Error when running HUMAnN tutorial

Microbial community profiling HUMAnN
1

I am a new user and get an error when running the HUMAnN tutorial. Specifically, running

	humann --input demo.fastq.gz --output demo_fastq

results in

	metaphlan: error: unrecognized arguments: --bowtie2out .../humann/demo_fastq/demo_humann_temp/demo_metaphlan_bowtie2.txt

The tutorial installs MetaPhlAn version 4.2.2 (4 Jun 2025) and humann v3.9, and the error may be related to this issue or possibly this one.

Can you please indicate which commands in the tutorial need to be updated? Thank you for your help.

2

The threads you found accurately describe the situation. This unfortunately cannot be resolved by a change to the tutorial commands - it’s something that needs to be updated with the software itself. The HUMAnN 3 tutorial is really designed for use with MetaPhlAn 3 + HUMAnN 3. There is also a relatively new HUMAnN 4 tutorial designed for use with MetaPhlAn 4 + HUMAnN 4, but again not MetaPhlAn 4.2 (for the reasons described in the linked threads):

3

Thank you for your response, Eric—it got me to the right answer. In case it helps other new users, here are the commands that worked for me:

Installation

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

This installs the correct version of MetaPhlAn (4.1); the one installed by default doesn’t work. It also installs the correct version of the database (mpa_vOct22_CHOCOPhlAnSGB_202403).

Running tutorial data

conda activate biobakery4
metaphlan demo.fastq.gz --input_type fastq > demo.tsv -x mpa_vOct22_CHOCOPhlAnSGB_202403 -t rel_ab_w_read_stats
humann -i demo.fastq.gz --taxonomic-profile demo.tsv --output demo

This runs MetaPhlAn and HUMAnN sequentially through two separate commands. They are run in a single command in the tutorial, but that didn’t work for me. It also uses the correct version of the database (mpa_vOct22_CHOCOPhlAnSGB_202403).

I look forward to using MetaPhlAn and HUMAnN in a tool my lab is developing.

1 Like