# Extending the metaphlan4 database (Jan2025) to include subspecies **URL:** \ **Category:** MetaPhlAn\ **Created:** [February 10, 2026, 8:12pm UTC](https://forum.biobakery.org/t/extending-the-metaphlan4-database-jan2025-to-include-subspecies/8767 "2026-02-10T20:12:34Z")\ **Posts on this page:** 3\ **Page:** 1
**Author:** ![ryanbb](https://avatars.discourse-cdn.com/v4/letter/r/d9b06d/32.png) [@ryanbb](https://forum.biobakery.org/u/ryanbb)\ **Post date:** [February 10, 2026, 8:12pm UTC](https://forum.biobakery.org/t/extending-the-metaphlan4-database-jan2025-to-include-subspecies/8767/1 "2026-02-10T20:12:34Z")
I’m trying to extend the current version (Jan2025) of the metaphlan so that the tool will estimate the abundances of two Bifidobacterium longum subspecies (subsp. longum and subsp. infantis). I’m following some published methodology [here](https://www.nature.com/articles/s41467-024-45209-y) with code available [here](https://github.com/yassourlab/MetaPhlAn-B.infantis/blob/main/metaphlan_longum_markers.py). I have successfully added the marker genes to the bowtie indexed database and updated the pkl file. My understanding is that the two subspecies are successfully added to the taxonomy and the marker genes are added to the markers with clades `t __subsp.infantis` and `t__ subsp.longum`. Then the original B. longum clade/SGB (`t__SGB17248`) is then removed and the marker genes are assigned to a new clade (`s__Bifidobacterium_longum`). However, when I run metaphlan4 the mapping is successful but I encounter an error with full traceback: `Traceback (most recent call last):` ` File "/opt/conda/bin/metaphlan", line 10, in ` ` sys.exit(main())` ` ~~~~ ^^` ` File "/opt/conda/lib/python3.13/site-packages/metaphlan/metaphlan.py", line 2318, in main` ` metaphlan_runner.run_metaphlan()` ` ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ^^` ` File "/opt/conda/lib/python3.13/site-packages/metaphlan/metaphlan.py", line 1919, in run_metaphlan` ` self.metaphlan_analysis.report_results(self.tree, self.total_metagenome, self.avg_read_length)` ` ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^` ` File "/opt/conda/lib/python3.13/site-packages/metaphlan/metaphlan.py", line 1385, in report_results` ` self.get_mapped_fraction()` ` ~~~~~~~~~~~~~~~~~~~~~~~~ ^^` ` File "/opt/conda/lib/python3.13/site-packages/metaphlan/metaphlan.py", line 1267, in get_mapped_fraction` ` self.mapped = self.tree.relative_abundances()` ` ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ^^` ` File "/opt/conda/lib/python3.13/site-packages/metaphlan/metaphlan.py", line 309, in relative_abundances` ` clade.compute_coverage()` ` ~~~~~~~~~~~~~~~~~~~~~~ ^^` ` File "/opt/conda/lib/python3.13/site-packages/metaphlan/metaphlan.py", line 179, in compute_coverage` ` self.coverage = sum([child.compute_coverage() for child in self.children.values()])` ` ~~~~~~~~~~~~~~~~~~~~~~ ^^` ` File "/opt/conda/lib/python3.13/site-packages/metaphlan/metaphlan.py", line 179, in compute_coverage` ` self.coverage = sum([child.compute_coverage() for child in self.children.values()])` ` ~~~~~~~~~~~~~~~~~~~~~~ ^^` ` File "/opt/conda/lib/python3.13/site-packages/metaphlan/metaphlan.py", line 179, in compute_coverage` ` self.coverage = sum([child.compute_coverage() for child in self.children.values()])` ` ~~~~~~~~~~~~~~~~~~~~~~ ^^` ` [Previous line repeated 4 more times]` ` File "/opt/conda/lib/python3.13/site-packages/metaphlan/metaphlan.py", line 184, in compute_coverage` ` rat_nreads = self.filter_markers_for_ext()` ` File "/opt/conda/lib/python3.13/site-packages/metaphlan/metaphlan.py", line 142, in filter_markers_for_ext` ` ext_markers2nreads = self.taxa2clades[ext].markers2nreads` ` ~~~~~~~~~~~~~~~~ ^^^^^` ` KeyError: 'SGB17248'` Obviously, at some point the clade (SGB17248) isn’t completely removed/reassigned from the database but I am struggling to find it (I can see the SGB ID is appended as part of the marker gene IDs but I have no idea whether this is used for anything). Is there anything in the error that will point to what I have missed in re-building the database? ---
**Author:** ![Claudia\_Mengoni](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/claudia_mengoni/32/2871_2.png) [@Claudia\_Mengoni](https://forum.biobakery.org/u/Claudia_Mengoni)\ **Post date:** [February 11, 2026, 9:51am UTC](https://forum.biobakery.org/t/extending-the-metaphlan4-database-jan2025-to-include-subspecies/8767/2 "2026-02-11T09:51:20Z")
Hi @ryanbb I am guessing the reason you encounter this issue is because within the database pickle file for each marker you have a list of external SGBs, which are SGBs different from the one that the marker is representing but that in a small percentage of cases has occurrences of that same marker. This means that the marker specifically doesn’t get used by MetaPhlAn when any of the external SGBs are present in the sample, to avoid false positive detections. What likely happened is that you are detecting an SGB which has SGB17248 as an external SGBs. In practical terms what this means is that you will need to remove the occurrence of SGB17248 from the list of external markers of all SGBs. To be safe you could add the two subclades instead. It should be something like: ```auto mpa_example=pkl.load(bz2.open(‘mpa_vJan25_CHOCOPhlAnSGB_202503.pkl’,‘r’)) for m in mpa_example[‘markers’]: if ‘SGB17248’ in mpa_example[‘markers’][m][‘ext’]: mpa_example[‘markers’][m][‘ext’].remove(‘SGB17248’) mpa_example[‘markers’][m][‘ext’].append(‘subsp.longum’) mpa_example[‘markers’][m][‘ext’].append(‘subsp.infantis’) ``` ---
**Author:** ![ryanbb](https://avatars.discourse-cdn.com/v4/letter/r/d9b06d/32.png) [@ryanbb](https://forum.biobakery.org/u/ryanbb)\ **Post date:** [February 13, 2026, 9:42am UTC](https://forum.biobakery.org/t/extending-the-metaphlan4-database-jan2025-to-include-subspecies/8767/3 "2026-02-13T09:42:15Z")
Thanks @Claudia_Mengoni this was indeed the issue. There were 4 other markers with external links to the SGB of interest. I can complete runs of metaphlan now.