pick open reference otus

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Lekha Menon

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Jul 20, 2017, 8:08:16 AM7/20/17
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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

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Jul 24, 2017, 5:54:26 PM7/24/17
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Hi Lekha,

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

Thanks!

Yoshiki.

TonyWalters

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Jul 25, 2017, 1:19:09 AM7/25/17
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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

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Jul 27, 2017, 11:22:42 AM7/27/17
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Thank you.

Lekha Menon

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Jul 27, 2017, 11:23:29 AM7/27/17
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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. 
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