Hello, I am working on a differential expression analysis comparing samples with different tissue color. I have 10 samples from dark tissue and 6 samples from white tissue. I created my DESeq dataset as follows:
dds <- DESeqDataSetFromMatrix(countData = counts.in, colData = design.matrix, design = ~ seed_color + batch) row.names(dds) <- row.names(counts.in) keep <- rowSums(counts(dds) >= 10) >= 3 dds <- dds[keep,]
I tried to find differentially expressed genes using a lfc threshold and used apeglm for lfc shrinkage.
dds <- DESeq(dds) res <- results(dds, contrast = c("tissue_color","white","dark"), alpha = 0.01) res.shrunk <- lfcShrink(dds, res=res, coef = 2, lfcThreshold = 1, type = "apeglm") summary(res.shrunk, alpha=0.01)
I visualized the results in a volcano plot and found that the fsos rate is set to 0 for genes with negative lfcs. However, when I releved the tissue_color factor and used the other tissue color as reference level, I got a slightly different number of significant genes and again a number of genes with fsos rate 0 only for negative lfcs.
dds$tissue_color <- relevel(dds$tissue_color, "white") # before relevel: > summary(res.comp1, alpha=0.01) out of 19516 with nonzero total read count s-value < 0.01 LFC > 1.00 (up) : 2040, 10% LFC < -1.00 (down) : 1956, 10% # after relevel: > summary(res.comp2, alpha=0.01) out of 19516 with nonzero total read count s-value < 0.01 LFC > 1.00 (up) : 1960, 10% LFC < -1.00 (down) : 2039, 10%
The colored points show the genes more highly expressed in that tissue (before and after releveling), with fsos rate equal to 0 in the top left of the plot. I am wondering why I see only genes with negative lfc set to 0, even when changing the reference level of the analysis. I would have expected that only the lfc sign would change after releveling. Is this behaviour expected or is there something wrong with my analysis? Thank you kindly for any help.