No species identified and empty pathway files created

Microbial community profiling HUMAnN
1

Hello,

I am using HUMANN3 and have downloaded it fully and run the example files which have given me outputs. Now I am trying to use it for small fastq file as a test run on my data though am getting the error below:

o 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. running diamond … aligning to reference database: uniref90_201901b_full.dmnd aligning to reference database: uniref90_201901b_ec_filtered.dmnd total bugs after translated alignment: 0 total gene families after translated alignment: 0 unaligned reads after translated alignment: 100.0000000000 % computing gene families … computing pathways abundance and coverage …

I am unsure as to what the reason is since we ran another alignment tool on the same fastq and found microbial species though I wanted to use HUMANN to find the metabolic pathways

Thank you for your help,
Krupa

2

Hello, Are there any errors in the log? If not, it would be great to check the MetaPhlAn taxonomic profile to see what species are identified and what their abundances are. It might be possible these species are less then the min abundance filter in HUMAnN. If you can provide more information that would be great!

Thanks!
Lauren

3

Hi Lauren,

Thank you for replying! Here is the log below:

Run config settings:

DATABASE SETTINGS
nucleotide database folder = /SAN/colcc/CPI_data2/DATABASES/humann/chocophlan
protein database folder = /SAN/colcc/CPI_data2/DATABASES/humann/uniref
pathways database file 1 = /SAN/colcc/CPI_data3/KRUPA_PIPELINES/miniconda3/envs/humann/lib/python3.7/site-packages/humann/data/pathways/metacyc_reactions_level4ec_only.
uniref.bz2
pathways database file 2 = /SAN/colcc/CPI_data3/KRUPA_PIPELINES/miniconda3/envs/humann/lib/python3.7/site-packages/humann/data/pathways/metacyc_pathways_structured_filt
ered_v24
utility mapping database folder = /SAN/colcc/CPI_data3/KRUPA_PIPELINES/miniconda3/envs/humann/lib/python3.7/site-packages/humann/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
threads = 1

SEARCH MODE
search mode = uniref90
nucleotide identity threshold = 0.0
translated identity threshold = 80.0

ALIGNMENT SETTINGS
bowtie2 options = --very-sensitive
diamond options = --top 1 --outfmt 6
evalue threshold = 1.0
prescreen threshold = 0.01
translated subject coverage threshold = 50.0
translated query coverage threshold = 90.0
nucleotide subject coverage threshold = 50.0
nucleotide query coverage threshold = 90.0

PATHWAYS SETTINGS
minpath = on
xipe = off
gap fill = on

INPUT AND OUTPUT FORMATS
input file format = fasta
output file format = tsv
output max decimals = 10
remove stratified output = False
remove column description output = False
log level = DEBUG

04/19/2023 04:25:36 PM - humann.store - DEBUG: Initialize Alignments class instance to minimize memory use
04/19/2023 04:25:36 PM - humann.store - DEBUG: Initialize Reads class instance to minimize memory use
04/19/2023 04:25:56 PM - humann.humann - INFO: Load pathways database part 1: /SAN/colcc/CPI_data3/KRUPA_PIPELINES/miniconda3/envs/humann/lib/python3.7/site-packages/hu
mann/data/pathways/metacyc_reactions_level4ec_only.uniref.bz2
04/19/2023 04:25:56 PM - humann.humann - INFO: Load pathways database part 2: /SAN/colcc/CPI_data3/KRUPA_PIPELINES/miniconda3/envs/humann/lib/python3.7/site-packages/hu
mann/data/pathways/metacyc_pathways_structured_filtered_v24
04/19/2023 04:25:56 PM - humann.search.prescreen - INFO: Running metaphlan …
04/19/2023 04:25:56 PM - humann.utilities - DEBUG: Using software: /SAN/colcc/CPI_data3/KRUPA_PIPELINES/miniconda3/envs/metaphlan/bin/metaphlan
04/19/2023 04:25:56 PM - humann.utilities - INFO: Execute command: /SAN/colcc/CPI_data3/KRUPA_PIPELINES/miniconda3/envs/metaphlan/bin/metaphlan /SAN/colcc/CPI_data3/KT_
Project_Outputs/tracerx_microbiome/WGS/outputs/bam_out.fasta --bowtie2db /SAN/colcc/CPI_data2/DATABASES/mpa_v30_CHOCOPhlAn_201901 -x mpa_v30_CHOCOPhlAn_201901 -o /SAN/c
olcc/CPI_data3/KT_Project_Outputs/tracerx_microbiome/test_output/bam_out_humann_temp/bam_out_metaphlan_bugs_list.tsv --input_type fasta --bowtie2out /SAN/colcc/CPI_data
3/KT_Project_Outputs/tracerx_microbiome/test_output/bam_out_humann_temp/bam_out_metaphlan_bowtie2.txt
04/19/2023 04:27:15 PM - humann.utilities - DEBUG: b’\nDownloading Dropbox - File Deleted - Simplify your life U
nable to download Dropbox - File Deleted - Simplify your life’
04/19/2023 04:27:15 PM - humann.humann - INFO: TIMESTAMP: Completed prescreen : 78 seconds
04/19/2023 04:27:15 PM - humann.search.prescreen - INFO: Total species selected from prescreen: 0
04/19/2023 04:27:15 PM - humann.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.

