# Maaslin2 (v1.12.0) issue with metadata catagories

**URL:** <https://forum.biobakery.org/t/maaslin2-v1-12-0-issue-with-metadata-catagories/7613>\
**Category:** MaAsLin\
**Created:** [November 27, 2024, 12:21pm UTC](https://forum.biobakery.org/t/maaslin2-v1-12-0-issue-with-metadata-catagories/7613 "2024-11-27T12:21:14Z")\
**Posts on this page:** 3\
**Page:** 1

<div class="post-metadata">

**Author:** ![WedwardsKent](https://avatars.discourse-cdn.com/v4/letter/w/c67d28/32.png) [@WedwardsKent](https://forum.biobakery.org/u/WedwardsKent)\
**Post date:** [November 27, 2024, 12:21pm UTC](https://forum.biobakery.org/t/maaslin2-v1-12-0-issue-with-metadata-catagories/7613/1 "2024-11-27T12:21:14Z")

</div>

Hello!  
I am currently working with Maaslin2 on some 16s sequencing data and have been using Maaslin2 to look for differentially abundant taxa based on two covariates. The program runs amazingly when i use only one variable, however, when i use two fixed variables the analysis is run normally for the 1st variable and the second i see only the variable name not its individual categories.

variable categories  
-Parasite load: Negative, LowLoad, HighLoad  
-Health: HH, IH, DH

the results will show only: IH, DH, ParasiteLoad (not the actual categories of that variable)  
Could this be an issue with the format of my data, or with maaslin? I have included the run command below.

fit\_data = Maaslin2(  
input\_data = maslin.otu\_table.nr.species,  
input\_metadata = maslin.meta\_data.nr.s,  
output = “maslin.output\_2.nr.covariate”,  
fixed\_effects = c(“Health”,“ParasiteLoad”),  
normalization=“NONE”,  
random\_effects = “NONE”,  
reference = c(“Health,HH”,“ParasiteLoad,Negative”))

Any feedback is very much appreciated  
Will

---

<div class="post-metadata">

**Author:** ![WillNickols](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/willnickols/32/3223_2.png) [@WillNickols](https://forum.biobakery.org/u/WillNickols)\
**Post date:** [November 27, 2024, 6:29pm UTC](https://forum.biobakery.org/t/maaslin2-v1-12-0-issue-with-metadata-catagories/7613/2 "2024-11-27T18:29:52Z")

</div>

Hi Will,

It looks like your reference string should be `"Health,HH;ParasiteLoad,Negative"` (with a semicolon). If that doesn’t work, check that parasite load is a factor or character, not a numeric type, and that all the levels are actually present in your data. It’s possible that because of the way parasite load overlaps with health, your categories might be entirely overlapping. You can check this with `table(maslin.meta_data.nr.s$Health, maslin.meta_data.nr.s$ParasiteLoad)`.

Will

---

<div class="post-metadata">

**Author:** ![WedwardsKent](https://avatars.discourse-cdn.com/v4/letter/w/c67d28/32.png) [@WedwardsKent](https://forum.biobakery.org/u/WedwardsKent)\
**Post date:** [November 28, 2024, 8:46am UTC](https://forum.biobakery.org/t/maaslin2-v1-12-0-issue-with-metadata-catagories/7613/3 "2024-11-28T08:46:27Z")

</div>

Hi Will (snap!)  
thanks for the help, you were right there is very strong overlap (almost complete) between the two metadata variables so i guess there were no exclusive differently abundant taxa. even with the semicolon it did not show the separate categories but i guess that is why.

Kind regards  
Will