Inquiry regarding joint analysis of two breeds using fixed effects in methylKit

[Guilherme Oselame]

Hi,

I am using methylKit for a DNA methylation analysis and I would like to clarify the best way to include 'Breed' as a fixed effect in my model.

Currently, I have a dataset with 19 animals. My experimental design consists of two groups (Affected vs. Control). I am processing them together in methRead using the treatment vector to identify cases and controls. The samples are structured as follows:

• Affected Group (10 animals): 5 animals from Breed 1 and 5 animals from Breed 2.

• Control Group (9 animals): 5 animals from Breed 1 and 4 animals from Breed 2.

In my preliminary attempt, I added the breed information as a covariate using numeric values (1 and 2). However, I want to ensure that the model treats 'Breed' strictly as a fixed effect and not as a continuous numeric covariate, especially since the sample sizes per breed are slightly different between groups.

I am relatively new to this type of statistical analysis in bioinformatics and would appreciate help with the following:

1. Does calculateDiffMeth support treating categorical variables like 'Breed' as fixed effects?

2. Since I used numbers (1 and 2) to represent the breeds, should I explicitly convert this column to a factor in R before passing it to the covariates argument to ensure it is treated as a fixed effect?

3. Given my specific sample distribution (5+5 affected, 5+4 control), is the standard calculateDiffMeth approach with covariates the most robust way to handle this, or is there a better alternative in methylKit for multi-breed designs?

Thank you for your time and for the support!

Best regards,

Guilherme.