04/19/2023 04:27:15 PM - humann.humann - INFO: TIMESTAMP: Completed custom database creation : 0 seconds
04/19/2023 04:27:15 PM - humann.humann - DEBUG: Custom database is empty
04/19/2023 04:27:15 PM - humann.store - DEBUG: Initialize Reads class instance to minimize memory use
04/19/2023 04:27:15 PM - humann.search.translated - INFO: Running diamond …
04/19/2023 04:27:15 PM - humann.search.translated - INFO: Aligning to reference database: uniref90_201901b_full.dmnd
04/19/2023 04:27:15 PM - humann.utilities - DEBUG: Remove file: /SAN/colcc/CPI_data3/KT_Project_Outputs/tracerx_microbiome/test_output/bam_out_humann_temp/tmp5d1d_mua/d
iamond_m8_vjobrk9q
04/19/2023 04:27:15 PM - humann.utilities - DEBUG: Using software: /SAN/colcc/CPI_data3/KRUPA_PIPELINES/miniconda3/envs/humann/bin/diamond
04/19/2023 04:27:15 PM - humann.utilities - INFO: Execute command: /SAN/colcc/CPI_data3/KRUPA_PIPELINES/miniconda3/envs/humann/bin/diamond blastx --query /SAN/colcc/CPI
data3/KT_Project_Outputs/tracerx_microbiome/WGS/outputs/bam_out.fasta --evalue 1.0 --threads 1 --top 1 --outfmt 6 --db /SAN/colcc/CPI_data2/DATABASES/humann/uniref/uni
ref90_201901b_full --out /SAN/colcc/CPI_data3/KT_Project_Outputs/tracerx_microbiome/test_output/bam_out_humann_temp/tmp5d1d_mua/diamond_m8_vjobrk9q --tmpdir /SAN/colcc/
CPI_data3/KT_Project_Outputs/tracerx_microbiome/test_output/bam_out_humann_temp/tmp5d1d_mua
04/19/2023 04:44:36 PM - humann.utilities - DEBUG: b’diamond v2.0.15.153 (C) Max Planck Society for the Advancement of Science\nDocumentation, support and updates avail
able at http://www.diamondsearch.org\nPlease cite: Sensitive protein alignments at tree-of-life scale using DIAMOND | Nature Methods Nature Methods (2021)\n\n#CPU threads: 1\nScoring parameters: (Matrix=BL
OSUM62 Lambda=0.267 K=0.041 Penalties=11/1)\nTemporary directory: /SAN/colcc/CPI_data3/KT_Project_Outputs/tracerx_microbiome/test_output/bam_out_humann_temp/tmp5d1d_mua
\nPercentage range of top alignment score to report hits: 1\nOpening the database… [0.695s]\nDatabase: /SAN/colcc/CPI_data2/DATABASES/humann/uniref/uniref90_201901b
full.dmnd (type: Diamond database, sequences: 87296736, letters: 29247941583)\nBlock size = 2000000000\nOpening the input file… [0s]\nOpening the output file… [0.
027s]\nLoading query sequences… [0s]\nMasking queries… [0.003s]\nBuilding query seed set… [0.002s]\nAlgorithm: Query-indexed\nBuilding query histograms… [0s
]\nAllocating buffers… [0s]\nLoading reference sequences… [27.297s]\nInitializing dictionary… [0.057s]\nInitializing temporary storage… [0.284s]\nBuilding r
eference histograms… [21.724s]\nAllocating buffers… [0s]\nProcessing query block 1, reference block 1/15, shape 1/2.\nBuilding reference seed array… [11.203s]
nBuilding query seed array… [0.001s]\nComputing hash join… [0.552s]\nSearching alignments… [0.055s]\nProcessing query block 1, reference block 1/15, shape 2/2.
\nBuilding reference seed array… [11.044s]\nBuilding query seed array… [0s]\nComputing hash join… [0.548s]\nSearching alignments… [0.016s]\nDeallocating buf
fers… [0.002s]\nClearing query masking… [0s]\nOpening temporary output file… [0.015s]\nComputing alignments… [0.072s]\nDeallocating reference… [0.011s]\n
Loading reference sequences… [21.647s]\nInitializing dictionary… [0.002s]\nInitializing temporary storage… [0.401s]\nBuilding reference histograms… [21.601s
]\nAllocating buffers… [0s]\nProcessing query block 1, reference block 2/15, shape 1/2.\nBuilding reference seed array… [11.242s]\nBuilding query seed array… [
0s]\nComputing hash join… [0.561s]\nSearching alignments… [0.019s]\nProcessing query block 1, reference block 2/15, shape 2/2.\nBuilding reference seed array…
[11.067s]\nBuilding query seed array… [0s]\nComputing hash join… [0.562s]\nSearching alignments… [0.01s]\nDeallocating buffers… [0.002s]\nClearing query mas
king… [0s]\nOpening temporary output file… [0.018s]\nComputing alignments… [0.067s]\nDeallocating reference… [0.01s]\nLoading reference sequences… [21.09
8s]\nInitializing dictionary… [0.002s]\nInitializing temporary storage… [0.331s]\nBuilding reference histograms… [21.48s]\nAllocating buffers… [0s]\nProcess
