Error : PICRUSt2 Tutorial

[Khalid IBRAHIM]

Hi everyone

 I have managed to run Swapping in SEPP for read placement with q2 picrust2 and then q2 picrust2 Tutorial using 12GB RAM. Now I am trying to use PICRUSt2 Tutorial (v2.2.0 beta) by Gavin Douglas edited this page on 18 Dec 2019 · 14 revisions. I received an error when trying run

place_seqs.py -s ../seqs.fna -o out.tre -p 1 \

              --intermediate intermediate/place_seqs

Warning - 2 input sequences aligned poorly to reference sequences (--min_align option specified a minimum proportion of 0.8 aligning to reference sequences). These input sequences will not be placed and will be excluded from downstream steps.

This is the set of poorly aligned input sequences to be excluded: 8dc3f5267079bb1860d7af669963ac99, 054b9939937bd3d8323c3c85a66897f3

Error running this command:

epa-ng --tree /home/qiime2/miniconda/envs/qiime2-2019.10/lib/python3.6/site-packages/picrust2/default_files/prokaryotic/pro_ref/pro_ref.tre --ref-msa intermediate/place_seqs/ref_seqs_hmmalign.fasta --query intermediate/place_seqs/study_seqs_hmmalign.fasta --chunk-size 5000 -T 1 -m /home/qiime2/miniconda/envs/qiime2-2019.10/lib/python3.6/site-packages/picrust2/default_files/prokaryotic/pro_ref/pro_ref.model -w intermediate/place_seqs/epa_out --filter-acc-lwr 0.99 --filter-max 100

Standard output of the above failed command:

INFO Selected: Output dir: intermediate/place_seqs/epa_out/

INFO Selected: Query file: intermediate/place_seqs/study_seqs_hmmalign.fasta

INFO Selected: Tree file: /home/qiime2/miniconda/envs/qiime2-2019.10/lib/python3.6/site-packages/picrust2/default_files/prokaryotic/pro_ref/pro_ref.tre

INFO Selected: Reference MSA: intermediate/place_seqs/ref_seqs_hmmalign.fasta

INFO Selected: Filtering by accumulated threshold: 0.99

INFO Selected: Maximum number of placements per query: 100

INFO Selected: Automatic switching of use of per rate scalers

INFO Selected: Preserving the root of the input tree

INFO Selected: Specified model file: /home/qiime2/miniconda/envs/qiime2-2019.10/lib/python3.6/site-packages/picrust2/default_files/prokaryotic/pro_ref/pro_ref.model

INFO    Rate heterogeneity: GAMMA (4 cats, mean),  alpha: 0.453141 (user),  weights&rates: (0.25,0.0250674) (0.25,0.220229) (0.25,0.782933) (0.25,2.97177) 

        Base frequencies (user): 0.229585 0.22008 0.298596 0.251739 

        Substitution rates (user): 1.00319 2.79077 1.5301 0.87441 3.83966 1

INFO Selected: Reading queries in chunks of: 5000

INFO Selected: Using threads: 1

INFO     ______ ____   ___           _   __ ______

        / ____// __ \ /   |         / | / // ____/

       / __/  / /_/ // /| | ______ /  |/ // / __  

      / /___ / ____// ___ |/_____// /|  // /_/ /  

     /_____//_/    /_/  |_|      /_/ |_/ \____/ (v0.3.6)

INFO Output file: intermediate/place_seqs/epa_out/epa_result.jplace

Could anyone help me to dissolve the error at the end? Thanks in advance.

Cheers

Khalid 

[Gavin Douglas]

Hey Khalid,

How much RAM do you have? EPA-ng usually fails at that step of the pipeline due to insufficient RAM, which you can get around by running SEPP instead with the QIIME 2 plugin instead, like you mentioned.

Cheers,

Gavin

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[Khalid IBRAHIM]

Dear Gavin 

Thank you so much for quickly replying.

 I am using Oracle VM VirtualBox (QIIME 2 Core system 11404MB) in Windows 10 system of 16GB.I managed to run  Swapping in SEPP for read placement with q2 picrust2 and I am trying to continuous running the PICRUSt2 pipeline to Generate metagenome predictions.

Cheers

Khalid

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[Khalid IBRAHIM]

Dear Gavin 

Thank you so much for quickly replying.

