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Combat normalization returns negative values

I am trying to correct batch effect using combat.

About 40% of my genes ends up having at least one negative result. If just drop those genes the resulting normalized PCA plot clusters neatly (but I loose 40 % of genes):

I've tried turning them into zeros, but that makes a really bad PCA clustering (especially at the PC1):

Is there a way to not loose almost half the data bit without distorting it too much?

sva rna-seq batch-correction r combat
• 10,311 views • link
updated by Ram Ram • written by lahat.albert • lahat.albert

Hi Lahat,

It would be useful to know littlle background of your samples, how you are using ComBat for batch normalization ; before and after boxplot of each sample.

Regards,
Mamun

• 0 views • link
updated by Ram Ram • written by mamunbabu2001 • mamunbabu2001

Hi. the samples are mice RNAseq data from several treatments. The sequencing was done in three batches. two batches (1 and 2 was done using truseq method), and batch 3 was done using gencore method.

Without normalization there is a very strong batch effect between methodologies:

Here is the code:

dat = read.table('200genes/counts.count',header=TRUE,row.names=1)
sif = read.table('200genes/batches',header=TRUE,sep='\t')
batch = as.character(unlist(sif['Batch']))
modcombat = model.matrix(~1, data = dat)
dat = as.matrix(dat)
library(sva)

dat_filtred = (dat[(rowVars(dat)) > 0,]) #removes 0 variance
print('this are the top genes removed (they should be zero)')
head(sort(rowMeans(dat[!( rowVars(dat) > 0),]),decreasing=TRUE))
combat_edata = ComBat(dat=dat_filtred,batch=batch,mod=NULL,par.prior=TRUE,prior.plots=FALSE)
combat_edata = ifelse(combat_edata<0,0,combat_edata) # converts negative normalized into 0
write.table(combat_edata,'200genes/counts.NORM.count')
• 0 views • link
updated by Ram Ram • written by lahat.albert • lahat.albert

1 answer

Hi There,

In RNASeq data some transcripts may have 0 FPKM/RPKM count. When you are using combat, have you already converted the data to log10 scale? Remember to add 1 before transforming the data back to log scale.

Not sure if ComBat is the best way to remove batch effect from RNASeq data. svaseq might be a better option.

Another approach :: Instead of correcting for the batch effect, why not include batch label as a factor in the design matrix in a multi-factorial analysis in DESeq or edgeR.

There is an elaborated discussion in this thread regarding this issue.

Hope this helps.

Mamun

• 0 views • link
updated by Ram Ram • written by mamunbabu2001 • mamunbabu2001

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