HUMANn 4 + MetaPhlAn 4

Microbial community profiling HUMAnN
1

I fixed this… SEE SOLUTION BELOW

Hi all sorry to bother you again,

MetaPhlAn version 4.1.1 (11 Mar 2024)
humann v4.0.0.alpha.1

I need to run MetaPhlAn 4 + HUMANn 4 (I want to run StrainPhlAn after that).

I had to install diamond, bowtie2, and glpk
I also provided the route to the databases with humann_config
I am using a SRR file from a human metagenome as a demo.

I used this for MetaPhlAn 4

metaphlan RAW/SRR14076335_1.fastq.gz --input_type fastq -s SAMS/SRR14076335.sam.bz2 --bowtie2out BOWTIE2/SRR14076335.bowtie2.bz2 -o BUGS/SRR14076335_profile.tsv --add_viruses --unclassified_estimation  --index mpa_vOct22_CHOCOPhlAnSGB_202403 --bowtie2db ./CHOCO/mpa_vOct22_CHOCOPhlAnSGB_202403

Then I uncompressed the sam file

bzip2 -d ./SAMS/SRR*

then I run humann

humann --input ./SAMS/SRR14076335.sam.bz2 --output ./OUT/SRR14076335 --threads 22 --metaphlan-options "--bowtie2db ./CHOCO" --nucleotide-database "./HUMANn/chocophlan"

I got this after that in the gene families file

Gene Family HUMAnN v4.0.0.alpha.1 Adjusted CPMs SRR14076335
READS_UNMAPPED 461337.0000000000

Edited to add the solution

My fault on this side I added -t rel_ab_w_read_stats and removed the --metaphlan-options from the humann run

metaphlan RAW/SRR14076335_1.fastq.gz --input_type fastq -s SAMS/SRR14076335.sam.bz2 --bowtie2out BOWTIE2/SRR14076335.bowtie2.bz2 -o BUGS/SRR14076335_profile.tsv --add_viruses --unclassified_estimation  --index mpa_vOct22_CHOCOPhlAnSGB_202403 --bowtie2db ./CHOCO/ -t rel_ab_w_read_stats --nproc 6 

humann --input ./RAW/SRR14076335_1.fastq.gz  --taxonomic-profile ./BUGS/SRR14076335_profile.tsv  --output ./FromBUGS/  --nucleotide-database "./HUMANn/chocophlan" --threads 2
3

Thanks for following up with your solution!

4

Hi,

I have a problem with running the demofile as provided by HUMANn v4.0.0.alpha.1 while using MetaPhlan 4.2 and using the newest database: mpa_vJan25_CHOCOPhlAnSGB_202503_VSG

When I summitted the job i got an error, because the flag --bowtie2out is changed to --mapout in metaphlan, but this is not yet updated in HUMANn. I have adjusted the prescreen.py code to correct for that. So that is working now.

However, now i received the following error:

PM - humann.search.prescreen - ERROR: The relative abundance and coverage were not found in the MetaPhlAn taxonomic profile

It seems that HUMANn expects different headers do differ when I run the demo file for both MetaPhlan and HUMANn independently:

Output:
#clade_name NCBI_tax_id relative_abundance additional_species

Expected:
#clade_name clade_taxid relative_abundance coverage estimated_number_of_reads_from_the_clade

the flag -t rel_ab_w_read_stats within MetaPhlan helps that error as described above:

humann --input ./05_HUMAnN/demo_humann_v4.fastq --output ./output_humann_demo_humann_2025DB_flag2 --metaphlan-options "--db_dir ./python3.9/site-packages/metaphlan/metaphlan_databases -t rel_ab_w_read_stats"

Next, It seems that the newest database 2025, does not work well with HUMANn as it expects the db:

humann.search.prescreen - ERROR: The MetaPhlAn taxonomic profile provided does not contain the database version vOct22_CHOCOPhlAnSGB_202403 in any of its header lines.

Anyone has an idea on how to solve this?

5

The HUMAnN 4 alpha is designed to work with MetaPhlAn’s vOct22_CHOCOPhlAnSGB_202403 marker set. It sounds like there is also an interface change in MetaPhlAn 4.2 that we will accommodate in the next HUMAnN 4 release. For now I would stick with an earlier MetaPhlAn 4 + the aforementioned marker database when running HUMAnN 4.

6

Just trying to get to grips with this too. Metaphlan 4 can use the new database for taxonomy but humann 4 uses the old Oct22 database and so I cant use metaphlan.tsv files that were generated from running metaphlan? I would have to run it all again using the fastq files for humann and let humann 4 reference to the Oct22 metaphlan database? I have been trying this for a couple of weeks now since i returned from the biobakery workshop. When I went, i was using an older metaphlan and humann, everything worked, I upgraded both and now they dont seem to work together.

7

I have replied to the new post you created here: