Metaphlan 4 using with Humann3.9

Microbial community profiling HUMAnN
1

Hello everyone,

I’m running into a version compatibility error when trying to use a taxonomic profile from the latest version of MetaPhlAn 4.2.2 as input for HUMAnN 3.9. I would appreciate any advice on the best practice for this workflow.

My Workflow

First, I successfully generated a taxonomic profile using MetaPhlAn 4 with the latest database:

Bash

metaphlan /mnt/d/leopardWGS/host_removed_alignment_results/02_M1_10K_combined.fastq.gz \
    --input_type fastq \
    --db_dir /mnt/d/humann_databases/chocophlan \
    -x mpa_vJan25_CHOCOPhlAnSGB_202503 \
    --nproc 6 \
    -o /mnt/d/leopardWGS/humann_output_M1_10K/taxonomic_profile.txt

Next, I tried to use this profile for functional analysis with HUMAnN 3.9:

Bash

humann --input /mnt/d/leopardWGS/host_removed_alignment_results/02_M1_10K_combined.fastq.gz \
    --output /mnt/d/leopardWGS/humann_output_M1_10K \
    --taxonomic-profile /mnt/d/leopardWGS/humann_output_M1_10K/taxonomic_profile.txt \
    --threads 6 \
    --protein-database /mnt/d/humann_databases/uniref/uniref90_201901b

The Error

This results in the following error, indicating a version mismatch:

ERROR: The MetaPhlAn taxonomic profile provided was not generated with the expected database version. Please update your version of MetaPhlAn to at least v3.0.

My Questions

  1. Is there a way to make the MetaPhlAn 4.x version profile compatible with HUMAnN 3.9, or is this combination not supported?

  2. If not, is the recommended approach to downgrade and use a MetaPhlAn 3 database (mpa_v30_CHOCOPhlAn_201901) to maintain compatibility with HUMAnN 3.9?

  3. For those using MetaPhlAn 4.x version for taxonomic profiling, what is the current best practice for the subsequent functional analysis? Are you using the HUMAnN 4 alpha version?

Thank you for your help!

2

The recent HUMAnN 3.X releases were each made to be compatible with the most recent MetaPhlAn 3 marker database or a specific MetaPhlAn 4 database. HUMAnN 3.9 was the most recent such compatibility release and was designed for the MetaPhlAn 4 Jun23 markers, which are now one step behind the most recent markers (Jan25). We don’t currently have a plan to keep issuing HUMAnN 3.X compatibility releases.

If you’re going for backward compatibility with existing profiles, I would use MetaPhlAn 3 + HUMAnN 3. Otherwise I’d suggest switching to MetaPhlAn 4 + HUMAnN 4 (alpha), with the alpha currently based on the Oct22 markers (with Jan25 in development). Note that the most recent MetaPhlAn 4.2+ is not compatible with the alpha, but we are working on that.