Hello, I'm doing some bulk RNA-Seq analysis to identify different genes in two condition (Braccio) but before that I want to identify some not known variable and delete the batch effect (RIN) of my variable. So I'm moving in this way:
dds_campione <- DESeqDataSetFromMatrix(countData=raw,
colData=condition_breakfast,
design=~ 1)
smallestGroupSize <- 3
keep <- rowSums(counts(dds_campione) >= 10) >= smallestGroupSize
dds_campione <- dds_campione[keep,]
dds_campione <- estimateSizeFactors(dds_campione)
matriceFiltrata_campione <- counts(dds_campione,normalized=TRUE)
mm_campione <- model.matrix(~ Braccio, colData(dds_campione))
mm0_campione <- model.matrix(~1, colData(dds_campione))
fit_campione <- svaseq(matriceFiltrata_campione, mod=mm_campione, mod0=mm0_campione)
dds_campione$SV1 <- fit_campione$sv[,1]
dds_campione$SV2 <- fit_campione$sv[,2]
dds_campione$SV3 <- fit_campione$sv[,3]
dds_campione$SV4 <- fit_campione$sv[,4]
dds_campione$SV5 <- fit_campione$sv[,5]
dds_campione$SV6 <- fit_campione$sv[,6]
dds_campione$SV7 <- fit_campione$sv[,7]
dds_campione$SV8 <- fit_campione$sv[,8]
design(dds_campione) <- ~ SV1 + SV2 + SV3 + SV4 + SV5 + SV6 + SV7 + SV8 + Braccio
vsd_campione <- vst(dds_campione, blind=FALSE)
assay(vsd_campione) <- limma::removeBatchEffect(assay(vsd_campione),
batch = condition_breakfast$RIN, design=mm_campione)
plotPCA(vsd_campione,intgroup=c( "Braccio"))
dds_campione <- DESeq(dds_campione)
res_campione <- results(dds_campione)
data_campione <- as.data.frame(res_campione)
data_campione <- tibble::rownames_to_column(data_campione, "geneID")
My question is:
When I look at the pca, my variance is high so removing the batch effect and using the sva seems to work but than, when I go one with the analysis in DESeq2, I do not see so much variabilites and that's because in "dds_campione <- DESeq(dds_campione)" I do not have the matix adjusted but the original one, how i can do? Because this matrix is with integers so I can not use it in the command. How I can use the adjusted matrix that I plot with the pca for the analysis?
I also try to insert the batch in the design
dds_campione <- DESeqDataSetFromMatrix(countData=raw,
colData=condition_breakfast,
design=~ RIN + Braccio)
But nothing really changes
Thank you!
