Error with PICRUST2

Hi,

I am having some issues with running PICRUST2. I am running it on a conda environment of its own (not as a Qiime2 plug-in since I could not install it as a plug-in).

I ran the following command:

picrust2_pipeline.py -s rep-set-dna-sequences.fasta -i feature-table.biom -o picrust2_out -p 1 --verbose

And I got the following output:

Placing sequences onto reference tree
place_seqs.py --study_fasta rep-set-dna-sequences.fasta --ref_dir /home/sam/anaconda3/envs/picrust2/lib/python3.6/site-packages/picrust2/default_files/prokaryotic/pro_ref --out_tree picrust2_out/out.tre --processes 1 --intermediate picrust2_out/intermediate/place_seqs --min_align 0.8 --chunk_size 5000 --verbose

Error running this command:
place_seqs.py --study_fasta rep-set-dna-sequences.fasta --ref_dir /home/sam/anaconda3/envs/picrust2/lib/python3.6/site-packages/picrust2/default_files/prokaryotic/pro_ref --out_tree picrust2_out/out.tre --processes 1 --intermediate picrust2_out/intermediate/place_seqs --min_align 0.8 --chunk_size 5000 --verbose

Standard output of the above failed command:



Standard error of the above failed command:
hmmalign --trim --dna --mapali /home/sam/anaconda3/envs/picrust2/lib/python3.6/site-packages/picrust2/default_files/prokaryotic/pro_ref/pro_ref.fna.gz --informat FASTA -o picrust2_out/intermediate/place_seqs/query_align.stockholm /home/sam/anaconda3/envs/picrust2/lib/python3.6/site-packages/picrust2/default_files/prokaryotic/pro_ref/pro_ref.hmm rep-set-dna-sequences.fasta

Warning - 12 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: 1b50bc8d744a56e26b53e5d9489fba2c, cf8b49e0970433dcf019fb9aee7ddedd, b178a3acb08d937baa5b4f8fd33bd45e, dd9a9cf0c265ffc7ff979fb8a500693f, b210158e4831c2b2d711283f48ed7c93, 33cce47427ef83813a149cfa9c717324, 3964a219fae7f81d8c054f360b0b88de, 36eb1b3e09ef3fa2bd7dfe9b004451bf, 0f716a00781a7d1506bc1affc0ac0c02, 4a2eaace4bd703d66c5b77384d44354d, c590df39769ae453c7d4252f851d63b8, a62c2607b1dec09fe1e19bada0d9e8d9

Raw input sequences ranged in length from 250 to 425

epa-ng --tree /home/sam/anaconda3/envs/picrust2/lib/python3.6/site-packages/picrust2/default_files/prokaryotic/pro_ref/pro_ref.tre --ref-msa picrust2_out/intermediate/place_seqs/ref_seqs_hmmalign.fasta --query picrust2_out/intermediate/place_seqs/study_seqs_hmmalign.fasta --chunk-size 5000 -T 1 -m /home/sam/anaconda3/envs/picrust2/lib/python3.6/site-packages/picrust2/default_files/prokaryotic/pro_ref/pro_ref.model -w picrust2_out/intermediate/place_seqs/epa_out --filter-acc-lwr 0.99 --filter-max 100

Error running this command:
epa-ng --tree /home/sam/anaconda3/envs/picrust2/lib/python3.6/site-packages/picrust2/default_files/prokaryotic/pro_ref/pro_ref.tre --ref-msa picrust2_out/intermediate/place_seqs/ref_seqs_hmmalign.fasta --query picrust2_out/intermediate/place_seqs/study_seqs_hmmalign.fasta --chunk-size 5000 -T 1 -m /home/sam/anaconda3/envs/picrust2/lib/python3.6/site-packages/picrust2/default_files/prokaryotic/pro_ref/pro_ref.model -w picrust2_out/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/intermediate/place_seqs/epa_out/
INFO Selected: Query file: picrust2_out/intermediate/place_seqs/study_seqs_hmmalign.fasta
INFO Selected: Tree file: /home/sam/anaconda3/envs/picrust2/lib/python3.6/site-packages/picrust2/default_files/prokaryotic/pro_ref/pro_ref.tre
INFO Selected: Reference MSA: picrust2_out/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/sam/anaconda3/envs/picrust2/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: picrust2_out/intermediate/place_seqs/epa_out/epa_result.jplace

Could you please help me?

Cheers,
Pablo

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