Error in using Maaslin2


I am a new user of the Maaslin2 package. I had used my data and have this error:

fit_data = Maaslin2(
input_data = final_merged_for_Maaslin2_input,
input_metadata = df_metadata,
min_prevalence = 0,
normalization = “NONE”,
output = “demo_output”,
fixed_effects = c(“Genotype”, “Timepoint”),
random_effects = c(“Subject”),
reference = c(“WT,d00”))
[1] “Warning: Deleting existing log file: demo_output/maaslin2.log”
2022-08-17 14:28:11 INFO::Writing function arguments to log file
2022-08-17 14:28:11 INFO::Verifying options selected are valid
2022-08-17 14:28:11 INFO::Determining format of input files
2022-08-17 14:28:11 INFO::Input format is data samples as rows and metadata samples as rows
2022-08-17 14:28:11 INFO::Formula for random effects: expr ~ (1 | Subject)
2022-08-17 14:28:11 INFO::Formula for fixed effects: expr ~ Genotype + Timepoint
Error in Maaslin2(input_data = final_merged_for_Maaslin2_input, input_metadata = df_metadata, :
Please provide the reference for the variable ‘Timepoint’ which includes more than 2 levels: d00, d01, d02, d03, d04, d05, d06, d07, d08, d09, d10.

It will be great if anyone can help me with this.

Hi @iman,

The issue here appears to be that MaAsLin is not reading the reference line of the code correctly. I think if you change it to: reference = c("Timepoint,d00") it should fix this error.

I hope that helps, please let us know if this issue persists.


It finally worked with this. Thank you very much!