Dear Bioconductor community,

I'm trying to analyze a RNA-seq dataset with edgeR after importing the Kallisto counts with tximport and using the offset matrix. But I'm not sure if I'm using the offset matrix correctly. Can you kindly advise me? Below the code

data import and normalization

Txi_gene <- tximport(path, 
                     type = "kallisto", 
                     tx2gene = Tx2gene, 
                     txOut = FALSE,
                     ignoreAfterBar=TRUE,
                     ignoreTxVersion =TRUE)

myCounts <- Txi_gene$counts
myTPM <- Txi_gene$abundance
normMat <- Txi_gene$length
colnames(myCounts)<-c(sampleLabels)

#obtaining per-observation scaling factors for length
normMat <- normMat/exp(rowMeans(log(normMat)))
normCounts <- myCounts/normMat

#computing effective library sizes from scaled counts
eff.lib <- calcNormFactors(normCounts) * colSums(normCounts)

#combining effective library sizes with the length factors, and calculating offsets for a log-link GLM
normMat <- sweep(normMat, 2, eff.lib, "*")
normMat <- log(normMat)

edgeR: create a DGElist

myDGEList <- DGEList(counts= myCounts)
myDGEList <- scaleOffset(myDGEList, normMat)

#creating a matrix of CPMs and logCPMs
cpms <- edgeR::cpm(myDGEList, offset = myDGEList$offset, log = FALSE)
colSums(cpms)
log2.cpms <- edgeR::cpm(myDGEList, offset = myDGEList$offset, log=TRUE)

#set the design
group <- paste(myo, sex, sep="_")
design <- model.matrix(~0+group)

#filtering
keep<-filterByExpr(myDGEList, design)
F_myDGEList<- myDGEList[keep, , keep.lib.sizes=TRUE]

#NORMALIZATION (normLibSizes) IS NOT NECESSARY

estimate dispersion, set contrasts, fit the data

disp <- estimateDisp(F_myDGEList, design, robust=TRUE) 

### SHOULD I add here the offset??? or it is automatically "into" the DGElist? ### 

my.contrasts <- makeContrasts(SM_F = groupNormal_F - groupSM_F, 
                                 WS_F = groupNormal_F - groupWS_F,
                                 WB_F = groupNormal_F - groupWB_F,
                                 SM_M = groupNormal_M - groupSM_M, 
                                 WS_M = groupNormal_M - groupWS_M,
                                 WB_M = groupNormal_M - groupWB_M,
                                 levels=design)

fit_qlf<- glmQLFit(disp, design, robust=TRUE, legacy=FALSE) 

### SHOULD I add here the offset??? or it is automatically "into" the DGElist? ###

QLFtest (example for a single comparison)

QLFtest.SM_F<- glmQLFTest(fit_qlf, contrast=my.contrasts [, "SM_F"])
toplist.QLFtest.SM_F<-topTags(QLFtest.SM_F, n = Inf, sort.by = "p.value",adjust.method = "BH")
is.de.QLFtest.SM_F<- decideTestsDGE(QLFtest.SM_F)
summary (is.de.QLFtest.SM_F)

(sessionInfo)
R version 4.3.2 (2023-10-31 ucrt) -- "Eye Holes"
Platform: x86_64-w64-mingw32/x64 (64-bit) 
EdgeR v 4.0.1

Thank you to everyone who takes the time to respond to my question.

Best

Marianna

The instructions are in the tximport vignette. The offset is added to the DGEList. You don't add it anywhere else, it is automatically propagated.