Question about fix_formula and group in specific data

[martagarri]

Hello everyone. I have been reading about the arguments fix_formula and group of the function ancombc2 in R. However, I still do not know how to proceed with a specific data set.

I have data of cloacal bird microbiome (16S rRNA). These birds correspond to 6 different localities and 3 different habitat types: 2 natural habitats (1 in Seville and 1 in Huelva), 2 rural habitats (1 in Seville and 1 in Huelva) and 2 urban habitats (1 in Seville and 1 in Huelva). Some of the birds are infected by avian malaria parasites and some of them are uninfected. I separated the data by habitat, so I will run ancombc2 3 times. Then, each data subset consists of birds from one habitats that can be from 2 different provinces (Sevilla/Huelva) and can present 2 different infection status (infected/uninfected).

For each subset, I am interested in the taxa differently abundant by infection status. However, the province variable can also affect the microbiota. Then, I wonder if I have to include province in the fix_formula but not in the group argument, ej:

output= ancombc2(data = ps_filtered_rural, tax_level ="Family",
fix_formula = "infection+province", rand_formula = NULL,
p_adj_method = "holm", pseudo_sens = TRUE,
prv_cut = 0.10, lib_cut = 1000, s0_perc = 0.05,
group = "infection", struc_zero = TRUE, neg_lb = TRUE,
alpha = 0.05, n_cl = 2, verbose = TRUE,
iter_control = list(tol = 1e-5, max_iter = 20,
verbose = FALSE),
em_control = list(tol = 1e-5, max_iter = 100),
lme_control = NULL,
mdfdr_control = list(fwer_ctrl_method = "holm", B = 100),
trend_control = NULL)

Thank you in advance,
Marta