WGCNA - 實戰
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WGCNA - 實戰

WGCNA - 實戰

0. 數據結構

0.1 Expression matrix

Trinity script TPM result

ID1 ID2 Liver_CK Intest_CK Muscle_CK Liver_30 Intest_30 Muscle_30 Liver_75 Intest_75 Muscle_75
TRINITY_DN100000_c1_g1 TRINITY_DN100000_c1_g1_i1 2.1 4.3 1.32 1.04 4.49 2.85 3.59 4.27 1.7
TRINITY_DN100001_c0_g1 TRINITY_DN100001_c0_g1_i1 0.45 21.0 0.02 0.15 8.85 4.33 0.18 14.76 0.0
TRINITY_DN100002_c0_g1 TRINITY_DN100002_c0_g1_i1 8.39 2.01 1.08 11.32 3.2 1.1 7.83 6.23 2.91

0.2 Traits

ID Group Organ Weight
Liver_CK CK Liver 33.32
Intest_CK CK Intest 33.32
Muscle_CK CK Muscle 33.32
Liver_30 SPC30 Liver 31.3
Intest_30 SPC30 Intest 31.3
… … … …

1. Data Clean

1.1 Load and Filter

library(WGCNA)

A <- read.csv("All_isoform.TPM.matrix.anno.xls",sep='\t')

## rownames:sample; colnames: Transcripts
row.names(A) = A[[2]]
A = A[-(1:2)]
datExpr0 = t(A)


## Data filter
gsg = goodSamplesGenes(datExpr0, verbose = 3);
gsg$allOK

if (!gsg$allOK)
{
  # Optionally, print the gene and sample names that were removed:
  if (sum(!gsg$goodGenes)>0)
     printFlush(paste("Removing genes:", paste(names(datExpr0)[!gsg$goodGenes], collapse = ", ")));
  if (sum(!gsg$goodSamples)>0)
     printFlush(paste("Removing samples:", paste(rownames(datExpr0)[!gsg$goodSamples], collapse = ", ")));
  # Remove the offending genes and samples from the data:
  datExpr0 = datExpr0[gsg$goodSamples, gsg$goodGenes]
}

1.2 Clust

sampleTree = hclust(dist(datExpr0), method = "average");
##png(file = "sampleClustering_T45.png",wi=300, he=300)
par(cex = 0.6);
par(mar = c(0,4,2,0))
plot(sampleTree, main = "Sample clustering to detect outliers", sub="", xlab="", cex.lab = 1.5,
     cex.axis = 1.5, cex.main = 2)
##dev.off()

sampleClustering
相對來說， Liver30 異質性有點大， 不過， 根據實驗來說， 應該是正常現象，因此跳過刪除異質性項。

datExpr =  datExpr0
nGenes = ncol(datExpr)
nSamples = nrow(datExpr)

1.3 Loading Traits

traitData = read.csv("traits.csv")

allTraits = traitData[-c(2,3)]


Samples = rownames(datExpr);
traitRows = match(Samples, allTraits$ID);
datTraits = allTraits[traitRows, -1];
rownames(datTraits) = allTraits[traitRows, 1];

collectGarbage()

## Re-cluster samples
sampleTree2 = hclust(dist(datExpr), method = "average")
## Convert traits to a color representation: white means low, red means high, grey means missing entry
traitColors = numbers2colors(datTraits, signed = FALSE);
## Plot the sample dendrogram and the colors underneath.
## png("traits_heatmap.png")
plotDendroAndColors(sampleTree2, traitColors,
                    groupLabels = names(datTraits),
                    main = "Sample dendrogram and trait heatmap")

