pick open reference otus

[Lekha Menon]

Hi,

We did the chimera removal using identify_chimeric_seqs.py -m usearch61 -i split_library_AN/seqs.fna -r SILVA_128_QIIME_release/rep_set/rep_set_16S_only/97/97_otus_16S.fasta -o usearch_chimera_removal --threads 4.0 and got the following files:

chimeras.txt

identify_chimeric_seqs.log

non_chimeras.txt

seqs.fna_chimeras_denovo.log

 seqs.fna_chimeras_denovo.uchime

seqs.fna_chimeras_ref.log

seqs.fna_chimeras_ref.uchime

seqs.fna_consensus_fixed.fasta

 seqs.fna_consensus_with_abundance.fasta

seqs.fna_consensus_with_abundance.uc

seqs.fna_smallmem_clustered.log

For the next step, we used the seqs.fna_consensus_fixed.fasta file for  OTU picking. We them combined the seqs.fna_consensus_fixed.fasta files from all samples using cat command and obtained combined_chimera_AN_seqs.fna. We then ran the following command to pick OTUs:

pick_open_reference_otus.py -i combined_chimera_AN_seqs.fna -r SILVA_128_QIIME_release/rep_set/rep_set_16S_only/97/97_otus_16S.fasta --suppress_taxonomy_assignment -o otus_usearch61_AN -O 2 -a -m usearch61

We got the following error:

qiime_config values:

pick_otus_reference_seqs_fp /opt/miniconda3/envs/qiime1/lib/python2.7/site-packages/qiime_default_reference/gg_13_8_otus/rep_set/97_otus.fasta

sc_queue all.q

pynast_template_alignment_fp /opt/miniconda3/envs/qiime1/lib/python2.7/site-packages/qiime_default_reference/gg_13_8_otus/rep_set_aligned/85_otus.pynast.fasta

cluster_jobs_fp start_parallel_jobs.py

assign_taxonomy_reference_seqs_fp /opt/miniconda3/envs/qiime1/lib/python2.7/site-packages/qiime_default_reference/gg_13_8_otus/rep_set/97_otus.fasta

torque_queue friendlyq

jobs_to_start 1

denoiser_min_per_core 50

assign_taxonomy_id_to_taxonomy_fp /opt/miniconda3/envs/qiime1/lib/python2.7/site-packages/qiime_default_reference/gg_13_8_otus/taxonomy/97_otu_taxonomy.txt

temp_dir /tmp/

blastall_fp blastall

seconds_to_sleep 1

parameter file values:

parallel:jobs_to_start 1

Input file md5 sums:

combined_chimera_AN_seqs.fna: 5254fb3c38c61968c2227b244362814e

SILVA_128_QIIME_release/rep_set/rep_set_16S_only/97/97_otus_16S.fasta: d969a5a991b8e087bf0a0b9e1fb16066

Forcing --suppress_new_clusters as this is reference-based OTU picking.

Executing commands.

# Pick Reference OTUs command 

pick_otus.py -i combined_chimera_AN_seqs.fna -o otus_usearch61_AN/step1_otus -r SILVA_128_QIIME_release/rep_set/rep_set_16S_only/97/97_otus_16S.fasta -m usearch61_ref  --suppress_new_clusters

*** ERROR RAISED DURING STEP: Pick Reference OTUs

Command run was:

 pick_otus.py -i combined_chimera_AN_seqs.fna -o otus_usearch61_AN/step1_otus -r SILVA_128_QIIME_release/rep_set/rep_set_16S_only/97/97_otus_16S.fasta -m usearch61_ref  --suppress_new_clusters

Command returned exit status: 1

Stdout:

Stderr

Traceback (most recent call last):

  File "/opt/miniconda3/envs/qiime1/bin/pick_otus.py", line 4, in <module>

    __import__('pkg_resources').run_script('qiime==1.9.1', 'pick_otus.py')

  File "/opt/miniconda3/envs/qiime1/lib/python2.7/site-packages/setuptools-27.2.0-py2.7.egg/pkg_resources/__init__.py", line 744, in run_script

    

  File "/opt/miniconda3/envs/qiime1/lib/python2.7/site-packages/setuptools-27.2.0-py2.7.egg/pkg_resources/__init__.py", line 1499, in run_script

    

  File "/opt/miniconda3/envs/qiime1/lib/python2.7/site-packages/qiime-1.9.1-py2.7.egg-info/scripts/pick_otus.py", line 1004, in <module>

    main()

  File "/opt/miniconda3/envs/qiime1/lib/python2.7/site-packages/qiime-1.9.1-py2.7.egg-info/scripts/pick_otus.py", line 897, in main

    otu_prefix=otu_prefix, HALT_EXEC=False)

  File "/opt/miniconda3/envs/qiime1/lib/python2.7/site-packages/qiime/pick_otus.py", line 1800, in __call__

    HALT_EXEC=HALT_EXEC

  File "/opt/miniconda3/envs/qiime1/lib/python2.7/site-packages/burrito_fillings-0.1.1-py2.7.egg/bfillings/usearch.py", line 1822, in usearch61_ref_cluster

  File "/opt/miniconda3/envs/qiime1/lib/python2.7/site-packages/burrito_fillings-0.1.1-py2.7.egg/bfillings/usearch.py", line 2504, in merge_clusters_dereplicated_seqs

KeyError: 'centroid=ANT6_18_joined/AN_6_18_43204'

Logging stopped at 12:53:07 on 20 Jul 2017

Would appreciate all the help. 

Thanks,

Lekha

[Yoshiki Vázquez Baeza]

Hi Lekha,

I've forwarded your question to someone else in the forum, I'll let them answer.

Thanks!

Yoshiki.

[TonyWalters]

Hello Lekha,

You want to use some different steps to filter out the chimeric reads. You'll filter the seqs.fna file (output from split_libraries folder) and the chimeras.txt file from your identify_chimeric_seqs output.

See: http://qiime.org/tutorials/chimera_checking.html#usearch-6-1

for the exact the commands you want to call.

[Lekha Menon]

Thank you.

[Lekha Menon]

Thank you Tony. That worked. I just realised that we were using a wrong input for filter fasta which messed up the pick otu step. Thanks once again.