 I am using Oracle VM VirtualBox (QIIME 2 Core system 11404MB) in Windows 10 system of 16GB RAM.I managed to run  Swapping in SEPP for read placement with q2 picrust2 and I am trying to continuous running the PICRUSt2 pipeline to Generate metagenome predictions.

Cheers

Khalid

[Gavin Douglas]

Hey Khalid,

You typically need at least 16 GB of RAM to run the default pipeline. I would stick with running SEPP and the qiime 2 plugin to avoid the high RAM usage. Just to be clear, in that case SEPP is used instead of EPA-ng. If you have the output tree from SEPP then you can start from the “hsp.py” step of the standalone tutorial (or alternatively you can just use the QIIME 2 plugin to create the default outputs).

Cheers,

Gavin

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[Khalid IBRAHIM]

Hi Gavin

Thanks so much for replying,

Yes I used  SEPP  and I have got both  tree.qza and placements.qza in  the output file. Just I want to tell you that  I am new to q2p2 and I need a bit clarification of PICRUSt2 pipeline. Particularly, how can I start the “hsp.py” step of the standalone tutorial (or alternatively you can just use the QIIME 2 plugin to create the default outputs).

Cheers

Khalid 

[Gavin Douglas]

Hey there Khalid,

To use the standalone version of PICRUSt2 (i.e. outside of QIIME 2) you would need to export the tree QZA file produced by QIIME 2 and then start at the hsp.py step of the tutorial here: https://github.com/picrust/picrust2/wiki/PICRUSt2-Tutorial-(v2.3.0-beta)

To use the QIIME 2 plugin to get standard outputs you could use "qiime picrust2 custom-tree-pipeline" as described here: https://github.com/picrust/picrust2/wiki/Swapping-in-SEPP-for-read-placement-with-q2-picrust2

Cheers,

Gavin

To view this discussion on the web visit https://groups.google.com/d/msgid/picrust-users/1ff6bb9a-f1d5-43e0-ab4f-e7dd51265868n%40googlegroups.com.

[Khalid IBRAHIM]

Hi Gavin,

Thank you for replying and Sorry for asking again,

I have got the below error (ValueError: No sequence ids overlap between all three of the input files.) when I runned the  to run 

metagenome_pipeline.py -i ../table.biom -m marker_predicted_and_nsti.tsv.gz -f EC_predicted.tsv.gz \

                       -o EC_metagenome_out --strat_out

First, I used (tree.nwk instead of out.tre) which extracted from tree.qza to run both 

hsp.py -i 16S -t out.tre -o marker_predicted_and_nsti.tsv.gz -p 1 -n

hsp.py -i EC -t out.tre -o EC_predicted.tsv.gz -p 1

Second, I used the feature-table.biom (instead of table.biom) from previously (ec_metagenome.qza)

qiime tools export \

   --input-path q2-picrust2_output/ec_metagenome.qza \

   --output-path pathabun_exported_ec

The commands error:

$ metagenome_pipeline.py -i feature-table.biom -m marker_predicted_and_nsti.tsv.gz -f EC_predicted.tsv.gz                        -o EC_metagenome_out --strat_out

0 of 976 ASVs were above the max NSTI cut-off of 2.0 and were removed.

Traceback (most recent call last):

  File "/home/qiime2/miniconda/envs/qiime2-2019.10/bin/metagenome_pipeline.py", line 122, in <module>

    main()

  File "/home/qiime2/miniconda/envs/qiime2-2019.10/bin/metagenome_pipeline.py", line 104, in main

    skip_norm=args.skip_norm)

  File "/home/qiime2/miniconda/envs/qiime2-2019.10/lib/python3.6/site-packages/picrust2/metagenome_pipeline.py", line 66, in run_metagenome_pipeline

    pred_marker)

  File "/home/qiime2/miniconda/envs/qiime2-2019.10/lib/python3.6/site-packages/picrust2/util.py", line 372, in three_df_index_overlap_sort

    "input files.")

ValueError: No sequence ids overlap between all three of the input files.

Cheers

Khalid 

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[Gavin Douglas]

Hey Khalid,

Would you mind sending me the file “feature-table.biom” too so I can help troubleshoot the ids? That should be the ASV abundance table just so we’re clear. The biom file you sent me was an abundance table of EC gene  families, which is the output of the metagenome prediction converted from the QIIME 2 plugin I think.

Cheers,

Gavin

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<marker_predicted_and_nsti.tsv.gz><EC_predicted.tsv.gz><tree.nwk><feature-table.biom_ec.tsv>