Running without reference


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?

Elliston Vallarino

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.

Jacob Nearing

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?