WGCNA

Question

[hellokwmin]

The code for WGCNA analysis is like this:

vsd = varianceStabilizingTransformation(dds.filter)

wpn_vsd = getVarianceStabilizedData(dds.filter)
rv_wpn = rowVars(wpn_vsd)
summary(rv_wpn)

q75_wpn <- quantile( rowVars(wpn_vsd), .75)  # <= original
q95_wpn <- quantile( rowVars(wpn_vsd), .95)
expr_normalized <- wpn_vsd[ rv_wpn > q95_wpn, ]
expr_normalized[1:5,1:10]
dim(expr_normalized)

expr_normalized_df <- data.frame(expr_normalized) %>%
  mutate(
    Gene_id = row.names(expr_normalized)
  ) %>%
  pivot_longer(-Gene_id)

expr_normalized_df %>% ggplot(., aes(x = name, y = value)) +
  geom_violin() +
  geom_point() +
  theme_bw() +
  theme(
    axis.text.x = element_text( angle = 90)
  ) +
  ylim(0, NA) +
  labs(
    title = "Normalized and 95 quantile Expression",
    x = "treatment",
    y = "normalized expression"
  )



### WGCNA
input_mat = t(expr_normalized)

allowWGCNAThreads() 

powers = c(c(1:10), seq(from = 12, to = 50, by = 2))

# Call the network topology analysis function
sft = pickSoftThreshold(
  input_mat,             # <= Input data
  #blockSize = 30,
  powerVector = powers,
  verbose = 5
)

par(mfrow = c(1,2));
cex1 = 0.9;

plot(sft$fitIndices[, 1],
     -sign(sft$fitIndices[, 3]) * sft$fitIndices[, 2],
     xlab = "Soft Threshold (power)",
     ylab = "Scale Free Topology Model Fit, signed R^2",
     main = paste("Scale independence")
)
text(sft$fitIndices[, 1],
     -sign(sft$fitIndices[, 3]) * sft$fitIndices[, 2],
     labels = powers, cex = cex1, col = "red"
)
abline(h = 0.90, col = "red")
plot(sft$fitIndices[, 1],
     sft$fitIndices[, 5],
     xlab = "Soft Threshold (power)",
     ylab = "Mean Connectivity",
     type = "n",
     main = paste("Mean connectivity")
)
text(sft$fitIndices[, 1],
     sft$fitIndices[, 5],
     labels = powers,
     cex = cex1, col = "red")


picked_power = 30
temp_cor <- cor       
cor <- WGCNA::cor         # Force it to use WGCNA cor function (fix a namespace conflict issue)

netwk <- blockwiseModules(input_mat,                # <= input here

                          # == Adjacency Function ==
                          power = picked_power,                # <= power here
                          networkType = "signed",

                          # == Tree and Block Options ==
                          deepSplit = 2,
                          pamRespectsDendro = F,
                          # detectCutHeight = 0.75,
                          minModuleSize = 30,
                          maxBlockSize = 11000,

                          # == Module Adjustments ==
                          reassignThreshold = 0,
                          mergeCutHeight = 0.25,

                          # == TOM == Archive the run results in TOM file (saves time)
                          saveTOMs = T,
                          saveTOMFileBase = "ER",

                          # == Output Options
                          numericLabels = T,
                          verbose = 3)

cor <- temp_cor     # Return cor function to original namespace


module_eigengenes = netwk$MEs

##### print out a preview

head(module_eigengenes)

## get number of genes for each module

table(netwk$colors)

# Convert labels to colors for plotting
mergedColors = labels2colors(netwk$colors)
# Plot the dendrogram and the module colors underneath
plotDendroAndColors(
  netwk$dendrograms[[1]],
  mergedColors[netwk$blockGenes[[1]]],
  "Module colors",
  dendroLabels = FALSE,
  hang = 0.03,
  addGuide = TRUE,
  guideHang = 0.05 )

netwk$colors[netwk$blockGenes[[1]]]
table(netwk$colors)

module_df <- data.frame(
  gene_id = names(netwk$colors),
  colors = labels2colors(netwk$colors)
)

module_df[1:5,]

write_delim(module_df,
            file = "gene_modules_antarctica_OTC.txt",
            delim = "\t")

# Get Module Eigengenes per cluster
MEs0 <- moduleEigengenes(input_mat, mergedColors)$eigengenes

# Reorder modules so similar modules are next to each other
MEs0 <- orderMEs(MEs0)
module_order = names(MEs0) %>% gsub("ME","", .)

# Add treatment names
MEs0$treatment = row.names(MEs0)

# tidy & plot data
mME = MEs0 %>%
  pivot_longer(-treatment) %>%
  mutate(
    name = gsub("ME", "", name),
    name = factor(name, levels = module_order)
  )

mME_plot <- mME %>%
  ggplot(aes(x = treatment, y = name, fill = value, label = sprintf("%.2f", value))) +
  geom_tile() +
  geom_text(size = 3, color = "black", show.legend = FALSE) +  
  theme_bw() +
  scale_fill_gradient2(
    low = "blue",
    high = "red",
    mid = "white",
    midpoint = 0,
    limit = c(-1, 1)
  ) +
  theme(axis.text.x = element_text(angle = 90)) +
  labs(
    title = "Module-trait Relationships",
    x = "Treatments",
    y = "Modules",
    fill = "Correlation"
  )

After running last code, the result is like this:

And, my question is can I put any p-value as numeric or aesterick? Many tutorial for WGCNA calculated p-values based on trait data which they are interested. But, I am dealing with RNA-seq data wherein only treatment names and corresponding Gene and Gene expression data. I cannot calculate any p-values using my data?

[jv]

The treatment is a sample trait. But do you really not have any additional metadata about your samples?