The bioBakery help forum

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 12:54:38 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]
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Allocating buffers… [0.004273s]
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Initializing temporary storage… [0.01668s]
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Computing alignments… [2.42973s]
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Computing alignments… [2.51166s]
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Building seed filter… [0.035789s]
Searching alignments… [0.045675s]
Processing query chunk 0, reference chunk 4, shape 1, index chunk 1.
Building reference index… [0.135742s]
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

Hi Jamie, Thank you for the detailed post! It looks like there might be some formatting issues with a sequence in your fastq input file. This causes an error running MetaPhlAn (which runs the prescreen) and because of this error no species are identified so no custom nucleotide database is generated and so the nucleotide search portion of HUMAnN is not run. If you would double check your input file, more specifically the sequence that seems to have different lengths of bases vs quality values, resolving this formatting issue should resolve the error. I will copy the sequence error below for your reference which includes the sequence id.

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).

Thank you,
Lauren