06/25/2020 01:07:55 AM - humann2.humann2 - INFO: Running humann2 v2.8.1 06/25/2020 01:07:55 AM - humann2.humann2 - INFO: Output files will be written to: /public/home/nx_lfs/metaphlan2_analysis/demo_fastq 06/25/2020 01:07:55 AM - humann2.humann2 - INFO: Writing temp files to directory: /public/home/nx_lfs/metaphlan2_analysis/demo_fastq/demo_humann2_temp 06/25/2020 01:07:55 AM - humann2.utilities - INFO: File ( /public/home/nx_lfs/metaphlan2_analysis/demo.fastq ) is of format: fastq 06/25/2020 01:07:55 AM - humann2.utilities - DEBUG: Check software, bowtie2, for required version, 2.2 06/25/2020 01:07:55 AM - humann2.utilities - INFO: Using bowtie2 version 2.3 06/25/2020 01:07:55 AM - humann2.humann2 - INFO: Search mode set to uniref90 because a uniref90 translated search database is selected 06/25/2020 01:07:55 AM - humann2.utilities - DEBUG: Check software, diamond, for required version, 0.8.22 06/25/2020 01:07:55 AM - humann2.utilities - INFO: Using diamond version 0.9.34 06/25/2020 01:07:55 AM - humann2.config - INFO: Run config settings: DATABASE SETTINGS nucleotide database folder = /public/home/nx_lfs/db/chocophlan protein database folder = /public/home/nx_lfs/db/uniref pathways database file 1 = /public/home/nx_lfs/anaconda2/envs/metagenome_env/lib/python2.7/site-packages/humann2/data/pathways/metacyc_reactions_level4ec_only.uniref.bz2 pathways database file 2 = /public/home/nx_lfs/anaconda2/envs/metagenome_env/lib/python2.7/site-packages/humann2/data/pathways/metacyc_pathways_structured_filtered utility mapping database folder = /public/home/nx_lfs/anaconda2/envs/metagenome_env/lib/python2.7/site-packages/humann2/data/misc RUN MODES resume = False verbose = False bypass prescreen = False bypass nucleotide index = False bypass nucleotide search = False bypass translated search = False translated search = diamond pick frames = off threads = 1 SEARCH MODE search mode = uniref90 identity threshold = 90.0 ALIGNMENT SETTINGS evalue threshold = 1.0 prescreen threshold = 0.01 translated subject coverage threshold = 50.0 translated query coverage threshold = 90.0 PATHWAYS SETTINGS minpath = on xipe = off gap fill = on INPUT AND OUTPUT FORMATS input file format = fastq output file format = tsv output max decimals = 10 remove stratified output = False remove column description output = False log level = DEBUG 06/25/2020 01:07:55 AM - humann2.store - DEBUG: Initialize Alignments class instance to minimize memory use 06/25/2020 01:07:55 AM - humann2.store - DEBUG: Initialize Reads class instance to minimize memory use 06/25/2020 01:08:02 AM - humann2.humann2 - INFO: Load pathways database part 1: /public/home/nx_lfs/anaconda2/envs/metagenome_env/lib/python2.7/site-packages/humann2/data/pathways/metacyc_reactions_level4ec_only.uniref.bz2 06/25/2020 01:08:19 AM - humann2.humann2 - INFO: Load pathways database part 2: /public/home/nx_lfs/anaconda2/envs/metagenome_env/lib/python2.7/site-packages/humann2/data/pathways/metacyc_pathways_structured_filtered 06/25/2020 01:08:19 AM - humann2.utilities - DEBUG: Check software, metaphlan2.py, for required version, 2.6 06/25/2020 01:08:20 AM - humann2.utilities - INFO: Using metaphlan2.py version 2.7 06/25/2020 01:08:20 AM - humann2.search.prescreen - INFO: Running metaphlan2.py ........ 06/25/2020 01:08:20 AM - humann2.utilities - DEBUG: Using software: /public/home/nx_lfs/anaconda2/envs/metagenome_env/bin/metaphlan2.py 06/25/2020 01:08:20 AM - humann2.utilities - INFO: Execute command: /public/home/nx_lfs/anaconda2/envs/metagenome_env/bin/metaphlan2.py /public/home/nx_lfs/metaphlan2_analysis/demo.fastq -t rel_ab -o /public/home/nx_lfs/metaphlan2_analysis/demo_fastq/demo_humann2_temp/demo_metaphlan_bugs_list.tsv --input_type multifastq --bowtie2out /public/home/nx_lfs/metaphlan2_analysis/demo_fastq/demo_humann2_temp/demo_metaphlan_bowtie2.txt 06/25/2020 01:09:25 AM - humann2.utilities - DEBUG: Help message for read_fastx.py 06/25/2020 01:09:25 AM - humann2.humann2 - INFO: TIMESTAMP: Completed prescreen : 66 seconds 06/25/2020 01:09:25 AM - humann2.search.prescreen - INFO: Found g__Bacteroides.s__Bacteroides_dorei : 59.40% of mapped reads 06/25/2020 01:09:25 AM - humann2.search.prescreen - INFO: Found g__Bacteroides.s__Bacteroides_vulgatus : 40.60% of mapped reads 06/25/2020 01:09:25 AM - humann2.search.prescreen - INFO: Total species selected from prescreen: 2 06/25/2020 01:09:25 AM - humann2.search.prescreen - DEBUG: Adding file to database: g__Bacteroides.s__Bacteroides_vulgatus.centroids.v0.1.1.ffn.gz 06/25/2020 01:09:25 AM - humann2.search.prescreen - DEBUG: Adding file to database: g__Bacteroides.s__Bacteroides_dorei.centroids.v0.1.1.ffn.gz 06/25/2020 01:09:25 AM - humann2.search.prescreen - INFO: Creating custom ChocoPhlAn database ........ 06/25/2020 01:09:25 AM - humann2.utilities - DEBUG: Using software: /usr/bin/gunzip 06/25/2020 01:09:25 AM - humann2.utilities - INFO: Execute command: /usr/bin/gunzip -c /public/home/nx_lfs/db/chocophlan/g__Bacteroides.s__Bacteroides_vulgatus.centroids.v0.1.1.ffn.gz /public/home/nx_lfs/db/chocophlan/g__Bacteroides.s__Bacteroides_dorei.centroids.v0.1.1.ffn.gz 06/25/2020 01:09:26 AM - humann2.humann2 - INFO: TIMESTAMP: Completed custom database creation : 0 seconds 06/25/2020 01:09:26 AM - humann2.search.nucleotide - INFO: Running bowtie2-build ........ 06/25/2020 01:09:26 AM - humann2.utilities - DEBUG: Using software: /public/home/nx_lfs/anaconda2/envs/metagenome_env/bin/bowtie2-build 06/25/2020 01:09:26 AM - humann2.utilities - INFO: Execute command: /public/home/nx_lfs/anaconda2/envs/metagenome_env/bin/bowtie2-build -f /public/home/nx_lfs/metaphlan2_analysis/demo_fastq/demo_humann2_temp/demo_custom_chocophlan_database.ffn /public/home/nx_lfs/metaphlan2_analysis/demo_fastq/demo_humann2_temp/demo_bowtie2_index 06/25/2020 01:09:30 AM - humann2.humann2 - INFO: TIMESTAMP: Completed database index : 4 seconds 06/25/2020 01:09:30 AM - humann2.search.nucleotide - DEBUG: Nucleotide input file is of type: fastq 06/25/2020 01:09:30 AM - humann2.utilities - DEBUG: Using software: /public/home/nx_lfs/anaconda2/envs/metagenome_env/bin/bowtie2 06/25/2020 01:09:30 AM - humann2.utilities - INFO: Execute command: /public/home/nx_lfs/anaconda2/envs/metagenome_env/bin/bowtie2 -q -x /public/home/nx_lfs/metaphlan2_analysis/demo_fastq/demo_humann2_temp/demo_bowtie2_index -U /public/home/nx_lfs/metaphlan2_analysis/demo.fastq -S /public/home/nx_lfs/metaphlan2_analysis/demo_fastq/demo_humann2_temp/demo_bowtie2_aligned.sam --very-sensitive 06/25/2020 01:09:33 AM - humann2.utilities - DEBUG: 21000 reads; of these: 21000 (100.00%) were unpaired; of these: 4655 (22.17%) aligned 0 times 6018 (28.66%) aligned exactly 1 time 10327 (49.18%) aligned >1 times 77.83% overall alignment rate 06/25/2020 01:09:33 AM - humann2.humann2 - INFO: TIMESTAMP: Completed nucleotide alignment : 3 seconds 06/25/2020 01:09:34 AM - humann2.search.nucleotide - DEBUG: Total nucleotide alignments not included based on small percent identities: 189 06/25/2020 01:09:34 AM - humann2.search.nucleotide - DEBUG: Keeping sam file 06/25/2020 01:09:34 AM - humann2.humann2 - INFO: TIMESTAMP: Completed nucleotide alignment post-processing : 1 seconds 06/25/2020 01:09:34 AM - humann2.humann2 - INFO: Total bugs from nucleotide alignment: 2 06/25/2020 01:09:34 AM - humann2.humann2 - INFO: g__Bacteroides.s__Bacteroides_vulgatus: 7336 hits g__Bacteroides.s__Bacteroides_dorei: 8820 hits 06/25/2020 01:09:34 AM - humann2.humann2 - INFO: Total gene families from nucleotide alignment: 3685 06/25/2020 01:09:34 AM - humann2.humann2 - INFO: Unaligned reads after nucleotide alignment: 23.0666666667 % 06/25/2020 01:09:34 AM - humann2.search.translated - INFO: Running diamond ........ 