Results Changed

[ShashTrip1]

I ran the same code (listed below) on two different occasions and got two very different results with certain idents switching from being up in group A to being up in Group B.

alldata_milo = Milo(as.SingleCellExperiment(alldata)) alldata_milo <- buildGraph(alldata_milo, k = 20, d = 30) alldata_milo <- makeNhoods(alldata_milo, prop = 0.05, k = 20, d=30, refined = TRUE) alldata_milo <- countCells(alldata_milo, meta.data = data.frame(colData(alldata_milo)), samples="orig.ident") alldata_design <- data.frame(colData(alldata_milo))[,c("orig.ident", "Type")] alldata_design <- distinct(alldata_design) rownames(alldata_design) <- alldata_design$orig.ident alldata_design <- alldata_design[colnames(nhoodCounts(alldata_milo)), , drop=FALSE] alldata_design alldata_milo <- calcNhoodDistance(alldata_milo, d=30) rownames(alldata_design) <- alldata_design$orig.ident da_results <- testNhoods(alldata_milo, design = ~ Type, design.df = alldata_design) da_results <- annotateNhoods(alldata_milo, da_results, coldata_col = "ident")

da_results$ident <- ifelse(da_results$ident_fraction < 0.7, "Mixed", da_results$ident)

plotDAbeeswarm(da_results, group.by = "ident")

[MikeDMorgan]

Did you use set.seed()?

[ShashTrip1]

No, I did not. But I would be surprised if that changed the results that dramatically where only certain idents change which group is upregulated.

For example two of my annotated groups (radial glial cells and angio-macros) switched from logFC of -5 to 5 while the other groups stayed similar (I understand some changes due to different random number).