Dear all,

I'm trying to run limma voom to find DE genes between females and males, but my results don't seem to make sense. I'm sure it's just some lame mistake. How should I make the contrast design to only compare males and females? There should definitely be DE genes. Here's my code:

> library(limma)
> library(edgeR)
>
> counts <- read.csv("...", header=FALSE, sep=",")
>
> genes <- read.csv("...", header=TRUE)
> genes <- data.frame(genes)
>
> pheno <- read.csv("...", header=TRUE, sep=",")
> samplenames <- pheno$sampleid
>
> pheno
                           sampleid    sex
1   GTEX-1192X-1026-SM-5H12P_RNA_XY   male
2   GTEX-11DXY-0526-SM-5EGGQ_RNA_XY   male
...
135  GTEX-ZYY3-0626-SM-5NQ6W_RNA_XX female
136  GTEX-ZZPU-0426-SM-5GZYH_RNA_XX female
>
> sex <- factor(pheno$sex, levels=c("female", "male"))
>
> design <- model.matrix(~sex-1)
> colnames(design) <- levels(sex)
> design
    female male
1        0    1
2        0    1
3        0    1
...
134      0    1
135      1    0
136      1    0
attr(,"assign")
[1] 1 1
attr(,"contrasts")
attr(,"contrasts")$sex
[1] "contr.treatment"

> dge <- DGEList(counts=counts, genes=genes)
> colnames(dge) <- samplenames
> dge$samples$sex <- sex
> dge$samples$samplename <- samplenames
> keep <- rowSums(cpm(dge)>2) >= 4
> dge <- dge[keep,]
> v <- voom(dge, design, normalize="quantile")
> fit <- lmFit(v, design)
> contrast_design <- makeContrasts(female-male, levels=design)
> contrast_design
        Contrasts
Levels   female - male
  female             1
  male              -1
>
> vfit <- contrasts.fit(fit, contrasts=contrast_design)
> efit <- eBayes(vfit)
> topTable(efit)
                    gene logFC AveExpr    t P.Value adj.P.Val     B
47425 ENSG00000141556.20 -0.19     5.8 -3.7 0.00028      0.93 -0.61
12256 ENSG00000123933.15 -0.31     5.3 -3.6 0.00042      0.93 -0.82
34809 ENSG00000175215.10 -0.30     6.7 -3.6 0.00037      0.93 -0.97
34421  ENSG00000123268.8  0.20     3.7  3.6 0.00047      0.93 -1.11
4440  ENSG00000143493.12  0.28     4.3  3.5 0.00073      1.00 -1.23
1815  ENSG00000198160.14  0.21     5.0  3.4 0.00085      1.00 -1.27
37434  ENSG00000139826.5  0.21     3.9  3.5 0.00067      1.00 -1.27
27189 ENSG00000157654.17  0.43     4.0  3.3 0.00132      1.00 -1.65
2441  ENSG00000162775.14  0.27     3.9  3.3 0.00134      1.00 -1.69
50848 ENSG00000126464.13 -0.26     3.7 -3.3 0.00127      1.00 -1.76

> summary(decideTests(efit))
   female - male
-1             0
0          15868
1              1

R version 3.4.1 (2017-06-30)
Platform: x86_64-w64-mingw32/x64 (64-bit)
Running under: Windows >= 8 x64 (build 9200)

Matrix products: default

locale:
[1] LC_COLLATE=English_United States.1252  LC_CTYPE=English_United States.1252    LC_MONETARY=English_United States.1252
[4] LC_NUMERIC=C                           LC_TIME=English_United States.1252    

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
[1] gplots_3.0.1   pheatmap_1.0.8 edgeR_3.18.1   limma_3.32.5  

loaded via a namespace (and not attached):
 [1] locfit_1.5-9.1     Rcpp_0.12.12       lattice_0.20-35    gtools_3.5.0       bitops_1.0-6       grid_3.4.1         plyr_1.8.4        
 [8] gtable_0.2.0       scales_0.4.1       KernSmooth_2.23-15 gdata_2.18.0       RColorBrewer_1.1-2 tools_3.4.1        munsell_0.4.3     
[15] compiler_3.4.1     colorspace_1.3-2   caTools_1.17.1