traits_heatmap

ummm, 並不能看出什麼來。只是個普通的熱圖加樹衛而已

2. Network construction and module detection

2.1 Pick soft-thresdholding power

powers = c(c(1:10), seq(from = 12, to=20, by=2))
## Call the network topology analysis function
sft = pickSoftThreshold(datExpr, powerVector = powers, verbose = 5)
## Plot the results:
sizeGrWindow(9, 5)
##png("sorft_Thread.png",wi=630,he=350)
## Scale-free topology fit index as a function of the soft-thresholding power
## png("T45_Soft_th.png",width=500,height=300)
cex1 = 0.9;
par(mfrow = c(1,2));
plot(sft$fitIndices[,1], -sign(sft$fitIndices[,3])*sft$fitIndices[,2],
     xlab="Soft Threshold (power)",ylab="Scale Free Topology Model Fit,signed R^2",type="n",
     main = paste("Scale independence"));
text(sft$fitIndices[,1], -sign(sft$fitIndices[,3])*sft$fitIndices[,2],
     labels=powers,cex=cex1,col="red");
## this line corresponds to using an R^2 cut-off of h
abline(h=0.90,col="red")
## Mean connectivity as a function of the soft-thresholding power
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")
##dev.off()

sorft_Thread

ummm… 等了半天，就這。。。這種垃圾數據的話， 還是放棄把。

3. 刪除不必要reads，再來做一遍

想法： 刪除低hit的reads， 減少計算量，減少垃圾數據的污染

Counts <- read.csv("/media/ken/Data/Yan/RNA-seq/report/4.exprs//All_isoform.COUNT.matrix.anno.xls",sep='\t')
Counts$Sum = rowSums(Counts[-c(1:2)])
Counts_sub = Counts[which(Counts$Sum>10),]
paste(round((nrow(Counts_sub)/nrow(Counts))*100,2),"%",sep="")

A_sub <- A[match(Counts_sub[[2]],rownames(A)),]

然後， 重複上面步驟, 從1.1 datExpr0 = t(A)替換成datExpr0 = t(A_sub)開始Soft_th2
鐺鐺鐺鐺鐺! 這次結果就好看多了把！雖然沒有 Tutorial 那樣標緻，但是，至少給了我繼續下去的勇氣。數據不好，就調高閾值把， 更具這條線，大概是要選9了

4. One-step NetWork

net = blockwiseModules(datExpr, power = 9,
                       TOMType = "unsigned", minModuleSize = 30,
                       reassignThreshold = 0, mergeCutHeight = 0.25,
                       numericLabels = TRUE, pamRespectsDendro = FALSE,
                       saveTOMs = TRUE,
                       saveTOMFileBase = "femaleMouseTOM",
                       verbose = 3)

這一步等待時間和你電腦計算能力，基因數量成正相關


moduleLabels = net$colors
moduleColors = labels2colors(net$colors)
MEs = net$MEs;
geneTree = net$dendrograms[[1]]
## open a graphics window
sizeGrWindow(12, 9)
## png('modules_tree.png',width=480, height=320)
## Convert labels to colors for plotting
mergedColors = labels2colors(net$colors)
## Plot the dendrogram and the module colors underneath
plotDendroAndColors(net$dendrograms[[1]], mergedColors[net$blockGenes[[1]]],
                    "Module colors",
                    dendroLabels = FALSE, hang = 0.03,
                    addGuide = TRUE, guideHang = 0.05)
##dev.off()

modules_tree

ummmm… 模塊似乎有點寬， 分的不均勻- - 感覺很爛的樣子。 要不再， 篩一下？

Modules 統計

library(ggplot2)
library(reshape2)
Color_TB = melt(table(labels2colors(net$colors)))
Color_TB$Var1 = factor(Color_TB$Var1, levels = Color_TB$Var1[order(Color_TB$value,decreasing = T)])
ggplot(Color_TB) + geom_bar(aes(x=Var1,y=value), stat = 'identity',fill=Color_TB$Var1)+
  theme_light() +theme(axis.text.x = element_text(angle = 90,hjust=1))

