CRITICAL: Can not find file sample_616_metaphlan_bugs_list.tsv

Microbial community profiling HUMAnN
1

Hello,
I am running Humann3.9 together with Metaphlan4 and keep getting: " Can not find file sample_616_metaphlan_bugs_list.tsv".

When inspecting the log file, the first error is related to the version of the database:
humann.search.prescreen - ERROR: The MetaPhlAn taxonomic profile provided was not generated with the database version v3 or vJun23 . Please update your version of MetaPhlAn to at least v3.0 or if you are using MetaPhlAn v4 please use the database vJun23.

I then dowloaded vJun23 and specified in the parameters but still keep getting a similar error:
humann.utilities - DEBUG: b’WARNING: It seems that you do not have Internet access.\nERROR: Cannot find a local database. Please run MetaPhlAn using option “-x <database_name>”.\n You can download the MetaPhlAn database from \n Index of /biobakery4/metaphlan_databases \n \n’
04/17/2024 04:55:41 PM - humann.utilities - CRITICAL: Can not find file sample_616_metaphlan_bugs_list.tsv

The command I used and got this last error was the following:
humann -i sample_616.fastq -o /humann3-output --input-format fastq --nucleotide-database /metaphlan_database_v31 --metaphlan-options=“–stat_q 0.01 -t rel_ab_w_read_stats --tax_lev g --ignore_eukaryotes --ignore_archaea -x mpa_vJun23_CHOCOPhlAnSGB_202307 --bowtie2db /metaphlan_database_vJun23_202307” --protein-database /humann3-database-uniref --threads 5

Any ideas on how to solve this, please?
This is the content of the /metaphlan_database_vJun23_202307 database folder I specified in the --bowtie2db parameter.
image

Thank you,

Thais

2

It sounds like your MetaPhlAn install is not working properly. I would see if you can get MetaPhlAn working with the vJun23 marker database outside of HUMAnN first.

3

Thanks for the reply!

I tried your suggestion with the following command and got the error below:

metaphlan --input_type fastq --stat_q 0.01 -t rel_ab_w_read_stats --tax_lev g --ignore_eukaryotes --ignore_archaea -x mpa_vJun23_CHOCOPhlAnSGB_202307 --bowtie2db /home/metaphlan_database_vJun23_202307 --bowtie2out sample_616.bowtie2.bz2

WARNING: It seems that you do not have Internet access.
ERROR: Cannot find a local database. Please run MetaPhlAn using option “-x <database_name>”.
** You can download the MetaPhlAn database from**
** Index of /biobakery4/metaphlan_databases**

It seems something is wrong recognizing this version of the database. It tells me to include the “-x” option although I already included it.

It’s worth mentioning I am working on a multiuser HPC for the analysis above.

To troubleshoot, I additionally tried running a Metaphlan4 installation I have in my own user (independent of the Humann3.9 installation) with this vJun23 database, and the same error as above appears. But, when I use a different database version - like the mpa_vJan21_CHOCOPhlAnSGB_202103 - it runs perfectly.

So it’s definitely something connected to this vJun23 database. On the other hand, Metaphlan within Humann3.9 does not let me run with a different version.

Any tips how to troubleshoot, please?

4

Sorry for the slow reply. This definitely feels like a MetaPhlAn problem: you are specifying a database it can’t find so it’s trying to download it, but since it doesn’t have internet access it can’t. You can try reposting this more recent issue under the MetaPhlAn topic and see if the moderators there can suggest a fix.

As far as HUMAnN is concerned, yes, different 3.X versions are tied to specific MetaPhlAn 4 databases for now. Also, note that with the command you’re running above you’re only requesting genus-level abundances, whereas HUMAnN will require species-level abundances (the default MetaPhlAn output) to be able to select pangenomes to profile against. Hence, once you get your MetaPhlAn issue sorted out, this command might still not work in HUMAnN.

Can you help me understand why you’re changing MetaPhlAn defaults / using non-default databases here?

5

Hello,

I’m trying to use HUMAnN 3.9 together with a MetaPhlAn 4.2.2 profile that was generated using the latest mpa_vJun23_CHOCOPhlAnSGB_202307 database.

MetaPhlAn runs fine and produces taxonomic output. However, when I use the resulting taxonomy file (*_metaphlan.txt) as input to HUMAnN via the --taxonomic-profile option, I encounter this error:

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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.

This is confusing because:

  • I am using MetaPhlAn v4.2.2, the latest release.
  • I used the vJun23 marker database.
  • HUMAnN and MetaPhlAn are both installed in the same Conda environment.
  • The .txt file is valid and contains relative abundances.

I’ve checked all paths and tested different versions, but still get this error. I’ve been stuck on this for over a month now. I really appreciate any help or insight.

HUMAnN is an exciting and powerful tool, but the database/version compatibility is very hard to manage.

Thanks in advance!

6

HUMAnN 3.9 provides forward compatibility to MetaPhlAn 4.1 and the Jun23 SGB database. We have not made a compatibility release yet for HUMAnN 3 and MetaPhlAn 4.2 + the Jan25 SGBs yet. I’m not 100% sure this will happen yet since we’re transitioning to HUMAnN 4 at this point, and the next version of HUMAnN 4 will be based on MetaPhlAn 4.2 + the Jan25 SGBs, so there will be less need for HUMAnN 3.x releases.