ing query block 1, reference block 3/15, shape 1/2.\nBuilding reference seed array… [11.15s]\nBuilding query seed array… [0s]\nComputing hash join… [0.549s]\nS
earching alignments… [0.018s]\nProcessing query block 1, reference block 3/15, shape 2/2.\nBuilding reference seed array… [10.931s]\nBuilding query seed array…
[0s]\nComputing hash join… [0.527s]\nSearching alignments… [0.01s]\nDeallocating buffers… [0.002s]\nClearing query masking… [0s]\nOpening temporary output
file… [0.035s]\nComputing alignments… [0.073s]\nDeallocating reference… [0.011s]\nLoading reference sequences… [25.082s]\nInitializing dictionary… [0.002
s]\nInitializing temporary storage… [0.419s]\nBuilding reference histograms… [21.421s]\nAllocating buffers… [0s]\nProcessing query block 1, reference block 4/1
5, shape 1/2.\nBuilding reference seed array… [11.171s]\nBuilding query seed array… [0.001s]\nComputing hash join… [0.547s]\nSearching alignments… [0.019s]
nProcessing query block 1, reference block 4/15, shape 2/2.\nBuilding reference seed array… [10.917s]\nBuilding query seed array… [0s]\nComputing hash join… [0
.526s]\nSearching alignments… [0.009s]\nDeallocating buffers… [0.002s]\nClearing query masking… [0s]\nOpening temporary output file… [0.003s]\nComputing ali
gnments… [0.066s]\nDeallocating reference… [0.011s]\nLoading reference sequences… [22.881s]\nInitializing dictionary… [0.008s]\nInitializing temporary stora
ge… [0.295s]\nBuilding reference histograms… [21.433s]\nAllocating buffers… [0s]\nProcessing query block 1, reference block 5/15, shape 1/2.\nBuilding referenc
e seed array… [11.135s]\nBuilding query seed array… [0s]\nComputing hash join… [0.547s]\nSearching alignments… [0.026s]\nProcessing query block 1, reference
block 5/15, shape 2/2.\nBuilding reference seed array… [10.922s]\nBuilding query seed array… [0s]\nComputing hash join… [0.527s]\nSearching alignments… [0.
009s]\nDeallocating buffers… [0.002s]\nClearing query masking… [0s]\nOpening temporary output file… [0.033s]\nComputing alignments… [0.071s]\nDeallocating r
eference… [0.011s]\nLoading reference sequences… [20.48s]\nInitializing dictionary… [0.007s]\nInitializing temporary storage… [3.783s]\nBuilding reference h
istograms… [21.436s]\nAllocating buffers… [0s]\nProcessing query block 1, reference block 6/15, shape 1/2.\nBuilding reference seed array… [11.143s]\nBuilding
query seed array… [0.001s]\nComputing hash join… [0.547s]\nSearching alignments… [0.019s]\nProcessing query block 1, reference block 6/15, shape 2/2.\nBuilding
reference seed array… [10.926s]\nBuilding query seed array… [0s]\nComputing hash join… [0.527s]\nSearching alignments… [0.016s]\nDeallocating buffers… [
0.002s]\nClearing query masking… [0s]\nOpening temporary output file… [0.016s]\nComputing alignments… [0.069s]\nDeallocating reference… [0.011s]\nLoading re
ference sequences… [21.245s]\nInitializing dictionary… [0.007s]\nInitializing temporary storage… [0.282s]\nBuilding reference histograms… [21.405s]\nAllocat
ing buffers… [0s]\nProcessing query block 1, reference block 7/15, shape 1/2.\nBuilding reference seed array… [11.187s]\nBuilding query seed array… [0s]\nCompu
ting hash join… [0.546s]\nSearching alignments… [0.019s]\nProcessing query block 1, reference block 7/15, shape 2/2.\nBuilding reference seed array… [10.927s]
nBuilding query seed array… [0s]\nComputing hash join… [0.527s]\nSearching alignments… [0.009s]\nDeallocating buffers… [0.002s]\nClearing query masking…
[0s]\nOpening temporary output file… [0.141s]\nComputing alignments… [0.218s]\nDeallocating reference… [0.011s]\nLoading reference sequences… [22.719s]\nIni
tializing dictionary… [0.002s]\nInitializing temporary storage… [0.344s]\nBuilding reference histograms… [21.47s]\nAllocating buffers… [0s]\nProcessing quer
y block 1, reference block 8/15, shape 1/2.\nBuilding reference seed array… [11.151s]\nBuilding query seed array… [0s]\nComputing hash join… [0.559s]\nSearchin
g alignments… [0.02s]\nProcessing query block 1, reference block 8/15, shape 2/2.\nBuilding reference seed array… [10.947s]\nBuilding query seed array… [0.001s
