Hi, I’m using Maaslin2 to analyze Humann2 output (genefamilies and pathabundance tables) to assess differences between treatment (Bb) and control (C) samples (5 biological replicates).
I used humann_renorm to obtain CPM values and the sample-specific tables were finally joined.
Then in R:
head(genefamilies)
head_genefamilies.csv (821 Bytes)
head(pathabundance)
head_pathabundance.csv (729 Bytes)
metadata
metadata.csv (177 Bytes)
I’ve first tried LOG transform on both files with the following command:
fit_data2 = Maaslin2(normalization = “NONE”,
input_data = df_input_data,
input_metadata = df_input_metadata,
output = “output2”,
fixed_effects = c(“condition”),
transform = “LOG”)
And then I’ve tried AST transformation:
fit_data2 = Maaslin2(normalization = “NONE”,
input_data = df_input_data,
input_metadata = df_input_metadata,
output = “output2”,
fixed_effects = c(“condition”),
transform = “AST”)
LOG results are ok, but identified few significantly abundant features:
number of significant genefamilies = 6
number of significant pathways = 6
AST results identified 160 genefamilies, while for pathways resulted in the following error (with no output):
Error in contrasts<-
(*tmp*
, value = contr.funs[1 + isOF[nn]]) :
contrasts can be applied only to factors with 2 or more levels
2021-10-29 10:26:27 WARNING::Fitting problem for feature 123 returning NA
Moreover, when I compare the results of gene familes (LOG vs AST), the upregulated gene families identified using LOG transformation are NOT present in AST gene families result table.
I would like to ask:
- Am I using the correct methods, considering that in my data zero-count is frequent for many features in many samples?
- Considering the discordance in the gene families output, in which method (LOG or AST) I should trust?
3)What’s the reason of such error when I use AST on pathways? It depends by the many zeros in my dataset?
Thank you in advance
Andrea
3)