How to load Seurat Object into WGCNA Tutorial Format
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Entering edit mode
9 weeks ago

As far as I can find, there is only one tutorial about loading Seurat objects into WGCNA (https://ucdavis-bioinformatics-training.github.io/2019-single-cell-RNA-sequencing-Workshop-UCD_UCSF/scrnaseq_analysis/scRNA_Workshop-PART6.html). I am really new to programming so it's probably just my inexperience, but I am not sure how to load my Seurat object into a format that works with WGCNA's tutorials (https://horvath.genetics.ucla.edu/html/CoexpressionNetwork/Rpackages/WGCNA/Tutorials/).

Here is what I have tried thus far:

This tries to replicate datExpr and datTraits from part I.1:

library(WGCNA)
library(Seurat)

#example Seurat object -----------------------------------------------

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$ClusterName = factor(sample(0:1,ncol(sobj),replace=TRUE))
sobj$Patient = paste0("Patient", 1:500)

sobj = AddModuleScore(object = sobj, features = tnbclist, 
                      name = "TNBC_List",ctrl=5)
sobj = AddModuleScore(object = sobj, features = ERlist, 
                      name = "ER_List",ctrl=5) 

#WGCNA -----------------------------------------------------------------

sobjwgcna <- sobj

sobjwgcna <- FindVariableFeatures(sobjwgcna, selection.method = "vst", nfeatures = 2000,
                                  verbose = FALSE, assay = "RNA")
options(stringsAsFactors = F)
sobjwgcnamat <- GetAssayData(sobjwgcna)
datExpr <- t(sobjwgcnamat)[,VariableFeatures(sobjwgcna)]

datTraits <- sobjwgcna@meta.data
datTraits = subset(datTraits, select = -c(nCount_RNA, nFeature_RNA))

I then copy-paste the code as written in the WGCNA I.2a tutorial (https://horvath.genetics.ucla.edu/html/CoexpressionNetwork/Rpackages/WGCNA/Tutorials/FemaleLiver-02-networkConstr-auto.pdf), and that all works until I get to this line in the I.3 tutorial (https://horvath.genetics.ucla.edu/html/CoexpressionNetwork/Rpackages/WGCNA/Tutorials/FemaleLiver-03-relateModsToExt.pdf):

MEList = moduleEigengenes(datExpr, colors = moduleColors)
Error in t.default(expr[, restrict1]) : argument is not a matrix

I tried converting both moduleColors and datExpr into a matrix with as.matrix(), but the error still persists.

Hopefully this makes sense, and thanks for reading!

seurat wgcna • 184 views
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1
Entering edit mode
8 weeks ago

So doing as.matrix(datExpr) right after

datExpr <- t(sobjwgcnamat)[,VariableFeatures(sobjwgcna)]

worked! I had been trying it right before

MEList = moduleEigengenes(datExpr, colors = moduleColors)

and that didn't work. Seems simple but order matters I guess.

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