No pathways detected with --input-format genetable

Microbial community profiling HUMAnN
1

I am trying to launch a test run of HUMAnN on an abundance table and while I get no errors in the log, I also am not successfuly with annotating the pathways.

A fragment of the file that I’m using is:


#UniRef90	fraction
UniRef90_A0A3Q0NAS4|g__Listeria.s__Listeria_monocytogenes	0.0013392857142857143
UniRef90_Q8Y8R7|g__Listeria.s__Listeria_monocytogenes	0.0008928571428571428
UniRef50|g__Listeria.s__Listeria_monocytogenes	0.0004464285714285714
UniRef90_Q8Y5Z9|g__Listeria.s__Listeria_monocytogenes	0.0004464285714285714
UniRef90_A0AK14|g__Listeria.s__Listeria_monocytogenes	0.0004464285714285714
UniRef90_A0A3T2GR97|g__Listeria.s__Listeria_monocytogenes	0.0004464285714285714
UniRef90_A0A2Z5BXQ6|g__Listeria.s__Listeria_monocytogenes	0.0004464285714285714
UniRef90_A0A5M1KZY5|g__Listeria.s__Listeria_monocytogenes	0.0004464285714285714
UniRef90_A0A3D7VE02|g__Listeria.s__Listeria_monocytogenes	0.0004464285714285714
UniRef90_A0AK19|g__Listeria.s__Listeria_monocytogenes	0.0004464285714285714
UniRef90_A0A2Z5BXC5|g__Listeria.s__Listeria_monocytogenes	0.0004464285714285714
UniRef90_A0A0B8QYN6|g__Listeria.s__Listeria_monocytogenes	0.0004464285714285714
...
UniRef90_Q8Y8M1|g__Listeria.s__Listeria_monocytogenes	0.0004464285714285714
UniRef90_Q8Y8M2|g__Listeria.s__Listeria_monocytogenes	0.0004464285714285714
UniRef90_Q8Y8M3|g__Listeria.s__Listeria_monocytogenes	0.0004464285714285714
UniRef90_A0A9P2EQU0|g__Listeria.s__Listeria_monocytogenes	0.0004464285714285714
UniRef90_Q8Y8M5|g__Listeria.s__Listeria_monocytogenes	0.0004464285714285714
UniRef90_A0A5D5U8N4|g__Listeria.s__Listeria_monocytogenes	0.0004464285714285714

The pipeline run:
humann -i data/cds_gb_uniref90_abundance_table.tsv -o data/cgd_gb_uniref90_abudnance_table_out --input-format genetable

Log file:

12/16/2024 12:54:20 PM - humann.humann - INFO: Running humann v3.9
12/16/2024 12:54:20 PM - humann.humann - INFO: Output files will be written to: /home/bioinfo-tools/functional-annotation/hmmer_utils/data/cgd_gb_uniref90_abudnance_table_out
12/16/2024 12:54:20 PM - humann.humann - INFO: Writing temp files to directory: /home/bioinfo-tools/functional-annotation/hmmer_utils/data/cgd_gb_uniref90_abudnance_table_out/cds_gb_uniref90_abundance_table_humann_temp
12/16/2024 12:54:20 PM - humann.config - INFO: 
Run config settings: 

DATABASE SETTINGS
nucleotide database folder = /data/my.user/chocophlan
protein database folder = /data/my.user/uniref
pathways database file 1 = /home/my.user/.local/lib/python3.10/site-packages/humann/data/pathways/metacyc_reactions_level4ec_only.uniref.bz2
pathways database file 2 = /home/my.user/.local/lib/python3.10/site-packages/humann/data/pathways/metacyc_pathways_structured_filtered_v24_subreactions
utility mapping database folder = /data/my.user/utility_mapping

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 = genetable
output file format = tsv
output max decimals = 10
remove stratified output = False
remove column description output = False
log level = DEBUG

