蛋白质差异表达分析：无显著差异问题排查

我想用limma工具对一组蛋白质做差异表达分析，两组样本分别是C和D（样本名称来自colnames(mat)的子串），但分析结果显示无任何差异表达蛋白。我的分析目标是筛选出上调（up）或下调（down）的蛋白，但结果里1725个蛋白全是无显著差异（NotSig），没法得到可用的子集。

使用的R代码 #

library(limma)

group <- factor(as.factor(gsub("_.*", "", colnames(mat))), levels=c("C", "D"))
design <- model.matrix(~ 1 + group)
colnames(design) <- c("C", "D")
contrasts <- makeContrasts(C - D, levels=colnames(design))
mat.log <- log2(mat)
mat <- na.omit(mat)
dge <- edgeR::DGEList(counts=mat, group=group)
keep <- edgeR::filterByExpr(dge)
v <- voom(mat, design, plot=TRUE)
vfit <- contrasts.fit(lmFit(v, design), contrasts=contrasts)
efit <- eBayes(vfit, proportion=0.1)
summary(decideTests(efit))

分析结果 #

> summary(decideTests(efit))
       C - D
Down       0
NotSig  1725
Up         0

数据示例 #

> dput(mat[1:10,])
structure(c(1.0275, 1.05, 0.55875, 0.78375, 0.7175, 0.76875, 
0.70875, 0.63, 0.73125, 0.76875, 0.8675, 0.74, 0.54875, 0.71375, 
0.6775, 0.64875, 0.61875, 0.63, 0.63125, 0.91875, 0.6775, 0.9, 
0.64875, 0.70375, 0.6975, 0.69875, 0.64875, 0.76, 0.65125, 0.82875, 
1.3075, 1.03, 0.81875, 1.06375, 0.8375, 0.91875, 0.99875, 1.03, 
0.98125, 1.05875, 0.7225, 0.89, 0.59125, 0.70625, 0.6925, 0.64125, 
0.63125, 0.64, 0.62875, 0.89125, 0.9625, 0.8, 0.60125, 0.77625, 
0.7325, 0.72125, 0.76125, 0.8, 0.75875, 0.78125, 1.2925, 1.05, 
0.75125, 0.91625, 0.8425, 0.86125, 0.86125, 0.83, 0.87875, 1.02125, 
0.9025, 0.98, 0.63125, 0.86625, 0.6625, 0.81125, 0.72125, 0.78, 
0.72875, 0.88125), dim = c(10L, 8L), dimnames = list(c("AAAS", 
"AAK1", "AAMDC", "AARS", "AASDHPPT", "ABCD3", "ABCE1", "ABCF1", 
"ABHD10", "ABI1"), c("C_1", "C_2", "C_3", "C_4", "D_5", "D_6", 
"D_7", "D_8")))

内容的提问来源于stack exchange，提问作者Anon