Problem in pick_open_reference.py_ ERROR

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David Seminario

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Apr 12, 2017, 9:54:08 AM4/12/17
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Very good morning to all and mr. Greg caporasso.

First of all I wish to inform you that I wish to analyze the metagenomic data of fungi coming from IonTorrent sequencing.

I have run the command:

MacQIIME MacBook-Pro-of-David: qiime_bernabe $ pick_open_reference_otus.py -i Split_Library_ITS / seqs.fna -p silva-123-params.txt -r SILVA_128_QIIME_release / rep_set / rep_set_all / 99 / 99_otus.fasta -o or-silva123-out
Traceback (most recent call last):
  File "/macqiime/anaconda/bin/pick_open_reference_otus.py", line 453, in <module>
    Main ()
  File "/macqiime/anaconda/bin/pick_open_reference_otus.py", line 432, in main
    Minimum_failure_threshold = minimum_failure_threshold)
  File "/macqiime/anaconda/lib/python2.7/site-packages/qiime/workflow/pick_open_reference_otus.py", line 1030, in pick_subsampled_open_reference_otus
    Status_update_callback = status_update_callback)
  File "/macqiime/anaconda/lib/python2.7/site-packages/qiime/workflow/pick_open_reference_otus.py", line 232, in assign_tax
    Close_logger_on_success = close_logger_on_success)
  File "/macqiime/anaconda/lib/python2.7/site-packages/qiime/workflow/util.py", line 122, in call_commands_serially
    Raise WorkflowError (msg)
Qiime.workflow.util.WorkflowError:

*** ERROR RAISED DURING STEP: Assign taxonomy
Command run was:
 Assign_taxonomy.py -o or-silva123-out / uclust_assigned_taxonomy -i or-silva123-out / rep_set.fna --reference_seqs_fp SILVA_128_QIIME_release / rep_set / rep_set_all / 99 / 99_otus.fasta --id_to_taxonomy_fp SILVA_128_QIIME_release / taxonomy_all / 99 / taxonomy_7_levels.txt
Command returned exit status: 2
Stdout:

Stderr
Error in assign_taxonomy.py: option --id_to_taxonomy_fp: file does not exist: 'SILVA_128_QIIME_release / taxonomy_all / 99 / taxonomy_7_levels.txt'

But I get problems I wish they would help me; thank you very much

Colin Brislawn

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Apr 12, 2017, 1:12:02 PM4/12/17
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Good morning David,

Thanks for getting in touch with us!

Did you look through the error message for clues? I found this one:
Error in assign_taxonomy.py: option --id_to_taxonomy_fp: file does not exist: 'SILVA_128_QIIME_release / taxonomy_all / 99 / taxonomy_7_levels.txt'

I noticed that there are spaces within this file path. I think this will confuse the linux terminal. Try running this script without spaces in the file paths, and let me know how this works. For example:
pick_open_reference_otus.py -i Split_Library_ITS/seqs.fna -p silva-123-params.txt -r SILVA_128_QIIME_release/rep_set/rep_set_all/99/99_otus.fasta -o or-silva123-out

Colin

David Seminario

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Apr 14, 2017, 10:07:56 AM4/14/17
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Mate again I have done the command, but it is not a problem of the spaces that I have but I do thank you very much


Pick_open_reference_otus.py -i Split_Library_ITS / seqs.fna -p silva-123-params.txt -r SILVA_128_QIIME_release / rep_set / rep_set_all / 99 / 99_otus.fasta -o or-silva123-out

Traceback (most recent call last):
  File "/macqiime/anaconda/bin/pick_open_reference_otus.py", line 453, in <module>
    Main ()
  File "/macqiime/anaconda/bin/pick_open_reference_otus.py", line 432, in main
    Minimum_failure_threshold = minimum_failure_threshold)
  File "/macqiime/anaconda/lib/python2.7/site-packages/qiime/workflow/pick_open_reference_otus.py", line 1071, in pick_subsampled_open_reference_otus
    Status_update_callback = status_update_callback)
  File "/macqiime/anaconda/lib/python2.7/site-packages/qiime/workflow/pick_open_reference_otus.py", line 327, in align_and_tree
    Close_logger_on_success = close_logger_on_success)
  File "/macqiime/anaconda/lib/python2.7/site-packages/qiime/workflow/util.py", line 122, in call_commands_serially
    Raise WorkflowError (msg)
Qiime.workflow.util.WorkflowError:

*** ERROR RAISED DURING STEP: Filter alignment
Command run was:
 Filter_alignment.py -o example2 / pynast_aligned_seqs -i example2 / pynast_aligned_seqs / rep_set_aligned.fasta --allowed_gap_frac 0.80 --entropy_threshold 0.10 --suppress_lane_mask_filter
Command returned exit status: 1
Stdout:

Stderr
Traceback (most recent call last):
  File "/macqiime/anaconda/bin/filter_alignment.py", line 155, in <module>
    Main ()
  File "/macqiime/anaconda/bin/filter_alignment.py", line 108, in main
    Raise ValueError ("An empty fasta file was provided."
ValueError: An empty fasta file was provided. Did the alignment complete sucessfully? Did PyNAST discard all sequences due to too-stringent minimum length or minimum percent ID settings?

Colin Brislawn

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Apr 14, 2017, 12:39:25 PM4/14/17
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Hello David,

Ah OK. The spaces in the command just surprised me. 

ValueError: An empty fasta file was provided. Did the alignment complete sucessfully? Did PyNAST discard all sequences due to too-stringent minimum length or minimum percent ID settings?
This error can happen during the PyNAST step if none of your reads match the reference database above the similarity threshold. 

I noticed that your input folder is Split_Library_ITS. Are your amplicons from the ITS region? If so, you could try using the UNITE database as the reference. 
You can download UNITE here: https://unite.ut.ee/repository.php 

I also noticed that your database is silva-123. This consistent with the "Did PyNAST discard all sequences" error because few or none of the ITS reads should be able to align to the silva database. 

Let me know what you try next,
Colin

Jai Ram Rideout

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Apr 14, 2017, 5:36:10 PM4/14/17
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Hi Colin and David,

ITS reads are difficult to align, see these posts for more details and some workarounds:



Best,
Jai

David Seminario

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Apr 15, 2017, 1:34:18 AM4/15/17
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thank you very much; Jam.

I was able to make the recommendations indicated; Thanks to this I have been able to assign otus for ITS sequences. Besides performing alpha and beta diversity ..


many thanks. I have done these analyzes in ubuntu 16.04


Another question if it is not annoyance; I want to install all the necessary packages on laptop macos; Because so far I can not install blast in MACQIIME any help I would appreciate it very much.

Colin Brislawn

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Apr 15, 2017, 1:47:40 PM4/15/17
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Hello David,

I have qiime installed on my mac, using the conda package. Full documentation here: 

Have you tried this version? How far did it get before failing?

Colin

Greg Caporaso

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Apr 17, 2017, 7:07:27 PM4/17/17
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Hi David and Colin,
MacQIIME doesn't install BLAST, and neither does the QIIME 1 conda install. Jeff Werner (the MacQIIME developer) has posted these instructions about getting BLAST installed on OS X:

Greg
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