]\nComputing hash join… [0.529s]\nSearching alignments… [0.009s]\nDeallocating buffers… [0.002s]\nClearing query masking… [0s]\nOpening temporary output fil
e… [0.858s]\nComputing alignments… [2.303s]\nDeallocating reference… [0.011s]\nLoading reference sequences… [25.458s]\nInitializing dictionary… [0.002s]
nInitializing temporary storage… [0.349s]\nBuilding reference histograms… [21.492s]\nAllocating buffers… [0s]\nProcessing query block 1, reference block 9/15,
shape 1/2.\nBuilding reference seed array… [11.411s]\nBuilding query seed array… [0.001s]\nComputing hash join… [0.573s]\nSearching alignments… [0.021s]\nPr
ocessing query block 1, reference block 9/15, shape 2/2.\nBuilding reference seed array… [11.102s]\nBuilding query seed array… [0s]\nComputing hash join… [0.52
8s]\nSearching alignments… [0.011s]\nDeallocating buffers… [0.002s]\nClearing query masking… [0s]\nOpening temporary output file… [0.286s]\nComputing alignm
ents… [0.292s]\nDeallocating reference… [0.012s]\nLoading reference sequences… [24.378s]\nInitializing dictionary… [0.007s]\nInitializing temporary storage.
… [0.34s]\nBuilding reference histograms… [21.598s]\nAllocating buffers… [0s]\nProcessing query block 1, reference block 10/15, shape 1/2.\nBuilding reference s
eed array… [11.159s]\nBuilding query seed array… [0s]\nComputing hash join… [0.548s]\nSearching alignments… [0.02s]\nProcessing query block 1, reference blo
ck 10/15, shape 2/2.\nBuilding reference seed array… [10.934s]\nBuilding query seed array… [0s]\nComputing hash join… [0.528s]\nSearching alignments… [0.009
s]\nDeallocating buffers… [0.002s]\nClearing query masking… [0s]\nOpening temporary output file… [0.099s]\nComputing alignments… [0.483s]\nDeallocating refe
rence… [0.012s]\nLoading reference sequences… [21.966s]\nInitializing dictionary… [0.006s]\nInitializing temporary storage… [0.366s]\nBuilding reference his
tograms… [22.384s]\nAllocating buffers… [0s]\nProcessing query block 1, reference block 11/15, shape 1/2.\nBuilding reference seed array… [11.623s]\nBuilding q
uery seed array… [0.001s]\nComputing hash join… [0.601s]\nSearching alignments… [0.021s]\nProcessing query block 1, reference block 11/15, shape 2/2.\nBuilding
reference seed array… [11.4s]\nBuilding query seed array… [0.001s]\nComputing hash join… [0.577s]\nSearching alignments… [0.011s]\nDeallocating buffers…
[0.002s]\nClearing query masking… [0s]\nOpening temporary output file… [0.041s]\nComputing alignments… [0.061s]\nDeallocating reference… [0.013s]\nLoading
reference sequences… [28.895s]\nInitializing dictionary… [0.006s]\nInitializing temporary storage… [0.41s]\nBuilding reference histograms… [21.837s]\nAlloca
ting buffers… [0s]\nProcessing query block 1, reference block 12/15, shape 1/2.\nBuilding reference seed array… [11.733s]\nBuilding query seed array… [0s]\nCom
puting hash join… [0.568s]\nSearching alignments… [0.032s]\nProcessing query block 1, reference block 12/15, shape 2/2.\nBuilding reference seed array… [11.27s
]\nBuilding query seed array… [0s]\nComputing hash join… [0.543s]\nSearching alignments… [0.01s]\nDeallocating buffers… [0.002s]\nClearing query masking…
[0s]\nOpening temporary output file… [0.003s]\nComputing alignments… [0.062s]\nDeallocating reference… [0.011s]\nLoading reference sequences… [29.224s]\nIn
itializing dictionary… [0.005s]\nInitializing temporary storage… [0.34s]\nBuilding reference histograms… [22.558s]\nAllocating buffers… [0s]\nProcessing que
ry block 1, reference block 13/15, shape 1/2.\nBuilding reference seed array… [11.348s]\nBuilding query seed array… [0.001s]\nComputing hash join… [0.592s]\nSe
arching alignments… [0.027s]\nProcessing query block 1, reference block 13/15, shape 2/2.\nBuilding reference seed array… [11.175s]\nBuilding query seed array…
[0.001s]\nComputing hash join… [0.55s]\nSearching alignments… [0.011s]\nDeallocating buffers… [0.002s]\nClearing query masking… [0s]\nOpening temporary out
put file… [0.27s]\nComputing alignments… [0.073s]\nDeallocating reference… [0.012s]\nLoading reference sequences… [28.988s]\nInitializing dictionary… [0.
006s]\nInitializing temporary storage… [0.374s]\nBuilding reference histograms… [22.005s]\nAllocating buffers… [0s]\nProcessing query block 1, reference block
