07/24/2020 12:54:38 AM - humann2.humann2 - INFO: Running humann2 v2.8.1
07/24/2020 12:54:38 AM - humann2.humann2 - INFO: Output files will be written to: /home/bio/temp
07/24/2020 12:54:38 AM - humann2.humann2 - INFO: Writing temp files to directory: /home/bio/temp/AH1_humann2_temp
07/24/2020 12:54:38 AM - humann2.utilities - INFO: File ( /home/bio/temp/concat/AH1.fq ) is of format: fastq
07/24/2020 12:54:38 AM - humann2.utilities - DEBUG: Check software, bowtie2, for required version, 2.2
07/24/2020 12:54:38 AM - humann2.utilities - INFO: Using bowtie2 version 2.3
07/24/2020 12:54:38 AM - humann2.humann2 - INFO: Search mode set to uniref90 because a uniref90 translated search database is selected
07/24/2020 12:54:38 AM - humann2.utilities - DEBUG: Check software, diamond, for required version, 0.8.22
07/24/2020 12:54:38 AM - humann2.utilities - INFO: Using diamond version 0.8.36
07/24/2020 12:54:38 AM - humann2.config - INFO:
Run config settings:
DATABASE SETTINGS
nucleotide database folder = /home/bio/db/humann2/chocophlan
protein database folder = /home/bio/db/humann2
pathways database file 1 = /home/bio/miniconda3/envs/meta/lib/python2.7/site-packages/humann2/data/pathways/metacyc_reactions_level4ec_only.uniref.bz2
pathways database file 2 = /home/bio/miniconda3/envs/meta/lib/python2.7/site-packages/humann2/data/pathways/metacyc_pathways_structured_filtered
utility mapping database folder = /home/bio/miniconda3/envs/meta/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 = 88
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
07/24/2020 12:54:38 AM - humann2.store - DEBUG: Initialize Alignments class instance to minimize memory use
07/24/2020 12:54:38 AM - humann2.store - DEBUG: Initialize Reads class instance to minimize memory use
07/24/2020 12:54:44 AM - humann2.humann2 - INFO: Load pathways database part 1: /home/bio/miniconda3/envs/meta/lib/python2.7/site-packages/humann2/data/pathways/metacyc_reactions_level4ec_only.uniref.bz2
07/24/2020 12:54:56 AM - humann2.humann2 - INFO: Load pathways database part 2: /home/bio/miniconda3/envs/meta/lib/python2.7/site-packages/humann2/data/pathways/metacyc_pathways_structured_filtered
07/24/2020 12:54:56 AM - humann2.utilities - DEBUG: Check software, metaphlan2.py, for required version, 2.6
07/24/2020 12:54:57 AM - humann2.utilities - INFO: Using metaphlan2.py version 2.7
07/24/2020 12:54:57 AM - humann2.search.prescreen - INFO: Running metaphlan2.py …
07/24/2020 12:54:57 AM - humann2.utilities - DEBUG: Using software: /home/bio/miniconda3/envs/meta/bin/metaphlan2.py
07/24/2020 12:54:57 AM - humann2.utilities - INFO: Execute command: /home/bio/miniconda3/envs/meta/bin/metaphlan2.py /home/bio/temp/concat/AH1.fq -t rel_ab -o /home/bio/temp/AH1_humann2_temp/AH1_metaphlan_bugs_list.tsv --input_type multifastq --bowtie2out /home/bio/temp/AH1_humann2_temp/AH1_metaphlan_bowtie2.txt --nproc 88
07/24/2020 12:56:03 AM - humann2.utilities - DEBUG: Help message for read_fastx.py
Traceback (most recent call last):
File “/home/bio/miniconda3/envs/meta/bin/read_fastx.py”, line 123, in
read_and_write_raw(f, opened=False, min_len=min_len)
File “/home/bio/miniconda3/envs/meta/bin/read_fastx.py”, line 89, in read_and_write_raw
read_and_write_raw_int(inf, min_len=min_len)
File “/home/bio/miniconda3/envs/meta/bin/read_fastx.py”, line 74, in read_and_write_raw_int
for record in SeqIO.parse(fd, fmt):
File “/home/bio/miniconda3/envs/meta/lib/python2.7/site-packages/Bio/SeqIO/init.py”, line 600, in parse
for r in i:
File “/home/bio/miniconda3/envs/meta/lib/python2.7/site-packages/Bio/SeqIO/QualityIO.py”, line 1031, in FastqPhredIterator
for title_line, seq_string, quality_string in FastqGeneralIterator(handle):
File “/home/bio/miniconda3/envs/meta/lib/python2.7/site-packages/Bio/SeqIO/QualityIO.py”, line 951, in FastqGeneralIterator
% (title_line, seq_len, len(quality_string)))
ValueError: Lengths of sequence and quality values differs for A00920:121:HGNNYDRXX:2:2278:12391:36918:N:0:TACAAGTA+AGTCAACA#0/1 (250 and 1229240).