12/16/2024 12:54:20 PM - humann.store - DEBUG: Initialize Alignments class instance to minimize memory use
12/16/2024 12:54:20 PM - humann.store - DEBUG: Initialize Reads class instance to minimize memory use
12/16/2024 12:54:28 PM - humann.humann - INFO: Load pathways database part 1: /home/my.user/.local/lib/python3.10/site-packages/humann/data/pathways/metacyc_reactions_level4ec_only.uniref.bz2
12/16/2024 12:54:28 PM - humann.humann - INFO: Load pathways database part 2: /home/my.user/.local/lib/python3.10/site-packages/humann/data/pathways/metacyc_pathways_structured_filtered_v24_subreactions
12/16/2024 12:54:28 PM - humann.humann - INFO: Process the gene table ...
12/16/2024 12:54:28 PM - humann.humann - INFO: TIMESTAMP: Completed 	processing gene table 	:	 0	 seconds
12/16/2024 12:54:28 PM - humann.humann - INFO: Computing pathways abundance and coverage ...
12/16/2024 12:54:28 PM - humann.quantify.modules - DEBUG: Write flat reactions to pathways file for Minpath
12/16/2024 12:54:28 PM - humann.quantify.modules - INFO: Compute reaction scores for bug: g__Listeria.s__Listeria_monocytogenes
12/16/2024 12:54:29 PM - humann.quantify.modules - INFO: Run MinPath on g__Listeria.s__Listeria_monocytogenes
12/16/2024 12:54:29 PM - humann.utilities - DEBUG: Using python module : /home/my.user/.local/lib/python3.10/site-packages/humann/quantify/MinPath12hmp.py
12/16/2024 12:54:29 PM - humann.utilities - INFO: Execute command: /usr/bin/python3 /home/my.user/.local/lib/python3.10/site-packages/humann/quantify/MinPath12hmp.py -any /home/bioinfo-tools/functional-annotation/hmmer_utils/data/cgd_gb_uniref90_abudnance_table_out/cds_gb_uniref90_abundance_table_humann_temp/tmprtlevoha/tmpzdwhnhsp -map /home/bioinfo-tools/functional-annotation/hmmer_utils/data/cgd_gb_uniref90_abudnance_table_out/cds_gb_uniref90_abundance_table_humann_temp/tmprtlevoha/tmpvq575ovh -report /home/bioinfo-tools/functional-annotation/hmmer_utils/data/cgd_gb_uniref90_abudnance_table_out/cds_gb_uniref90_abundance_table_humann_temp/tmprtlevoha/tmp51ikauqn -details /home/bioinfo-tools/functional-annotation/hmmer_utils/data/cgd_gb_uniref90_abudnance_table_out/cds_gb_uniref90_abundance_table_humann_temp/tmprtlevoha/tmpzbo0mo8o -mps /home/bioinfo-tools/functional-annotation/hmmer_utils/data/cgd_gb_uniref90_abudnance_table_out/cds_gb_uniref90_abundance_table_humann_temp/tmprtlevoha/tmpc_ufhr0v
12/16/2024 12:54:29 PM - humann.quantify.modules - DEBUG: Compute pathway abundance for bug: g__Listeria.s__Listeria_monocytogenes
12/16/2024 12:54:29 PM - humann.quantify.modules - DEBUG: Compute pathway coverage for bug: g__Listeria.s__Listeria_monocytogenes
12/16/2024 12:54:29 PM - humann.quantify.modules - DEBUG: Print pathways _Abundance
12/16/2024 12:54:29 PM - humann.quantify.modules - DEBUG: Print pathways _Coverage
12/16/2024 12:54:29 PM - humann.humann - INFO: TIMESTAMP: Completed 	computing pathways 	:	 1	 seconds
12/16/2024 12:54:29 PM - humann.humann - INFO: 
Output files created: 
/home/bioinfo-tools/functional-annotation/hmmer_utils/data/cgd_gb_uniref90_abudnance_table_out/cds_gb_uniref90_abundance_table_pathabundance.tsv
/home/bioinfo-tools/functional-annotation/hmmer_utils/data/cgd_gb_uniref90_abudnance_table_out/cds_gb_uniref90_abundance_table_pathcoverage.tsv

12/16/2024 12:54:29 PM - humann.utilities - DEBUG: Remove directory: /home/bioinfo-tools/functional-annotation/hmmer_utils/data/cgd_gb_uniref90_abudnance_table_out/cds_gb_uniref90_abundance_table_humann_temp/tmprtlevoha

And the resulting output:

# Pathway	cds_gb_uniref90_abundance_table_Abundance
UNMAPPED	0.0000000000
UNINTEGRATED	0.0000000000

Is there something I’m doing wrong with preparing the input file or launching HUMAnN?

2

That input file looks a little weird, e.g. mix of UniRef50s and UniRef90s, and it’s clearly been filtered to a single species (losing the original stratified structure). It is possible that something in there is causing the pathway quantification to fail.

3

Indeed the data from a single species have been used to have a small test dataset. Good catch with the loset UniRef50 ID, I completely missed that - thanks!