14/15, shape 1/2.\nBuilding reference seed array… [11.415s]\nBuilding query seed array… [0.001s]\nComputing hash join… [0.577s]\nSearching alignments… [0.02
s]\nProcessing query block 1, reference block 14/15, shape 2/2.\nBuilding reference seed array… [11.156s]\nBuilding query seed array… [0.001s]\nComputing hash joi
n… [0.582s]\nSearching alignments… [0.011s]\nDeallocating buffers… [0.002s]\nClearing query masking… [0s]\nOpening temporary output file… [0.721s]\nCompu
ting alignments… [1.301s]\nDeallocating reference… [0.012s]\nLoading reference sequences… [21.072s]\nInitializing dictionary… [0.001s]\nInitializing tempora
ry storage… [0.419s]\nBuilding reference histograms… [13.786s]\nAllocating buffers… [0s]\nProcessing query block 1, reference block 15/15, shape 1/2.\nBuilding
reference seed array… [7.06s]\nBuilding query seed array… [0s]\nComputing hash join… [0.348s]\nSearching alignments… [0.014s]\nProcessing query block 1, re
ference block 15/15, shape 2/2.\nBuilding reference seed array… [6.914s]\nBuilding query seed array… [0.001s]\nComputing hash join… [0.338s]\nSearching alignme
nts… [0.006s]\nDeallocating buffers… [0.001s]\nClearing query masking… [0s]\nOpening temporary output file… [0.016s]\nComputing alignments… [0.059s]\nDea
llocating reference… [0.007s]\nLoading reference sequences… [0s]\nDeallocating buffers… [0s]\nJoining output blocks… Joining output blocks… [0.021s]\n [0.
055s]\nDeallocating queries… [0.002s]\nLoading query sequences… [0s]\nClosing the input file… [0s]\nClosing the output file… [0s]\nCleaning up… [0s]\nTot
al time = 1041.46s\nReported 15 pairwise alignments, 15 HSPs.\n4 queries aligned.\n’
04/19/2023 04:44:36 PM - humann.search.translated - INFO: Aligning to reference database: uniref90_201901b_ec_filtered.dmnd
04/19/2023 04:44:36 PM - humann.utilities - DEBUG: Remove file: /SAN/colcc/CPI_data3/KT_Project_Outputs/tracerx_microbiome/test_output/bam_out_humann_temp/tmp5d1d_mua/d
iamond_m8_jkiows7f
04/19/2023 04:44:36 PM - humann.utilities - DEBUG: Using software: /SAN/colcc/CPI_data3/KRUPA_PIPELINES/miniconda3/envs/humann/bin/diamond
04/19/2023 04:44:36 PM - humann.utilities - INFO: Execute command: /SAN/colcc/CPI_data3/KRUPA_PIPELINES/miniconda3/envs/humann/bin/diamond blastx --query /SAN/colcc/CPI
data3/KT_Project_Outputs/tracerx_microbiome/WGS/outputs/bam_out.fasta --evalue 1.0 --threads 1 --top 1 --outfmt 6 --db /SAN/colcc/CPI_data2/DATABASES/humann/uniref/uni
ref90_201901b_ec_filtered --out /SAN/colcc/CPI_data3/KT_Project_Outputs/tracerx_microbiome/test_output/bam_out_humann_temp/tmp5d1d_mua/diamond_m8_jkiows7f --tmpdir /SAN
/colcc/CPI_data3/KT_Project_Outputs/tracerx_microbiome/test_output/bam_out_humann_temp/tmp5d1d_mua
04/19/2023 04:46:06 PM - humann.utilities - DEBUG: b’diamond v2.0.15.153 (C) Max Planck Society for the Advancement of Science\nDocumentation, support and updates avail
able at http://www.diamondsearch.org\nPlease cite: Sensitive protein alignments at tree-of-life scale using DIAMOND | Nature Methods Nature Methods (2021)\n\n#CPU threads: 1\nScoring parameters: (Matrix=BL
OSUM62 Lambda=0.267 K=0.041 Penalties=11/1)\nTemporary directory: /SAN/colcc/CPI_data3/KT_Project_Outputs/tracerx_microbiome/test_output/bam_out_humann_temp/tmp5d1d_mua
\nPercentage range of top alignment score to report hits: 1\nOpening the database… [0.605s]\nDatabase: /SAN/colcc/CPI_data2/DATABASES/humann/uniref/uniref90_201901b
ec_filtered.dmnd (type: Diamond database, sequences: 6191048, letters: 2497143558)\nBlock size = 2000000000\nOpening the input file… [0s]\nOpening the output file…
[0.012s]\nLoading query sequences… [0s]\nMasking queries… [0.003s]\nBuilding query seed set… [0.002s]\nAlgorithm: Query-indexed\nBuilding query histograms…
[0s]\nAllocating buffers… [0s]\nLoading reference sequences… [24.964s]\nInitializing dictionary… [0.029s]\nInitializing temporary storage… [0.366s]\nBuild
ing reference histograms… [21.643s]\nAllocating buffers… [0s]\nProcessing query block 1, reference block 1/2, shape 1/2.\nBuilding reference seed array… [11.57
