How to Keep Statistics with Reordered Combined ggplot2 Graph - r

Question

I'm using ggplot2 to make violin plots of module scores from Seurat, and am wanting to add statistics to it. I made the following violin plot, but I want to switch the violins around from reading "0" and "1" from left to right, to "1" and "0". (Thanks to #StupidWolf for the example!)
library(Seurat)
library(SeuratObject)
library(ggplot2)
library(ggpubr)
library(reshape2)
#add Seurat's module scores and create Seurat object from them =====================
ERlist <- list(c("CPB1", "RP11-53O19.1", "TFF1", "MB", "ANKRD30B",
"LINC00173", "DSCAM-AS1", "IGHG1", "SERPINA5", "ESR1",
"ILRP2", "IGLC3", "CA12", "RP11-64B16.2", "SLC7A2",
"AFF3", "IGFBP4", "GSTM3", "ANKRD30A", "GSTT1", "GSTM1",
"AC026806.2", "C19ORF33", "STC2", "HSPB8", "RPL29P11",
"FBP1", "AGR3", "TCEAL1", "CYP4B1", "SYT1", "COX6C",
"MT1E", "SYTL2", "THSD4", "IFI6", "K1AA1467", "SLC39A6",
"ABCD3", "SERPINA3", "DEGS2", "ERLIN2", "HEBP1", "BCL2",
"TCEAL3", "PPT1", "SLC7A8", "RP11-96D1.10", "H4C8",
"PI15", "PLPP5", "PLAAT4", "GALNT6", "IL6ST", "MYC",
"BST2", "RP11-658F2.8", "MRPS30", "MAPT", "AMFR", "TCEAL4",
"MED13L", "ISG15", "NDUFC2", "TIMP3", "RP13-39P12.3", "PARD68"))
tnbclist <- list(c("FABP7", "TSPAN8", "CYP4Z1", "HOXA10", "CLDN1",
"TMSB15A", "C10ORF10", "TRPV6", "HOXA9", "ATP13A4",
"GLYATL2", "RP11-48O20.4", "DYRK3", "MUCL1", "ID4", "FGFR2",
"SHOX2", "Z83851.1", "CD82", "COL6A1", "KRT23", "GCHFR",
"PRICKLE1", "GCNT2", "KHDRBS3", "SIPA1L2", "LMO4", "TFAP2B",
"SLC43A3", "FURIN", "ELF5", "C1ORF116", "ADD3", "EFNA3",
"EFCAB4A", "LTF", "LRRC31", "ARL4C", "GPNMB", "VIM",
"SDR16C5", "RHOV", "PXDC1", "MALL", "YAP1", "A2ML1",
"RP1-257A7.5", "RP11-353N4.6", "ZBTB18", "CTD-2314B22.3", "GALNT3",
"BCL11A", "CXADR", "SSFA2", "ADM", "GUCY1A3", "GSTP1",
"ADCK3", "SLC25A37", "SFRP1", "PRNP", "DEGS1", "RP11-110G21.2",
"AL589743.1", "ATF3", "SIVA1", "TACSTD2", "HEBP2"))
genes = c(unlist(c(ERlist,tnbclist)))
mat = matrix(rnbinom(500*length(genes),mu=500,size=1),ncol=500)
rownames(mat) = genes
colnames(mat) = paste0("cell",1:500)
sobj = CreateSeuratObject(mat)
sobj = NormalizeData(sobj)
sobj$ClusterName = factor(sample(0:1,ncol(sobj),replace=TRUE))
sobj = AddModuleScore(object = sobj, features = tnbclist,
name = "TNBC_List",ctrl=5)
sobj = AddModuleScore(object = sobj, features = ERlist,
name = "ER_List",ctrl=5)
sobjlists = FetchData(object = sobj, vars = c("ER_List1", "TNBC_List1", "ClusterName"))
#violin plot =======================================================
my_comparisons <- list( c("0", "1") )
ggplot(sobjlists,aes(x= ClusterName, y = ER_List1)) +
geom_violin(aes(fill=ClusterName)) +
geom_boxplot(width=0.1) + labs(y= "ER+ Signature", x = "ClusterName") + ggtitle(label = "Object") +
theme(plot.title = element_text(hjust = 0.5)) + stat_compare_means(comparisons = my_comparisons, method = "wilcox.test")+ # Add pairwise comparisons p-value
stat_compare_means(label.y = 0.75)
Using the solution from How to reorder plots in combined ggplot2 graph?, I can reorder the plots by adding
+ scale_x_discrete(limits = c("1", "0"))
to the very end of the code I have now. However, doing that, the "Wilcoxon, p = 0.13" disappears, and gives the following error:
ggplot(sobjlists,aes(x= ClusterName, y = ER_List1)) +
geom_violin(aes(fill=ClusterName)) +
geom_boxplot(width=0.1) + labs(y= "ER+ Signature", x = "ClusterName") + ggtitle(label = "Object") +
theme(plot.title = element_text(hjust = 0.5)) + stat_compare_means(comparisons = my_comparisons, method = "wilcox.test")+ # Add pairwise comparisons p-value
stat_compare_means(label.y = 0.75) + scale_x_discrete(limits = c("1", "0"))
Warning messages:
1: Unknown or uninitialised column: `p`.
2: Computation failed in `stat_compare_means()`:
argument "x" is missing, with no default
For a TL;DR, how do I keep the top statistic in and reorder the violins?
Thanks for reading!

Answer 1

Here is what worked for me (From https://www.datanovia.com/en/blog/how-to-change-ggplot-legend-order/)
sobjlists$ClusterName <- factor(sobjlists$ClusterName, levels = c("1", "0"))