I am trying to use the function findMarkers() from the package scran to find the marker genes of different cellt ypes.
My experiment / samples: I have 3 different cell types with 3 replicates each.
Data input: on the sequencing reads I have run DESeq2 and used varianceStabilizingTransformation() to get a matrix of normalized counts:
sampleinfo[,1:2] Sample Celltype AM1 AM1 Alveolarmacrophages AM2 AM2 Alveolarmacrophages AM3 AM3 Alveolarmacrophages IM1 IM1 Interstitialmacrophages IM2 IM2 Interstitialmacrophages IM3 IM3 Interstitialmacrophages T1 T1 Tcells T2 T2 Tcells T3 T3 T_cells
dds <- DESeqDataSetFromMatrix(countData = onlycounts, colData = sampleinfo, design= ~ Celltype) dds <- DESeq(dds) vsd <- varianceStabilizingTransformation(dds, blind=T) vsdmat <- assay(vsd)
head(vsd_mat) AM1 AM2 AM3 IM1 IM2 IM3 T1 T2 T3 let-7a-1-3p 10.3550933 9.8872116 10.5713000 8.4606248 8.8959434 8.641476 8.8700965 8.9457874 9.9124382 let-7a-2-3p -0.5877171 -0.5877171 -0.5877171 0.6988212 0.7829733 2.546719 -0.5877171 -0.5877171 -0.5877171 let-7a-5p 18.5178483 18.1865091 18.8431510 16.6119296 16.7121333 17.419055 18.6922011 18.8095962 18.8838528
Then I used the findMarkers() function
ncol(vsdmat)==length(sampleinfo$Celltype) #check if it´s T markergenes <- findMarkers(vsdmat, groups=sampleinfo$Cell_type, test.type="wilcox", pval.type="all")
No errors, but I was surprised that no transcripts had FDR < 0,05 for any of the cell types: I do not think this is right because in PCA plots and heatmaps the cell types are very nicely separated:
I have tried to use other systems for p-value:
markergenessome <- findMarkers(vsdmat, groups=sampleinfo$Celltype, test.type="wilcox", pval.type="some") markergenesany <- findMarkers(vsdmat, groups=sampleinfo$Celltype, test.type="wilcox", pval.type="any")
but this doesn´t improve the results either.
Do you know what could be going wrong? Or do you know any other package/functions to do this? I have googled but all similar functions I have found are for single-cell sequencing data. DESEq2 is also not useful because it doesn´t doesn´t do all the contrasts automatically, you need to type them one by one (I have now only 3 cell types, but later I will have more, so it´s not convenient).