Hi,

I'm trying to remove the batch effect from my dataset. I've used the following code:

create deseq object. In the design matrix, I have the patient variable with 'cancer' or 'healthy' samples. And the sex variable with male/M or female/F

dds<-DESeqDataSetFromMatrix(dataset,colData =metadata, design = ~Patientgroup+sex)
dds <- estimateSizeFactors(dds)
dds<-DESeq(dds)

create surrogate variable

dat  <- counts(dds, normalized = TRUE)
idx  <- rowMeans(dat) > 1
dat  <- dat[idx, ]
mod  <- model.matrix(~ Patient.group+sex, colData(dds))
mod0 <- model.matrix(~1, colData(dds))
svseq <- svaseq(dat, mod, mod0, n.sv = 1)
ddssva$SV1 <- svseq$sv[,1]
ddssva$SV2 <- svseq$sv[,2]
design(ddssva) <- ~ SV1 + Patient.group + sex

remove surrogate batch effect

rld <- vst(ddssva, blind=FALSE)
mat <- assay(rld)
mm <- model.matrix(~Patient.group + sex, colData(rld))
mat <- limma::removeBatchEffect(mat, batch=vsd$SV1, design=mm)

However, the 'mat line' at the end gives me this warning: Coefficients not estimable: batch173. Warning message: Partial NA coefficients for 3741 probe(s)

I believe this is because the 'mm' object looks like this, with patient group column only containing 1's. This is part of the 'mm' dataset:

                        (Intercept) Patient.groupcancer sexM
sample.001                        1                             1    0          
sample.002                        1                             1    1          
sample.003                        1                             1    1          
sample.004                        1                             1    0          
sample.005                        1                             1    0          
sample.007                        1                             1    1          
sample.008                        1                             1    0          

However, I don't understand why the patient group suddenly only has 1's. Can anyone explain?