# Running without reference

**URL:** <https://forum.biobakery.org/t/running-without-reference/7113>\
**Category:** MaAsLin\
**Created:** [May 30, 2024, 4:39pm UTC](https://forum.biobakery.org/t/running-without-reference/7113 "2024-05-30T16:39:29Z")\
**Posts on this page:** 3\
**Page:** 1

<div class="post-metadata">

**Author:** ![EllistonV](https://avatars.discourse-cdn.com/v4/letter/e/c0e974/32.png) [@EllistonV](https://forum.biobakery.org/u/EllistonV)\
**Post date:** [May 30, 2024, 4:39pm UTC](https://forum.biobakery.org/t/running-without-reference/7113/1 "2024-05-30T16:39:30Z")

</div>

Hello,

I ran a 16S sequencing, and I analyzed it via MOTHUR. I am trying to determine if any of the ASVs correlate with the presence of Legionella. I have 3 ‘levels’: high, low, and negative, but I do not have a reference.

Is there any way to run MAASLIN2 without a reference level like it was done with LEfSe?

Thanks,  
Elliston Vallarino

---

<div class="post-metadata">

**Author:** ![nearinj](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/nearinj/32/2135_2.png) [@nearinj](https://forum.biobakery.org/u/nearinj)\
**Post date:** [May 30, 2024, 9:45pm UTC](https://forum.biobakery.org/t/running-without-reference/7113/2 "2024-05-30T21:45:12Z")

</div>

Hi @EllistonV,

MaAsLin2 makes use of linear models which require a reference (or baseline) category to compare coefficients between. When there is only two levels within a category it will just pick one as the baseline. However, if more than two levels exist you need to determine which should be the baseline.

Thanks,  
Jacob Nearing

---

<div class="post-metadata">

**Author:** ![EllistonV](https://avatars.discourse-cdn.com/v4/letter/e/c0e974/32.png) [@EllistonV](https://forum.biobakery.org/u/EllistonV)\
**Post date:** [May 31, 2024, 10:51pm UTC](https://forum.biobakery.org/t/running-without-reference/7113/3 "2024-05-31T22:51:58Z")

</div>

Thank you so much for the answer!

So, this means that I must have reference data to use MaAslin2? If there are more than 2 levels, will changing the reference level affect the results?

Regards,  
Elliston