#pick_open_reference_otus.py -i '/home/eri/jf/DW/dw_ac_splib/dw_ac_all_tags.seqOut' -o '/home/eri/jf/DW/dw_ac_smr_sumac' -p '/home/eri/jf/q_params.txt' -m sortmerna_sumaclust Logging started at 17:23:09 on 15 May 2017 QIIME version: 1.9.1 qiime_config values: blastmat_dir /usr/share/ncbi/data pick_otus_reference_seqs_fp /home/eri/jf/MV_i/db_silva_128/rep_all_97_otus.fasta sc_queue all.q pynast_template_alignment_fp /home/eri/jf/MV_i/db_silva_128/core_alignment_SILVA128.fna assign_taxonomy_reference_seqs_fp /home/eri/jf/MV_i/db_silva_128/rep_all_97_otus.fasta torque_queue friendlyq jobs_to_start 22 denoiser_min_per_core 50 assign_taxonomy_id_to_taxonomy_fp /home/eri/jf/MV_i/db_silva_128/tax_all_97/majority_taxonomy_7_levels.txt temp_dir /tmp/ blastall_fp blastall seconds_to_sleep 45 parameter file values: alpha_diversity:metrics shannon,PD_whole_tree,chao1,observed_otus,simpson filter_alignment.py:allowed_gap_frac 0.80 filter_alignment.py:entropy_threshold 0.0005 pick_open_reference_otus.py:otu_picking_method sortmerna_sumaclust pick_open_reference_otus.py:min_otu_size 2 pick_open_reference_otus.py:reference_fp /home/eri/jf/MV_i/db_silva_128/rep_all_97_otus.fasta pick_open_reference_otus.py:percent_subsample 0.01 pick_open_reference_otus.py:jobs_to_start 20 pick_open_reference_otus.py:parallel True parallel:jobs_to_start 22 Input file md5 sums: /home/eri/jf/DW/dw_ac_splib/dw_ac_all_tags.seqOut: 39ce6b171adb22da8d32c17d73376922 /home/eri/jf/MV_i/db_silva_128/rep_all_97_otus.fasta: ceebb6b091e1d899ea6aef2e1104d7e4 Forcing --suppress_new_clusters as this is reference-based OTU picking. Executing commands. # Pick Reference OTUs command pick_otus.py -i /home/eri/jf/DW/dw_ac_splib/dw_ac_all_tags.seqOut -o dw_ac_smr_sumac/step1_otus -r /home/eri/jf/MV_i/db_silva_128/rep_all_97_otus.fasta -m uclust_ref --suppress_new_clusters Stdout: Stderr: # Generate full failures fasta file command filter_fasta.py -f /home/eri/jf/DW/dw_ac_splib/dw_ac_all_tags.seqOut -s dw_ac_smr_sumac/step1_otus/dw_ac_all_tags_failures.txt -o dw_ac_smr_sumac/step1_otus/failures.fasta Stdout: Stderr: Executing commands. # Pick rep set command pick_rep_set.py -i dw_ac_smr_sumac/step1_otus/dw_ac_all_tags_otus.txt -o dw_ac_smr_sumac/step1_otus/step1_rep_set.fna -f /home/eri/jf/DW/dw_ac_splib/dw_ac_all_tags.seqOut Stdout: Stderr: Executing commands. # Pick de novo OTUs on step1 failures command pick_otus.py -i dw_ac_smr_sumac/step1_otus/failures.fasta -o dw_ac_smr_sumac/step4_otus/ -m uclust --denovo_otu_id_prefix New.CleanUp.ReferenceOTU Stdout: Stderr: # Merge OTU maps command cat dw_ac_smr_sumac/step1_otus/dw_ac_all_tags_otus.txt dw_ac_smr_sumac/step4_otus//failures_otus.txt > dw_ac_smr_sumac/final_otu_map.txt Stdout: Stderr: # Pick representative set for subsampled failures command pick_rep_set.py -i dw_ac_smr_sumac/step4_otus//failures_otus.txt -o dw_ac_smr_sumac/step4_otus//step4_rep_set.fna -f dw_ac_smr_sumac/step1_otus/failures.fasta Stdout: Stderr: # Filter singletons from the otu map using API python -c "import qiime; qiime.filter.filter_otus_from_otu_map('/home/eri/jf/DW/dw_ac_smr_sumac/final_otu_map.txt', '/home/eri/jf/DW/dw_ac_smr_sumac/final_otu_map_mc2.txt', '2')" # Write non-singleton otus representative sequences from step1 to the final rep set file: dw_ac_smr_sumac/rep_set.fna # Copy the full input refseqs file to the new refseq file cp /home/eri/jf/MV_i/db_silva_128/rep_all_97_otus.fasta dw_ac_smr_sumac/new_refseqs.fna # Write non-singleton otus representative sequences from step 4 to the final representative set and the new reference set (dw_ac_smr_sumac/rep_set.fna and dw_ac_smr_sumac/new_refseqs.fna respectively) Executing commands. # Make the otu table command make_otu_table.py -i dw_ac_smr_sumac/final_otu_map_mc2.txt -o dw_ac_smr_sumac/otu_table_mc2.biom Stdout: Stderr: Executing commands. # Assign taxonomy command assign_taxonomy.py -o dw_ac_smr_sumac/uclust_assigned_taxonomy -i dw_ac_smr_sumac/rep_set.fna Stdout: Stderr: Executing commands. # Add taxa to OTU table command biom add-metadata -i dw_ac_smr_sumac/otu_table_mc2.biom --observation-metadata-fp dw_ac_smr_sumac/uclust_assigned_taxonomy/rep_set_tax_assignments.txt -o dw_ac_smr_sumac/otu_table_mc2_w_tax.biom --sc-separated taxonomy --observation-header OTUID,taxonomy Stdout: Stderr: Executing commands. # Align sequences command align_seqs.py -i dw_ac_smr_sumac/rep_set.fna -o dw_ac_smr_sumac/pynast_aligned_seqs Stdout: Stderr: # Filter alignment command filter_alignment.py -o dw_ac_smr_sumac/pynast_aligned_seqs -i dw_ac_smr_sumac/pynast_aligned_seqs/rep_set_aligned.fasta *** ERROR RAISED DURING STEP: Filter alignment Command run was: filter_alignment.py -o dw_ac_smr_sumac/pynast_aligned_seqs -i dw_ac_smr_sumac/pynast_aligned_seqs/rep_set_aligned.fasta Command returned exit status: 1 Stdout: Stderr Traceback (most recent call last): File "/usr/local/bin/filter_alignment.py", line 155, in main() File "/usr/local/bin/filter_alignment.py", line 148, in main entropy_threshold=opts.entropy_threshold): File "/usr/local/lib/python2.7/dist-packages/qiime/filter_alignment.py", line 87, in apply_lane_mask_and_gap_filter raise ValueError("Positional filtering resulted in removal of all " ValueError: Positional filtering resulted in removal of all alignment positions. Logging stopped at 17:48:36 on 15 May 2017