123

5. Traits

nGenes = ncol(datExpr);
nSamples = nrow(datExpr);
## Recalculate MEs with color labels
MEs0 = moduleEigengenes(datExpr, moduleColors)$eigengenes
MEs = orderMEs(MEs0)
moduleTraitCor = cor(MEs, datTraits, use = "p");
moduleTraitPvalue = corPvalueStudent(moduleTraitCor, nSamples);


sizeGrWindow(10,6)
## Will display correlations and their p-values
## png("T45_traits_heatmap.png",width=1000,height=600)
textMatrix =  paste(signif(moduleTraitCor, 2), "\n(",
                           signif(moduleTraitPvalue, 1), ")", sep = "");
dim(textMatrix) = dim(moduleTraitCor)
par(mar = c(6, 8.5, 3, 3));
## Display the correlation values within a heatmap plot
labeledHeatmap(Matrix = moduleTraitCor,
               xLabels = names(datTraits),
               yLabels = names(MEs),
               ySymbols = names(MEs),
               colorLabels = FALSE,
               colors = greenWhiteRed(50),
               textMatrix = textMatrix,
               setStdMargins = FALSE,
               cex.text = 0.5,
               zlim = c(-1,1),
               main = paste("Module-trait relationships"))

traits_heatmap
ummm, 能看個大概就好2333.還是有點意思的

6. Focus on Weight

weight = as.data.frame(datTraits$Weight)
modNames = substring(names(MEs), 3)

geneModuleMembership = as.data.frame(cor(datExpr, MEs, use = "p"));
MMPvalue = as.data.frame(corPvalueStudent(as.matrix(geneModuleMembership), nSamples));

names(geneModuleMembership) = paste("MM", modNames, sep="");
names(MMPvalue) = paste("p.MM", modNames, sep="");

geneTraitSignificance = as.data.frame(cor(datExpr, weight, use = "p"));
GSPvalue = as.data.frame(corPvalueStudent(as.matrix(geneTraitSignificance), nSamples));

names(geneTraitSignificance) = paste("GS.", names(weight), sep="");
names(GSPvalue) = paste("p.GS.", names(weight), sep="");


module = "grey"
column = match(module, modNames);
moduleGenes = moduleColors==module;

sizeGrWindow(7, 7);
## png("grey_weight.png")
par(mfrow = c(1,1));
verboseScatterplot(abs(geneModuleMembership[moduleGenes, column]),
                   abs(geneTraitSignificance[moduleGenes, 1]),
                   xlab = paste("Module Membership in", module, "module"),
                   ylab = "Gene significance for body weight",
                   main = paste("Module membership vs. gene significance\n"),
                   cex.main = 1.2, cex.lab = 1.2, cex.axis = 1.2, col = module)

grey_weight
ummmm… 有點少， 哈哈哈， 才18個，數了一下。和tutorial 差好多呀

因爲是非模式物種，所以無法直接用tutorial給的方法做註釋。直接調到後面的可視化把。

7. NetWork Visualizing

nGenes = ncol(datExpr)
nSamples = nrow(datExpr)

## Calculate topological overlap anew: this could be done more efficiently by saving the TOM
## calculated during module detection, but let us do it again here.
dissTOM = 1-TOMsimilarityFromExpr(datExpr, power = 9);
## Transform dissTOM with a power to make moderately strong connections more visible in the heatmap
plotTOM = dissTOM^7;
## Set diagonal to NA for a nicer plot
diag(plotTOM) = NA;
## Call the plot function
sizeGrWindow(9,9)
TOMplot(plotTOM, geneTree, moduleColors, main = "Network heatmap plot, all genes")

nSelect = 400
## For reproducibility, we set the random seed
set.seed(10);
select = sample(nGenes, size = nSelect);
selectTOM = dissTOM[select, select];
## There's no simple way of restricting a clustering tree to a subset of genes, so we must re-cluster.
selectTree = hclust(as.dist(selectTOM), method = "average")
selectColors = moduleColors[select];
## Open a graphical window
sizeGrWindow(9,9)
## Taking the dissimilarity to a power, say 10, makes the plot more informative by effectively changing
## the color palette; setting the diagonal to NA also improves the clarity of the plot
##png('123.png')
plotDiss = selectTOM^7;
diag(plotDiss) = NA;
TOMplot(plotDiss, selectTree, selectColors, main = "Network heatmap plot, selected genes")