07/24/2020 12:56:03 AM - humann2.humann2 - INFO: TIMESTAMP: Completed prescreen : 67 seconds
07/24/2020 12:56:03 AM - humann2.search.prescreen - INFO: Total species selected from prescreen: 0
07/24/2020 12:56:03 AM - humann2.search.prescreen - DEBUG:
No species were selected from the prescreen.
Because of this the custom ChocoPhlAn database is empty.
This will result in zero species-specific gene families and pathways.
07/24/2020 12:56:03 AM - humann2.humann2 - INFO: TIMESTAMP: Completed custom database creation : 0 seconds
07/24/2020 12:56:03 AM - humann2.humann2 - DEBUG: Custom database is empty
07/24/2020 12:56:03 AM - humann2.store - DEBUG: Initialize Reads class instance to minimize memory use
07/24/2020 12:56:09 AM - humann2.utilities - DEBUG: Remove file: /home/bio/temp/AH1_humann2_temp/tmpOXjg2v/tmpZDlKOt
07/24/2020 12:56:09 AM - humann2.search.translated - DEBUG: Convert unaligned reads fastq file to fasta
07/24/2020 12:56:14 AM - humann2.search.translated - INFO: Running diamond …
07/24/2020 12:56:14 AM - humann2.search.translated - INFO: Aligning to reference database: uniref90_annotated.1.1.dmnd
07/24/2020 12:56:14 AM - humann2.utilities - DEBUG: Remove file: /home/bio/temp/AH1_humann2_temp/tmpOXjg2v/diamond_m8_DyEiWu
07/24/2020 12:56:14 AM - humann2.utilities - DEBUG: Using software: /home/bio/miniconda3/envs/meta/bin/diamond
07/24/2020 12:56:14 AM - humann2.utilities - INFO: Execute command: /home/bio/miniconda3/envs/meta/bin/diamond blastx --query /home/bio/temp/AH1_humann2_temp/tmpOXjg2v/tmp2Emh_m --evalue 1.0 --threads 88 --max-target-seqs 20 --outfmt 6 --db /home/bio/db/humann2/uniref90_annotated.1.1 --out /home/bio/temp/AH1_humann2_temp/tmpOXjg2v/diamond_m8_DyEiWu --tmpdir /home/bio/temp/AH1_humann2_temp/tmpOXjg2v
07/24/2020 01:01:10 AM - humann2.utilities - DEBUG: diamond v0.8.36.98 | by Benjamin Buchfink buchfink@gmail.com
Check http://github.com/bbuchfink/diamond for updates.
