I am trying to adjust my RNA data using ComBat-Seq (from sva R package) since I realised that there are 3 batches that I need to adjust for:

• Place (2 levels: place 1, place 2)
• Library Preparation Date (16 levels - different dates)
• Type of tube (2 levels: A, B)

I have 960 samples and around 62000 genes.

In my biological matrix, I have: Age, Sex, Group (cases,controls..) and WBC counts.

biol_mat = model.matrix(~Age + as.factor(Sex) + as.factor(Group) + LYMPH + MONO + NEUT, data=phenotype)

In the tutorial of Combat-Seq appears how to adjust by 1 variable but it doesn't tell you how to adjust by more than 1.

I have seen a lot of posts using combat that the only way is to adjust by 1 variable and then, with those results, adjust again by the 2nd variable and so on.

That would be:

Adjust by library prep.

raw.cts_adjustedLibPrep <- ComBat_seq(counts = raw_cts_matrix, batch=batch_libraryprep, group=NULL, covar_mod = biol_mat)

Adjust by library prep + type of tube.

raw.cts_adjusted_LibPrep_TypeTube <- ComBat_seq(counts = raw.cts_adjustedLibPrep, batch=batch_type_tube, group=NULL, covar_mod = biol_mat)

Adjust by library prep + type of tube + place

 raw.cts_adjusted_LibPrep_TypeTube_Place <- ComBat_seq(counts = raw.cts_adjusted_LibPrep_TypeTube, batch=batch_place, group=NULL, covar_mod = biol_mat)

For the first adjustment (library prep) it takes around 15min, but for the second... it has been running for more than 2 days.. I stopped it and launch it again, changing the adjustment but I am not sure if it will work..

Does anybody have an idea about how to fix the problem?

Thanks in advance