ummm… 好像是內存爆了，直接退出。 試試不用radian看看失敗了- -放棄， 跳過把參考圖：
123

MEs = moduleEigengenes(datExpr, moduleColors)$eigengenes
## Isolate weight from the clinical traits
weight = as.data.frame(datTraits$Weight);
names(weight) = "weight"
## Add the weight to existing module eigengenes
MET = orderMEs(cbind(MEs, weight))
## Plot the relationships among the eigengenes and the trait
sizeGrWindow(5,7.5);
##png('T45_Tree_weight_module.png',width=400,height=600)
par(cex = 0.9)
plotEigengeneNetworks(MET, "", marDendro = c(0,4,1,2), marHeatmap = c(3,4,1,2), cex.lab = 0.8, xLabelsAngle = 90)

tTvZu9.png

這個圖還是可以畫的

8. 數據導出

modules = c("darkorange2", "grey","lightcyan");
inModule = is.finite(match(moduleColors, modules));

for(color in modules){
  write.table(colnames(t(net$colors[moduleColors==color])),paste("module_",color,'.list',sep="") ,row.names=F,quote=F,col.names=F)
}

probes = names(datExpr)

## Recalculate topological overlap if needed
TOM = TOMsimilarityFromExpr(datExpr, power = 9);
## Read in the annotation file
annot = read.csv(file = "GeneAnnotation.csv");
## Select modules
## Select module probes
modProbes = probes[inModule];
modGenes = annot$gene_symbol[match(modProbes, annot$substanceBXH)];
## Select the corresponding Topological Overlap
modTOM = TOM[inModule, inModule];
dimnames(modTOM) = list(modProbes, modProbes)
## Export the network into edge and node list files Cytoscape can read
cyt = exportNetworkToCytoscape(modTOM,
  edgeFile = paste("CytoscapeInput-edges-", paste(modules, collapse="-"), ".txt", sep=""),
  nodeFile = paste("CytoscapeInput-nodes-", paste(modules, collapse="-"), ".txt", sep=""),
  weighted = TRUE,
  threshold = 0.02,
  nodeNames = modProbes,
  altNodeNames = modGenes,
  nodeAttr = moduleColors[inModule]);

9. 如果 softThread 還是取6

Modules 分佈

library(ggplot2)
library(reshape2)
library(patchwork)
Color_TB = melt(table(labels2colors(net$colors)))
Color_TB$Var1 = factor(Color_TB$Var1, levels = Color_TB$Var1[order(Color_TB$value,decreasing = T)])
p<- ggplot(Color_TB) + geom_bar(aes(x=Var1,y=value), stat = 'identity',fill=Color_TB$Var1)+
  theme_light() +theme(axis.text.x = element_text(angle = 90,hjust=1))

SplitBar(p,8500,10000,27000,0,c(1,4))

畫圖函點擊SplitBar
123

S6_modules_tree

Optimal

A1. Reduce Input Transcripts

這裏， 我們把閾值設定在 45(9*5)

library(WGCNA)

A <- read.csv("All_isoform.TPM.matrix.anno.xls",sep='\t')
## rownames:sample; colnames: Transcripts
row.names(A) = A[[2]]
A = A[-(1:2)]

Counts <- read.csv("/media/ken/Data/Yan/RNA-seq/report/4.exprs//All_isoform.COUNT.matrix.anno.xls",sep='\t')
Counts$Sum = rowSums(Counts[-c(1:2)])
Counts_sub = Counts[which(Counts$Sum>45),]
paste(round((nrow(Counts_sub)/nrow(Counts))*100,2),"%",sep="")

A_sub <- A[match(Counts_sub[[2]],rownames(A)),]

datExpr0 = t(A_sub)

