Errors with humann-metaphlan

Microbial community profiling HUMAnN
1

Hello, all!

I would appreciate any help.

I have been strugling with this for a few days…

I want to use humann, so first i installed the newest version, and it kept giving me errors like - metaphlan: error: unrecognized arguments: --bowtie2out

So, I looked and found a few solutions, like here - metaphlan: error: unrecognized arguments: --bowtie2out - Error when running HUMAnN tutorial - #3 by tjhackmann

I used suggested solution

conda create --name biobakery4 python=3.12
conda activate biobakery4
conda config --add channels defaults
conda config --add channels bioconda
conda config --add channels conda-forge
conda config --add channels biobakery
conda install humann=4.0.0a1 metaphlan=4.1 -c biobakery
metaphlan --install --index mpa_vOct22_CHOCOPhlAnSGB_202403

But, when I tryied to run humann, it kept giving me the next error - The MetaPhlAn taxonomic profile provided does not contain the database version vOct22_CHOCOPhlAnSGB_202403 in any of its header lines.

It looked like I had the wrong version of metaphlan.

I had - humann v4.0.0.alpha.1 and MetaPhlAn version 4.1.1 (11 Mar 2024)

So, I updated the metaphlan and got the same error, as first time - metaphlan: error: unrecognized arguments: --bowtie2out /……/Humann_out_final/final.contigs_humann_temp/final.contigs_metaphlan_bowtie2.txt

I am lost, really, not sure what to do.

Probably I just don’t understand the root of these errors.

Updating the database I also saw - [Error] EnvironmentError “HTTP Error 404: Not Found”
Unable to download http://cmprod1.cibio.unitn.it/biobakery4/metaphlan_databases/mpa_vOct22_CHOCOPhlAnSGB_202403.nwk

I would appreciare any suggestions!

Thanks,

Best,

Alla

2

I have tried to reinstall humann

Every time I try to run it I got a new issues, they are presented below:

To install humann I used - Option 1: Latest Release (Recommended)

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then -humann_databases --download chocophlan full /path/chocophlan

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I tried to run it - humann --input /CLEAN_READS_metawrap/all/combined_reads_FP1.fastq --threads 20 --output /path/humann_raw/fp1/
Output files will be written to: /path/humann_raw/fp1
Removing spaces from identifiers in input file …

CRITICAL ERROR: The metaphlan executable can not be found. Please check the install.

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Ok, looks like there is no metaphlan, I have installed it using -

conda install bioconda::metaphlan

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Try number 2 - humann --input /CLEAN_READS_metawrap/all/combined_reads_FP1.fastq --threads 20 --output /path/humann_raw/fp1/ --metaphlan /path/miniconda3/bin/metaphlan
Output files will be written to: /path/humann_raw/fp1
Removing spaces from identifiers in input file …

ERROR: You are using the demo UniRef database with a non-demo input file. If you have not already done so, please run humann_databases to download the full UniRef database. If you have downloaded the full database, use the option --protein-database to provide the location. You can also run humann_config to update the default database location. For additional information, please see the HUMAnN User Manual.

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Ok, I did next -

humann_databases --download uniref uniref90_diamond /path/uniref_humann

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Try number 3 - humann --input /path/CLEAN_READS_metawrap/all/combined_reads_FP1.fastq --threads 20 --output /path/humann_raw/fp1/ --metaphlan /path/miniconda3/bin/metaphlan
Output files will be written to: /path/humann_raw/fp1
Removing spaces from identifiers in input file …

Running metaphlan …

CRITICAL ERROR: Error executing: /path/miniconda3/bin/metaphlan /path/humann_raw/fp1/combined_reads_FP1_humann_temp/tmp75imktmc/tmpxru3wz4b -t rel_ab -o /path/humann_raw/fp1/combined_reads_FP1_humann_temp/combined_reads_FP1_metaphlan_bugs_list.tsv --input_type fastq --bowtie2out /path/humann_raw/fp1/combined_reads_FP1_humann_temp/combined_reads_FP1_metaphlan_bowtie2.txt --nproc 20

metaphlan: error: unrecognized arguments: --bowtie2out /path/humann_raw/fp1/combined_reads_FP1_humann_temp/combined_reads_FP1_metaphlan_bowtie2.txt___________________________________________

Please, anyone can suggest what am I doing wrong?

I am just going circules here…

humann --version
humann v3.9

metaphlan --version
MetaPhlAn version 4.2.2 (4 Jun 2025)

Thanks

3

Humann requires specific versions of metaphlan (and its dbs). In the recent past, I could not get a functional install of humann 3.9.

However, I can say that humann 3.8 is compatible with metaphlan 4.1 ( Getting humann and metaphlan to integrate ; includes installation directions for both humann and metaphlan via conda and how to execute them together)

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