HUMAnN MinPath drastically reducing pathway counts and inconsistent results

Microbial community profiling HUMAnN
1

Hello,

I’m currently running HUMAnN, and when I enable the --minpath option, certain pathways almost completely disappear from the output.
For example, for PWY-7237: myo-, chiro- and scyllo-inositol degradation, when MinPath is OFF I see many strain-level entries (dozens), but when MinPath is ON, only a single entry remains.

Here’s what I have checked:

  1. Database integrity

    • metacyc_reactions_level4ec_only.uniref.bz2, metacyc_pathways_structured_filtered_v24_subreactions, map_metacyc-pwy_name.txt.gz, map_metacyc-rxn_name.txt.gz all have identical md5sum values compared to the other environment.

    • Verified via logs that both runs are using the same --pathways-database file: metacyc_pathways_structured_filtered_v24_subreactions.

  2. Environment variables / execution settings

    • Setting PYTHONHASHSEED=0, LC_ALL=C, and changing multi-threading settings made no difference in results.

  3. Additional observations

    • When MinPath is OFF, my results are identical to the comparison environment.

    • This means the discrepancy is introduced only during the MinPath step.

    • It seems MinPath is selecting a minimal set of pathways from RXNs, which is filtering out many strain-level pathway entries.

    • I’m wondering if noise filtering or gap-fill steps might be overly aggressive for certain pathways like PWY-7237.

Questions:

  • Is such a drastic reduction in pathways expected behavior when running MinPath, or could this indicate a database/mapping issue?

  • For cases like PWY-7237, what in the MinPath logic could cause this over-filtering?

  • Does the gap-fill process (and its related mapping files) have any impact on MinPath results?

Thank you in advance for your help.

2

MinPath tries to explain the reactions you saw using the smallest number of pathways possible, so if you turn it on you will 100% expect to see fewer pathways in the output! Imagine you have three pathways:

  1. A → B
  2. B → C
  3. A → B → C

If you see all three reactions in your sample (A + B + C), and MinPath is on, HUMAnN will only report pathway #3 since that is the simplest explanation of the three reactions. Whereas if you turn MinPath off then HUMAnN would report all three pathways.