#CPU threads: 88
Scoring parameters: (Matrix=BLOSUM62 Lambda=0.267 K=0.041 Penalties=11/1)
#Target sequences to report alignments for: 20
Temporary directory: /home/bio/temp/AH1_humann2_temp/tmpOXjg2v
Opening the database… [9e-06s]
Opening the input file… [1.4e-05s]
Opening the output file… [1.1e-05s]
Loading query sequences… [0.94992s]
Running complexity filter… [0.643231s]
Building query histograms… [0.181994s]
Allocating buffers… [0.004273s]
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Initializing temporary storage… [0.01668s]
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Opening temporary output file… [0.000104s]
Computing alignments… [2.42973s]
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Computing alignments… [1.88496s]
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Deallocating buffers… [0.319991s]
Opening temporary output file… [0.00011s]
Computing alignments… [2.33864s]
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Building reference histograms… [2.07839s]
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Building seed filter… [0.297354s]
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Building seed filter… [0.280215s]
Searching alignments… [0.720123s]
Deallocating buffers… [0.329535s]
Opening temporary output file… [0.000132s]
Computing alignments… [2.51166s]
Deallocating reference… [0.148843s]
Loading reference sequences… [0.163781s]
Building reference histograms… [0.188441s]
Allocating buffers… [0.004698s]
Initializing temporary storage… [0.019274s]
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Building seed filter… [0.034525s]
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Building seed filter… [0.033118s]
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Building seed filter… [0.036182s]
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Building query index… [0.101391s]
Building seed filter… [0.033177s]
Searching alignments… [0.155112s]
Processing query chunk 0, reference chunk 4, shape 1, index chunk 2.
Building reference index… [0.147998s]
Building query index… [0.100192s]
Building seed filter… [0.036285s]
Searching alignments… [0.442196s]
Processing query chunk 0, reference chunk 4, shape 1, index chunk 3.
Building reference index… [0.134996s]
Building query index… [0.095986s]
Building seed filter… [0.032232s]
Searching alignments… [0.2171s]
Processing query chunk 0, reference chunk 4, shape 2, index chunk 0.
Building reference index… [0.139522s]
Building query index… [0.095769s]
Building seed filter… [0.031706s]
Searching alignments… [0.346017s]
Processing query chunk 0, reference chunk 4, shape 2, index chunk 1.
Building reference index… [0.151409s]
Building query index… [0.099187s]
Building seed filter… [0.031547s]
Searching alignments… [0.281806s]
Processing query chunk 0, reference chunk 4, shape 2, index chunk 2.
Building reference index… [0.143743s]
Building query index… [0.096926s]
Building seed filter… [0.03628s]
Searching alignments… [0.064184s]
Processing query chunk 0, reference chunk 4, shape 2, index chunk 3.
Building reference index… [0.131663s]
Building query index… [0.089608s]
Building seed filter… [0.033461s]
Searching alignments… [0.033455s]
Processing query chunk 0, reference chunk 4, shape 3, index chunk 0.
Building reference index… [0.129791s]
Building query index… [0.0971s]
Building seed filter… [0.032715s]
Searching alignments… [0.081845s]
Processing query chunk 0, reference chunk 4, shape 3, index chunk 1.
Building reference index… [0.136792s]
Building query index… [0.095205s]
Building seed filter… [0.032075s]
Searching alignments… [0.037459s]
Processing query chunk 0, reference chunk 4, shape 3, index chunk 2.
Building reference index… [0.144873s]
Building query index… [0.097941s]
Building seed filter… [0.032173s]
Searching alignments… [0.402392s]
Processing query chunk 0, reference chunk 4, shape 3, index chunk 3.
Building reference index… [0.131428s]
Building query index… [0.088914s]
Building seed filter… [0.031901s]
Searching alignments… [0.040683s]
Deallocating buffers… [0.016488s]
Opening temporary output file… [0.000112s]
Computing alignments… [0.458893s]
Deallocating reference… [0.007812s]
Loading reference sequences… [2.3e-05s]
Deallocating buffers… [0.013108s]
Joining output blocks… [4.07241s]
Deallocating queries… [0.007177s]
Loading query sequences… [2.5e-05s]
Closing the input file… [4e-06s]
Closing the output file… [1.3e-05s]
Closing the database file… [3e-06s]
Total time = 295.506s
Reported 5541065 pairwise alignments, 5541065 HSSPs.
299682 queries aligned.
07/24/2020 01:01:10 AM - humann2.utilities - DEBUG: Using software: /usr/bin/cat
07/24/2020 01:01:10 AM - humann2.utilities - INFO: Execute command: /usr/bin/cat /home/bio/temp/AH1_humann2_temp/tmpOXjg2v/diamond_m8_DyEiWu
07/24/2020 01:01:10 AM - humann2.utilities - DEBUG: Remove file: /home/bio/temp/AH1_humann2_temp/tmpOXjg2v/tmp2Emh_m
07/24/2020 01:01:10 AM - humann2.humann2 - INFO: TIMESTAMP: Completed translated alignment : 308 seconds
07/24/2020 01:02:10 AM - humann2.search.blastx_coverage - INFO: Total alignments without coverage information: 0
07/24/2020 01:02:10 AM - humann2.search.blastx_coverage - INFO: Total proteins in blastx output: 101
07/24/2020 01:02:10 AM - humann2.search.blastx_coverage - INFO: Total proteins without lengths: 0
07/24/2020 01:02:10 AM - humann2.search.blastx_coverage - INFO: Proteins with coverage greater than threshold (50.0): 58
07/24/2020 01:03:13 AM - humann2.utilities - DEBUG: Total alignments where percent identity is not a number: 0
07/24/2020 01:03:13 AM - humann2.utilities - DEBUG: Total alignments where alignment length is not a number: 0
07/24/2020 01:03:13 AM - humann2.utilities - DEBUG: Total alignments where E-value is not a number: 0
07/24/2020 01:03:13 AM - humann2.utilities - DEBUG: Total translated alignments not included based on large e-value: 0
07/24/2020 01:03:13 AM - humann2.utilities - DEBUG: Total translated alignments not included based on small percent identity: 5149438
07/24/2020 01:03:13 AM - humann2.utilities - DEBUG: Total translated alignments not included based on small query coverage: 4094022
07/24/2020 01:03:13 AM - humann2.search.translated - DEBUG: Total translated alignments not included based on small subject coverage value: 4634
07/24/2020 01:03:19 AM - humann2.utilities - DEBUG: Remove file: /home/bio/temp/AH1_humann2_temp/tmpOXjg2v/tmpgPxyDE
07/24/2020 01:03:19 AM - humann2.humann2 - INFO: TIMESTAMP: Completed translated alignment post-processing : 129 seconds
07/24/2020 01:03:19 AM - humann2.humann2 - INFO: Total bugs after translated alignment: 1
07/24/2020 01:03:19 AM - humann2.humann2 - INFO:
unclassified: 42862 hits
07/24/2020 01:03:19 AM - humann2.humann2 - INFO: Total gene families after translated alignment: 58
07/24/2020 01:03:19 AM - humann2.humann2 - INFO: Unaligned reads after translated alignment: 92.4778511558 %
07/24/2020 01:03:19 AM - humann2.humann2 - INFO: Computing gene families …
07/24/2020 01:03:19 AM - humann2.quantify.families - DEBUG: Compute gene families
07/24/2020 01:03:19 AM - humann2.store - INFO:
Total gene families : 58
unclassified : 58 gene families
07/24/2020 01:03:40 AM - humann2.humann2 - INFO: TIMESTAMP: Completed computing gene families : 21 seconds
07/24/2020 01:03:40 AM - humann2.humann2 - INFO: Computing pathways abundance and coverage …
07/24/2020 01:03:40 AM - humann2.quantify.modules - DEBUG: Write flat reactions to pathways file for Minpath
07/24/2020 01:03:40 AM - humann2.quantify.modules - INFO: Compute reaction scores for bug: unclassified
07/24/2020 01:03:41 AM - humann2.quantify.modules - INFO: Compute reaction scores for bug: all
07/24/2020 01:03:41 AM - humann2.quantify.modules - DEBUG: Print pathways _Abundance
07/24/2020 01:03:41 AM - humann2.quantify.modules - DEBUG: Print pathways _Coverage
07/24/2020 01:03:41 AM - humann2.humann2 - INFO: TIMESTAMP: Completed computing pathways : 1 seconds
07/24/2020 01:03:41 AM - humann2.humann2 - INFO:
Output files created:
/home/bio/temp/AH1_genefamilies.tsv
/home/bio/temp/AH1_pathabundance.tsv
/home/bio/temp/AH1_pathcoverage.tsv
07/24/2020 01:03:41 AM - humann2.utilities - DEBUG: Remove directory: /home/bio/temp/AH1_humann2_temp/tmpOXjg2v
the above is the log when I tried to analysis my own data from swine after QC ,Could you tell me if it runs on the correct ways