Picrust2 novel user error

[we32i225]

Dear Picrust team
Firstly apologies if this is a common issue, i am trying to run picrust2 using conda (i have used microbiomeanalyst in the past). I have the FASTA and BIOM files however when i ran it, it did not run and i am not sure what the actual error here is.
Here is the whole of the epa_info.log file (which i believe is the error log?)

INFO Selected: Output dir: picrust2_out_biocharr/intermediate/place_seqs/epa_out/
INFO Selected: Query file: picrust2_out_biocharr/intermediate/place_seqs/study_seqs_hmmalign.fasta
INFO Selected: Tree file: /home/we32/anaconda3/envs/picrust2/lib/python3.6/site-packages/picrust2/default_files/prokaryotic/pro_ref/pro_ref.tre
INFO Selected: Reference MSA: picrust2_out_biocharr/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/we32/anaconda3/envs/picrust2/lib/python3.6/site-packages/picrust2/default_files/prokaryotic/pro_ref/pro_ref.model
INFO Selected: Reading queries in chunks of: 5000
INFO Selected: Using threads: 1
INFO ______ ____ ___ _ __ ______
/ // __ \ / | / | / // /
/ __/ / // // /| | ______ / |/ // / __
/ / / // ___ |/_____// /| // // /
/_____/// // || // |/ __/ (v0.3.8)

Below is the whole error message received

Error running this command:
place_seqs.py --study_fasta biochar_picrust2_files/biochar_OTU.fna --ref_dir /home/we32/anaconda3/envs/picrust2/lib/python3.6/site-packages/picrust2/default_files/prokaryotic/pro_ref --out_tree picrust2_out_biocharr/out.tre --processes 1 --intermediate picrust2_out_biocharr/intermediate/place_seqs --min_align 0.8 --chunk_size 5000 --placement_tool epa-ng

Standard error of the above failed command:
Warning - 64 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: ASV4213, ASV11622, ASV5913, ASV12498, ASV14688, ASV14305, ASV11074, ASV5819, ASV13298, ASV13677, ASV13027, ASV6359, ASV14776, ASV837, ASV12627, ASV5419, ASV11258, ASV14669, ASV14790, ASV14218, ASV12761, ASV13536, ASV12763, ASV3557, ASV249, ASV14677, ASV10936, ASV9234, ASV13004, ASV10852, ASV11520, ASV12770, ASV10605, ASV1739, ASV10810, ASV14774, ASV12750, ASV4263, ASV10341, ASV14387, ASV14277, ASV14887, ASV12892, ASV5304, ASV10366, ASV12528, ASV14496, ASV1090, ASV12876, ASV11557, ASV9615, ASV14826, ASV7586, ASV11615, ASV9862, ASV8001, ASV10645, ASV6357, ASV11306, ASV11849, ASV7909, ASV1290, ASV12731, ASV10696

Error running this command:
epa-ng --tree /home/we32/anaconda3/envs/picrust2/lib/python3.6/site-packages/picrust2/default_files/prokaryotic/pro_ref/pro_ref.tre --ref-msa picrust2_out_biocharr/intermediate/place_seqs/ref_seqs_hmmalign.fasta --query picrust2_out_biocharr/intermediate/place_seqs/study_seqs_hmmalign.fasta --chunk-size 5000 -T 1 -m /home/we32/anaconda3/envs/picrust2/lib/python3.6/site-packages/picrust2/default_files/prokaryotic/pro_ref/pro_ref.model -w picrust2_out_biocharr/intermediate/place_seqs/epa_out --filter-acc-lwr 0.99 --filter-max 100

Again apologies if this is a noob error
Kind regards
Will

[LY1989eco]

reduce the value of chunk_size. #376

[we32i225]

i have tried running the command reccomened by @R-Wright-1
place_seqs.py -s cleanfeature.fasta -o placed_seqs.tre -p 1
--intermediate placement_working --chunk_size 1000 (using my sequences and biom file)

no luck, however i think my issues might be more severe (or more stupid), i tried running the same pcirst2 pipeline command using the tutorial data and it also failed. error below

Error running this command:
place_seqs.py --study_fasta chemerin_16S/seqs.fna --ref_dir /home/we32/anaconda3/envs/picrust2/lib/python3.6/site-packages/picrust2/default_files/prokaryotic/pro_ref --out_tree picrust2_out_tutorial/out.tre --processes 1 --intermediate picrust2_out_tutorial/intermediate/place_seqs --min_align 0.8 --chunk_size 5000 --placement_tool epa-ng

Standard error of the above failed command:

Error running this command:
epa-ng --tree /home/we32/anaconda3/envs/picrust2/lib/python3.6/site-packages/picrust2/default_files/prokaryotic/pro_ref/pro_ref.tre --ref-msa picrust2_out_tutorial/intermediate/place_seqs/ref_seqs_hmmalign.fasta --query picrust2_out_tutorial/intermediate/place_seqs/study_seqs_hmmalign.fasta --chunk-size 5000 -T 1 -m /home/we32/anaconda3/envs/picrust2/lib/python3.6/site-packages/picrust2/default_files/prokaryotic/pro_ref/pro_ref.model -w picrust2_out_tutorial/intermediate/place_seqs/epa_out --filter-acc-lwr 0.99 --filter-max 100

Standard output of the above failed command:
INFO Selected: Output dir: picrust2_out_tutorial/intermediate/place_seqs/epa_out/
INFO Selected: Query file: picrust2_out_tutorial/intermediate/place_seqs/study_seqs_hmmalign.fasta
INFO Selected: Tree file: /home/we32/anaconda3/envs/picrust2/lib/python3.6/site-packages/picrust2/default_files/prokaryotic/pro_ref/pro_ref.tre
INFO Selected: Reference MSA: picrust2_out_tutorial/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/we32/anaconda3/envs/picrust2/lib/python3.6/site-packages/picrust2/default_files/prokaryotic/pro_ref/pro_ref.model
INFO Selected: Reading queries in chunks of: 5000
INFO Selected: Using threads: 1
INFO ______ ____ ___ _ __ ______
/ // __ \ / | / | / // /
/ __/ / // // /| | ______ / |/ // / __
/ / / // ___ |/_____// /| // // /
/_____/// // || // |/ __/ (v0.3.8)
INFO Using model parameters:
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 Output file: picrust2_out_tutorial/intermediate/place_seqs/epa_out/epa_result.jplace

so now im still not sure what my issue here is. Thanks for your help regardless!

[R-Wright-1]

Hi there,

Apologies for my incredibly slow reply. If you're willing to, could you send me the file that you are trying to run? I can see if this is an issue with your installation/your machine or your file.

I have also just added some general troubleshooting details to the top of the FAQ page, so you could also try running PICRUSt2 with the example data that I give there to see if it is an issue with your installation or your data.

Thanks,
Robyn

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