8s]\nBuilding query seed array… [0.015s]\nComputing hash join… [0.612s]\nSearching alignments… [0.073s]\nProcessing query block 1, reference block 1/2, shape 2
/2.\nBuilding reference seed array… [11.46s]\nBuilding query seed array… [0s]\nComputing hash join… [0.612s]\nSearching alignments… [0.015s]\nDeallocating b
uffers… [0.031s]\nClearing query masking… [0s]\nOpening temporary output file… [0.012s]\nComputing alignments… [0.104s]\nDeallocating reference… [0.012s]
\nLoading reference sequences… [4.43s]\nInitializing dictionary… [0s]\nInitializing temporary storage… [0.282s]\nBuilding reference histograms… [5.706s]\nAl
locating buffers… [0s]\nProcessing query block 1, reference block 2/2, shape 1/2.\nBuilding reference seed array… [2.942s]\nBuilding query seed array… [0s]\nCo
mputing hash join… [0.146s]\nSearching alignments… [0.008s]\nProcessing query block 1, reference block 2/2, shape 2/2.\nBuilding reference seed array… [2.907s]
\nBuilding query seed array… [0s]\nComputing hash join… [0.141s]\nSearching alignments… [0.007s]\nDeallocating buffers… [0.001s]\nClearing query masking…
[0s]\nOpening temporary output file… [0.023s]\nComputing alignments… [0.046s]\nDeallocating reference… [0.006s]\nLoading reference sequences… [0s]\nDealloc
ating buffers… [0s]\nJoining output blocks… Joining output blocks… [0.017s]\n [0.02s]\nDeallocating queries… [0s]\nLoading query sequences… [0s]\nClosing
the input file… [0s]\nClosing the output file… [0s]\nCleaning up… [0s]\nTotal time = 89.001s\nReported 3 pairwise alignments, 3 HSPs.\n2 queries aligned.\n’
04/19/2023 04:46:06 PM - humann.utilities - DEBUG: Using software: /bin/cat
04/19/2023 04:46:06 PM - humann.utilities - INFO: Execute command: /bin/cat /SAN/colcc/CPI_data3/KT_Project_Outputs/tracerx_microbiome/test_output/bam_out_humann_temp/t
mp5d1d_mua/diamond_m8_vjobrk9q /SAN/colcc/CPI_data3/KT_Project_Outputs/tracerx_microbiome/test_output/bam_out_humann_temp/tmp5d1d_mua/diamond_m8_jkiows7f
04/19/2023 04:46:06 PM - humann.humann - INFO: TIMESTAMP: Completed translated alignment : 1131 seconds
04/19/2023 04:46:06 PM - humann.utilities - DEBUG: Total alignments where percent identity is not a number: 0
04/19/2023 04:46:06 PM - humann.utilities - DEBUG: Total alignments where alignment length is not a number: 0
04/19/2023 04:46:06 PM - humann.utilities - DEBUG: Total alignments where E-value is not a number: 0
04/19/2023 04:46:06 PM - humann.utilities - DEBUG: Total alignments not included based on large e-value: 0
04/19/2023 04:46:06 PM - humann.utilities - DEBUG: Total alignments not included based on small percent identity: 1
04/19/2023 04:46:06 PM - humann.utilities - DEBUG: Total alignments not included based on small query coverage: 0
04/19/2023 04:46:06 PM - humann.search.blastx_coverage - INFO: Total alignments without coverage information: 0
04/19/2023 04:46:06 PM - humann.search.blastx_coverage - INFO: Total proteins in blastx output: 16
04/19/2023 04:46:06 PM - humann.search.blastx_coverage - INFO: Total proteins without lengths: 0
04/19/2023 04:46:06 PM - humann.search.blastx_coverage - INFO: Proteins with coverage greater than threshold (50.0): 0
04/19/2023 04:46:06 PM - humann.utilities - DEBUG: Total alignments where percent identity is not a number: 0
04/19/2023 04:46:06 PM - humann.utilities - DEBUG: Total alignments where alignment length is not a number: 0
04/19/2023 04:46:06 PM - humann.utilities - DEBUG: Total alignments where E-value is not a number: 0
04/19/2023 04:46:06 PM - humann.utilities - DEBUG: Total alignments not included based on large e-value: 0
04/19/2023 04:46:06 PM - humann.utilities - DEBUG: Total alignments not included based on small percent identity: 1
04/19/2023 04:46:06 PM - humann.utilities - DEBUG: Total alignments not included based on small query coverage: 0
04/19/2023 04:46:06 PM - humann.search.translated - DEBUG: Total translated alignments not included based on small subject coverage value: 17
04/19/2023 04:46:06 PM - humann.humann - INFO: TIMESTAMP: Completed translated alignment post-processing : 0 seconds
04/19/2023 04:46:06 PM - humann.humann - INFO: Total bugs after translated alignment: 0
04/19/2023 04:46:06 PM - humann.humann - INFO:

