Dear creators of ComBat-seq,

for experimental reasons, I was trying to run ComBat-seq on only two batches of the GFRN dataset provided (https://github.com/zhangyuqing/ComBat-seq/tree/master/real_data_application). I cropped the matrix accordingly (99 % sure it is not the issue). Everything works perfectly as long as I do not use the 'group' parameter. I made sure only to use the correct labels here. See my batch and group input:

batch <- factor(c(1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,2,2,2,2,2,2,2,2,2,2,2,2))

group <- factor(c("gfp", "gfp", "gfp", "gfp", "gfp", "gfp", "gfp", "gfp", "gfp", "gfp", "gfp", "gfp", "her2",
"her2", "her2", "her2", "her2", "gfp", "gfp", "gfp", "gfp", "gfp", "gfp", "egfr", "egfr",
"egfr", "egfr", "egfr", "egfr"))

Both have the same length of 29, which matches with my amount of samples/columns in the count dataset.

Be aware that I am a programmer myself and doublechecked everything to be correct. Everything should work, yet I get the following error:

The covariates are confounded! Please remove one or more of the covariates so the design is not confounded

Is there any hidden restriction on the covariates (groups) I use? With all three batches and respective metadata it works fine on the full GFRN dataset. In my example above, Batch 1 contains gfp and her2 and Batch 2 contains gfp and egfr.

I do need your input on this, I believe, since the chosen license prohibits me from altering and using it in a research and puclication setting.

Thank you in advance!

Simon Schlumbohm