## Data filter
gsg = goodSamplesGenes(datExpr0, verbose = 3);
gsg$allOK

if (!gsg$allOK)
{
  # Optionally, print the gene and sample names that were removed:
  if (sum(!gsg$goodGenes)>0)
     printFlush(paste("Removing genes:", paste(names(datExpr0)[!gsg$goodGenes], collapse = ", ")));
  if (sum(!gsg$goodSamples)>0)
     printFlush(paste("Removing samples:", paste(rownames(datExpr0)[!gsg$goodSamples], collapse = ", ")));
  # Remove the offending genes and samples from the data:
  datExpr0 = datExpr0[gsg$goodSamples, gsg$goodGenes]
}

剩下了"32.96%"的 Transcripts

接下來退回到1.2

T45_Soft_th
ummmm, 好像還沒之前的好看了- -，

還是取9把，soft

modules 29 個。
traits聚類：

Cluster1 Cluster2
T45_traits_heatmap tHgxTs.png

最亮的沒有上次那麼亮，= =相關係數有點低， 不高

modules 分佈情況:

tmp

B. Remove low-hits & Non-DEGs

library(WGCNA)

A <- read.csv("All_isoform.TPM.matrix.anno.xls",sep='\t')
## rownames:sample; colnames: Transcripts
row.names(A) = A[[2]]
A = A[-(1:2)]

Counts <- read.csv("/media/ken/Data/Yan/RNA-seq/report/4.exprs//All_isoform.COUNT.matrix.anno.xls",sep='\t')
Counts$Sum = rowSums(Counts[-c(1:2)])
Counts_sub = Counts[which(Counts$Sum>9),]
paste(round((nrow(Counts_sub)/nrow(Counts))*100,2),"%",sep="")

A_sub <- A[match(Counts_sub[[2]],rownames(A)),]

DEG1 <- read.table("../edgeR_Intest/diffExpr.P1e-3_C2.matrix",head=T)
DEG2 <- read.table("../edgeR_Liver/diffExpr.P1e-3_C2.matrix",head=T)
DEG3 <- read.table("../edgeR_Muscle/diffExpr.P1e-3_C2.matrix",head=T)

List = as.character(unique(DEG1[[1]], DEG2[[1]], DEG3[[1]]))

A_DEG = na.omit(A_sub[match(row.names(A_sub), List),])

datExpr0 = t(A_DEG)

## Data filter
gsg = goodSamplesGenes(datExpr0, verbose = 3);
gsg$allOK

if (!gsg$allOK)
{
  # Optionally, print the gene and sample names that were removed:
  if (sum(!gsg$goodGenes)>0)
     printFlush(paste("Removing genes:", paste(names(datExpr0)[!gsg$goodGenes], collapse = ", ")));
  if (sum(!gsg$goodSamples)>0)
     printFlush(paste("Removing samples:", paste(rownames(datExpr0)[!gsg$goodSamples], collapse = ", ")));
  # Remove the offending genes and samples from the data:
  datExpr0 = datExpr0[gsg$goodSamples, gsg$goodGenes]
}

跳回 1.2
ummmm, 這麼篩完以後， 剩下了1105個Transcripts 。。。

聚類 Soft圖
這次聚類結果，就很不一樣了 不過sortpower這個圖，基本還是和前文一樣沒怎麼變。取0.9以上的話， 又得取10了
tbo3UU.png tbo1ET.png

還是去9吧。 1000個，秒算完。先看看分組頻率 和樹圖這個圖，我就覺得，看着舒服多了- -唉

聚類 Soft圖
DEG_bar DEG_tree
第一次畫出Tomplot，激動 這個聚類，相關性都不太高呀
DEG_tomplot DEG_Tree_weight_module

DEG_traits_heatmap

相關性確實是太低了。 我們看一下這1000基因的表達趨勢把。

library(ggplotify)

A_DEG$Module = labels2colors(net$colors)
A_DEG$ID = row.names(A_DEG)

TB = melt(A_DEG,id.vars = c("ID","Module"))
TB$variable = factor(TB$variable,levels=sort(as.character(unique((TB$variable)))))