04/19/2023 04:46:06 PM - humann.humann - INFO: Total gene families after translated alignment: 0
04/19/2023 04:46:06 PM - humann.humann - INFO: Unaligned reads after translated alignment: 100.0000000000 %
04/19/2023 04:46:06 PM - humann.humann - INFO: Computing gene families …
04/19/2023 04:46:06 PM - humann.quantify.families - DEBUG: Compute gene families
04/19/2023 04:46:06 PM - humann.store - INFO:
Total gene families : 0

04/19/2023 04:46:19 PM - humann.humann - INFO: TIMESTAMP: Completed computing gene families : 14 seconds
04/19/2023 04:46:19 PM - humann.humann - INFO: Computing pathways abundance and coverage …
04/19/2023 04:46:19 PM - humann.quantify.modules - DEBUG: Write flat reactions to pathways file for Minpath
04/19/2023 04:46:19 PM - humann.quantify.modules - INFO: Compute reaction scores for bug: all
04/19/2023 04:46:21 PM - humann.quantify.modules - DEBUG: Print pathways _Abundance
04/19/2023 04:46:21 PM - humann.quantify.modules - DEBUG: Print pathways _Coverage
04/19/2023 04:46:21 PM - humann.humann - INFO: TIMESTAMP: Completed computing pathways : 2 seconds
04/19/2023 04:46:21 PM - humann.humann - INFO:
Output files created:
/SAN/colcc/CPI_data3/KT_Project_Outputs/tracerx_microbiome/test_output/bam_out_genefamilies.tsv
/SAN/colcc/CPI_data3/KT_Project_Outputs/tracerx_microbiome/test_output/bam_out_pathabundance.tsv
/SAN/colcc/CPI_data3/KT_Project_Outputs/tracerx_microbiome/test_output/bam_out_pathcoverage.tsv

