Problem in pick_open_reference.py_ ERROR

[David Seminario]

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]

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]

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]

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. 

http://qiime.org/home_static/dataFiles.html

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]

Hi Colin and David,

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

https://groups.google.com/d/msg/qiime-forum/VrPSwlw_5go/TK1sj6FFBwAJ

https://groups.google.com/d/msg/qiime-forum/TvPVbJ2o5lY/GDvIXeetnRQJ

Best,

Jai

[David Seminario]

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]

Hello David,

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

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

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

Colin

[Greg Caporaso]

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:

http://www.wernerlab.org/software/macqiime/macqiime-installation/installing-blast-in-os-x

Greg