06/25/2020 01:09:34 AM - humann2.search.translated - INFO: Aligning to reference database: uniref90_annotated_1_1_0.9.34.dmnd 06/25/2020 01:09:34 AM - humann2.utilities - DEBUG: Remove file: /public/home/nx_lfs/metaphlan2_analysis/demo_fastq/demo_humann2_temp/tmp1rXqxO/diamond_m8_DJdMVK 06/25/2020 01:09:34 AM - humann2.utilities - DEBUG: Using software: /public/home/nx_lfs/anaconda2/envs/metagenome_env/bin/diamond 06/25/2020 01:09:34 AM - humann2.utilities - INFO: Execute command: /public/home/nx_lfs/anaconda2/envs/metagenome_env/bin/diamond blastx --query /public/home/nx_lfs/metaphlan2_analysis/demo_fastq/demo_humann2_temp/demo_bowtie2_unaligned.fa --evalue 1.0 --threads 1 --max-target-seqs 20 --outfmt 6 --db /public/home/nx_lfs/db/uniref/uniref90_annotated_1_1_0.9.34 --out /public/home/nx_lfs/metaphlan2_analysis/demo_fastq/demo_humann2_temp/tmp1rXqxO/diamond_m8_DJdMVK --tmpdir /public/home/nx_lfs/metaphlan2_analysis/demo_fastq/demo_humann2_temp/tmp1rXqxO 06/25/2020 01:14:04 AM - humann2.utilities - CRITICAL: Error executing: /public/home/nx_lfs/anaconda2/envs/metagenome_env/bin/diamond blastx --query /public/home/nx_lfs/metaphlan2_analysis/demo_fastq/demo_humann2_temp/demo_bowtie2_unaligned.fa --evalue 1.0 --threads 1 --max-target-seqs 20 --outfmt 6 --db /public/home/nx_lfs/db/uniref/uniref90_annotated_1_1_0.9.34 --out /public/home/nx_lfs/metaphlan2_analysis/demo_fastq/demo_humann2_temp/tmp1rXqxO/diamond_m8_DJdMVK --tmpdir /public/home/nx_lfs/metaphlan2_analysis/demo_fastq/demo_humann2_temp/tmp1rXqxO Error message returned from diamond : diamond v0.9.34.135 (C) Max Planck Society for the Advancement of Science Documentation, support and updates available at http://www.diamondsearch.org #CPU threads: 1 Scoring parameters: (Matrix=BLOSUM62 Lambda=0.267 K=0.041 Penalties=11/1) Temporary directory: /public/home/nx_lfs/metaphlan2_analysis/demo_fastq/demo_humann2_temp/tmp1rXqxO Opening the database... [0.183s] #Target sequences to report alignments for: 20 Reference = /public/home/nx_lfs/db/uniref/uniref90_annotated_1_1_0.9.34.dmnd Sequences = 23876964 Letters = 8108002110 Block size = 2000000000 Opening the input file... [0s] Opening the output file... [0.001s] Loading query sequences... [0.014s] Masking queries... [0.251s] Building query seed set... [0.006s] Algorithm: Query-indexed Building query histograms... [0.001s] Allocating buffers... [0s] Loading reference sequences... [4.618s] Masking reference... [226.219s] Initializing temporary storage... [0.03s] Building reference histograms... [4.899s] Allocating buffers... [0s] Processing query block 0, reference block 0, shape 0, index chunk 0. Building reference seed array... [14.141s] Building query seed array... [0.007s] Computing hash join... [5.335s] Building seed filter... [0.347s] Searching alignments... [13.44s] Deallocating buffers... [0.357s] Opening temporary output file... [0.002s] Computing alignments... No such file or directory [0.001s] Error: Error writing file /public/home/nx_lfs/metaphlan2_analysis/demo_fastq/demo_humann2_temp/tmp1rXqxO/diamond-tmp-8eupgI 06/25/2020 01:14:04 AM - humann2.utilities - CRITICAL: TRACEBACK: Traceback (most recent call last): File "/public/home/nx_lfs/anaconda2/envs/metagenome_env/lib/python2.7/site-packages/humann2/utilities.py", line 735, in execute_command p_out = subprocess.check_output(cmd, stderr=subprocess.STDOUT) File "/public/home/nx_lfs/anaconda2/envs/metagenome_env/lib/python2.7/subprocess.py", line 223, in check_output raise CalledProcessError(retcode, cmd, output=output) CalledProcessError: Command '['/public/home/nx_lfs/anaconda2/envs/metagenome_env/bin/diamond', 'blastx', '--query', '/public/home/nx_lfs/metaphlan2_analysis/demo_fastq/demo_humann2_temp/demo_bowtie2_unaligned.fa', '--evalue', '1.0', '--threads', '1', '--max-target-seqs', '20', '--outfmt', '6', '--db', '/public/home/nx_lfs/db/uniref/uniref90_annotated_1_1_0.9.34', '--out', '/public/home/nx_lfs/metaphlan2_analysis/demo_fastq/demo_humann2_temp/tmp1rXqxO/diamond_m8_DJdMVK', '--tmpdir', '/public/home/nx_lfs/metaphlan2_analysis/demo_fastq/demo_humann2_temp/tmp1rXqxO']' returned non-zero exit status 1