04/19/2023 04:46:21 PM - humann.utilities - DEBUG: Remove directory: /SAN/colcc/CPI_data3/KT_Project_Outputs/tracerx_microbiome/test_output/bam_out_humann_temp/tmp5d1d_
mua

In terms of the taxonomic profile from metaphlan it comes up as as unknown and then no pathways are identified. I know the fasta file has Pseudomonas Auerginosa though not sure why HUMAnN cant identify it

4

Hi did you find the problem? I think it is because your files are stored in Dropbox. It says was unable to download your file.

/19/2023 04:27:15 PM - humann.utilities - DEBUG: b’\nDownloading Dropbox - File Deleted - Simplify your life U
nable to download Dropbox - File Deleted - Simplify your life’

5

Thank you for posting the log. That makes sense in that it looks like HUMAnN does not find any species that pass the threshold abundance in the MetaPhlAn output file. Do you see any errors in your MetaPhlAn run? If not, have you run your samples through a quality control step to remove low quality reads and host contaminate sequences?

Thank you,
Lauren

6

Hello,

I received this message
humann v3.7
MetaPhlAn version 3.0.9 (17 May 2021)
Bowtie2 version 2.2.3

home/kbrokordt/miniconda3/lib/python3.12/site-packages/humann/quantify/MinPath12hmp.py:804: SyntaxWarning: invalid escape sequence ‘\d’
m = re.match(‘[1]+(?P\d+)’, aline)
/home/kbrokordt/miniconda3/lib/python3.12/site-packages/humann/quantify/MinPath12hmp.py:804: SyntaxWarning: invalid escape sequence ‘\d’
m = re.match(‘[2]+(?P\d+)’, aline)
Output files will be written to: /home/kbrokordt/camaron/Pruebas/nonrRNA/OUTOUT

Running metaphlan …

Total species selected from prescreen: 0

Selected species explain 0.00% of predicted community composition

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.

Aligning to reference database: uniref90_201901b_full.dmnd

Total bugs after translated alignment: 1
unclassified: 5557552 hits

Total gene families after translated alignment: 14628

Unaligned reads after translated alignment: 97.5684571395 %

Any suggestion

  1. ^\d ↩︎

  2. ^\d ↩︎

7

I encountered the same SyntaxWarning message as Carlos. I am pretty sure that it’s due to change in Python 3.12 where it’s now required to use raw strings (r"…") for regular expressions.

For my dataset, HUMAnN did successfully find and quantify pathways despite emitting the warning.

I’m not confident, but I think it’s possible that that MinPath WriteReport function that contains the offending string isn’t even reached during a default HUMAnN run. It appears to be specific to KEGG pathways.

8

Thanks for the suggestion. We are usually not testing on the most recent Python release, so this is definitely possible. We will look into it.