  • the print_qiime_config.py -tf command shows this:

System information
==================
         Platform:    linux2
   Python version:    2.7.3 (default, Aug  1 2012, 05:14:39)  [GCC 4.6.3]
Python executable:    /usr/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:    2.5.0 (HDF5 version: 1.8.4)
                         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 [May 26 2015 15:28:37]
                                gdata:    Installed.
RDP Classifier version (if installed):    rdp_classifier-2.2.jar
          Java version (if installed):    1.6.0_34

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:    /home/ubuntu/qiime_software/blast-2.2.22-release/data
      pick_otus_reference_seqs_fp:    /usr/local/lib/python2.7/dist-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:    /usr/local/lib/python2.7/dist-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:    /usr/local/lib/python2.7/dist-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:    /usr/local/lib/python2.7/dist-packages/qiime_default_reference/gg_13_8_otus/taxonomy/97_otu_taxonomy.txt
                         temp_dir:    /tmp/
                     slurm_memory:    None
                      slurm_queue:    None
                      blastall_fp:    /home/ubuntu/qiime_software/blast-2.2.22-release/bin/blastall
                 seconds_to_sleep:    1


If you could help me I'd appreciate so much.


Constantino Schillebeeckx

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Aug 24, 2016, 2:41:09 PM8/24/16
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Did you install uclust?  Ensure you have v1.2.22 or greater.


Next, there are two non-python dependencies required for the QIIME base package. These should be installed by following their respective install instructions.

Jose Antonio Navas Molina

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Aug 25, 2016, 12:57:49 AM8/25/16
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Hello,

Can you post the output of running:

uclust --version


?


Thanks!


estefania...@gmail.com

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Aug 25, 2016, 6:10:56 PM8/25/16
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Hi,
I dont know if I i didnt install correctly the UCLUST, because it doesnt appear in the output of the  print_qiime_config.py command. that I've already posted.

The version of UCLUST currently instaled is uclust v1.2.22q
(C) Copyright 2009-10 Robert C. Edgar

Thanks for answering

Jose Antonio Navas Molina

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Aug 25, 2016, 10:06:29 PM8/25/16
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Hello,

I just re-read your original post and realized that you're using the QIIME AMI in Amazon AWS. Which instance size are you using? It is possible that the instance that you're using is not big enough, and uclust is failing with some uninformative error.

Thanks,

estefania...@gmail.com

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Sep 1, 2016, 12:19:52 AM9/1/16
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Hi,
I am using 3m.large size.
Thanks

Jose Antonio Navas Molina

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Sep 1, 2016, 12:36:33 AM9/1/16
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7.5 GB of memory may not be enough. You can probably give it a try with a m3.xlarge.
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