TB$ID = factor(TB$ID, levels = row.names(A_DEG)[order(A_DEG$Module)])

p2 <- ggplot( TB, aes( variable, ID)) + theme(panel.grid.major = element_blank())+
    theme(legend.key=element_blank(), axis.text.y = element_blank(),
          axis.title=element_blank(),axis.ticks.y=element_blank(),
          axis.text.x=element_text(angle=90,hjust=1))+
    geom_tile(aes(fill=log(1+value)))+
    scale_fill_gradient2(low="steelblue",mid="white",high="red",midpoint =1)

A_DEG$ID = factor(A_DEG$ID, levels = row.names(A_DEG)[order(A_DEG$Module)])
p1 <-ggplot(A_DEG[10:11], aes( x="Module", y=ID)) + theme(panel.grid.major = element_blank())+
    theme(axis.text.y = element_blank(), legend.position =  'left',
        axis.title=element_blank(),axis.ticks.y=element_blank(),
        axis.text.x=element_text(angle=90,hjust=1))+
    geom_tile(fill=A_DEG$Module)

p1+p2 +  plot_layout(design = 'ABBBBBBBBBB')

熱圖還是，可以的，就是配色有點麻煩- -
co_heatmap
只管來看， 聚類效果還是OK的把。不過correlation值太低了 = = 有什麼之後再去深究把。

添加樹打包兩個函數

PLOT2 <- function(dendr){
  ggplot() +
  geom_segment(data=segment(dendr), aes(x=x, y=y, xend=xend, yend=yend)) +
  geom_text(data=label(dendr), aes(x, y, label=label, hjust=0),
           size=3) +
  coord_flip() + scale_y_reverse(expand=c(0.2, 0)) +
  theme(axis.line.y=element_blank(),
        axis.ticks.y=element_blank(),
        axis.text.y=element_blank(),
        axis.title.y=element_blank(),
        panel.background=element_rect(fill="white"),
        panel.grid=element_blank())
}

LS_judg<- function(TB){
  if (TB['x'] == TB['xend']){
    R = 'v'
  }else{
    R = 'h'
  }
  return(R)
}

TreePlot <- function(dendr, Width_tree){
  dendr$labels$x = Width_tree[dendr$labels$x]
  dendr$segments = dendr$segments[order(dendr$segments$x),]
  # 做個list
  List = c()
  for(i in c(1:length(Width_tree))){
    List = c(List, which(dendr$segments$x==i))
  }
  # 調整豎線
  for( i in c(1:length(dendr$labels$label))){
  Dif_tmp = Width_tree[i]- dendr$segments$x[List[i]]
  dendr$segments$x[List[i]:nrow(dendr$segments)] = dendr$segments$x[List[i]:nrow(dendr$segments)]+Dif_tmp
  dendr$segments$xend[List[i]:nrow(dendr$segments)] = dendr$segments$xend[List[i]:nrow(dendr$segments)]+Dif_tmp
  }
  # 刪除添加新的橫線
  dendr$segments = dendr$segments[dendr$segments$x == dendr$segments$xend,]
  Y_list = dendr$segments$y[duplicated(dendr$segments$y)]
  for(i in Y_list){  
  tmp = (dendr$segments$x[dendr$segments$y  == i])
  dendr$segments = rbind(dendr$segments, data.frame(x=tmp[1],y=i,xend=tmp[2],yend=i))
  }
  Seg = dendr$segments
  for( i in c(nrow(dendr$segments):1)){
    if(LS_judg(dendr$segments[i,]) == 'h'){
      tmpy = dendr$segments[i,]$y
      tmpTB = Seg[Seg$yend== tmpy,]
        for( ii in c(1:nrow(tmpTB))){
        if(LS_judg(tmpTB[ii,]) == 'v'){
          Num = rownames(tmpTB[1,])
          dendr$segments[match(Num, row.names(dendr$segments)),][c(1,3)] = sum(Seg[i,c('x','xend')])/2
        }
      }
    }
  }
  # 重画横线：
  dendr$segments = dendr$segments[dendr$segments$x == dendr$segments$xend,]
  Y_list = dendr$segments$y[duplicated(dendr$segments$y)]
  for(i in Y_list){  
  tmp = (dendr$segments$x[dendr$segments$y  == i])
  dendr$segments = rbind(dendr$segments, data.frame(x=tmp[1],y=i,xend=tmp[2],yend=i))
  }
 return(dendr)
}

