Hi everyone,
I am new to RNA-seq and have I recently performed RNA-seq (Illumina NovaSeq 6000; 25M reads; PE, 100bp; Mus_musculus:mm10). I am currently performing a DE on my samples using EdgeR and noticed that my read counts do not change after normalization. Based on my understanding of how EdgeR normalization works, the read counts should change. Should I use a different normalization approach? Can someone help determine what I am doing incorrectly? Below I've included the R commands I've used to perform the analysis and the output.
COMMAND LINES IN R:
head(fc_res)
row.names(fc_res) <- fc_res$Gene
fc_res <- fc_res[-c(1,20)]
group <- factor(c(1,1,1,2,2,2,3,3,3,4,4,4,5,5,5,6,6,6))
y <- DGEList(counts=fc_res, group=group)
y$samples
keep <- filterByExpr(y, design)
filt1 <- y[keep,,keep.lib.sizes=FALSE]
dim(filt1)
ynorm <- calcNormFactors(filt1)
ynorm$samples
plotMDS(ynorm)
OUTPUT:
group lib.size norm.factors
S1_REP1 1 21780263 0.9964533
S1_REP2 1 19985191 0.9930817
S1_REP3 1 22149855 0.9885835
S2_REP1 2 23139231 0.9778646
S2_REP2 2 23631094 0.9808015
S3_REP3 2 25284278 0.9769698
S3_REP1 3 20292937 0.9350821
S3_REP2 3 25694663 0.9335373
S3_REP3 3 20825373 0.9310621
S4_REP1 4 23995722 0.9514329
S4_REP2 4 23429701 0.9364913
S4_REP3 4 22528422 0.9643743
S5_REP1 5 27466471 1.1070687
S5_REP2 5 29807414 1.0291615
S5_REP3 5 20450417 1.0667385
S6_REP1 6 24783768 1.0259821
S6_REP2 6 21733797 1.1276330
S6_REP3 6 18539629 1.1109447
Thank you, David