画图

library(ggplot2)
library(ggdendro)
library(reshape2)
library(patchwork)
library(RColorBrewer)

colorRampPalette(rev(brewer.pal(n = 7,name = "RdYlBu"))) -> cc

TB_color = (data.frame(net$colors,color=labels2colors(net$colors)))
A_DEG$ID = row.names(A_DEG)
A_DEG$Module = TB_color$color[match(row.names(A_DEG), row.names(TB_color))]

## Samples dendrogram
hc <- hclust(dist(t(A_DEG[1:9])))
dendr_head <- dendro_data(hc, type="rectangle") # convert for ggplot


## Module dendrogram
hc <- hclust(dist(t(MEs)))
dendr <- dendro_data(hc, type="rectangle") # convert for ggplot

### 1. order and list
Module_list = as.character(dendr$labels$label[order(dendr$labels$x)])
Module_or = c()
for( i in strsplit(Module_list,'ME')){
  Module_or = c(Module_or, i[2])
}

### 2. Axis location
TB_mbar = data.frame(table(A_DEG$Module))
TB_mbar = TB_mbar = data.frame(table(A_DEG$Module))
TB_mbar$hei = TB_mbar$Freq[1]/2

for(i in c(2:nrow(TB_mbar))){
  TB_mbar$hei[i] =sum(TB_mbar$Freq[1:i-1])+ (TB_mbar$Freq[i]/2)
}

Width_tree = TB_mbar$hei

AA <- TreePlot(dendr, Width_tree)


## Module Bar

### acquiring module list

## order module
A_DEG$Module = factor(A_DEG$Module, levels = Module_or)
## order ID
A_DEG$ID = factor(A_DEG$ID, levels = row.names(A_DEG)[order(A_DEG$Module)])

## Transcripts Heatmap

primary_data = as.matrix(A_DEG[1:9])
##transformations
data = log2(primary_data+1)
data = as.matrix(data) # convert to matrix
## Centering rows
data = data.frame(t(scale(t(data), scale=F)))

TB = melt(cbind(data,A_DEG[c("ID","Module")]),id.vars = c("ID","Module"))
TB$ID = factor(TB$ID, levels = row.names(A_DEG)[order(A_DEG$Module)])
TB$variable = factor(TB$variable, levels =levels(dendr_head$labels$label))


p0 <- ggplot() +
      geom_segment(data=segment(AA), aes(x=x, y=y, xend=xend, yend=yend)) +
      geom_text(data=label(AA), aes(x, y, label=label, hjust=0),
               size=3) +
      coord_flip() + scale_y_reverse(expand=c(0.7, 0)) +
      theme(axis.line.y=element_blank(),
            axis.ticks.y=element_blank(),
            axis.text.y=element_blank(),
            axis.title =element_blank(),
            panel.background=element_rect(fill="white"),
            panel.grid=element_blank())

p1 <-ggplot(A_DEG[10:11], aes( x="Module", y=ID)) +
    theme(panel.grid.major = element_blank())+
    theme(axis.text.y = element_blank(), legend.position =  'left',
        axis.title=element_blank(),
        axis.ticks=element_blank(),
        panel.background=element_rect(fill="white"),
        axis.text.x=element_text(angle=90,hjust=1))+
    geom_tile(fill=A_DEG$Module)

p2 <- ggplot( TB, aes( variable, ID)) +
    theme(panel.grid.major = element_blank())+
    theme(legend.key=element_blank(), axis.text.y = element_blank(),
    axis.title=element_blank(),axis.ticks.y=element_blank(),
    axis.text.x=element_text(angle=90,hjust=1))+
    geom_tile(aes(fill=value))+
    scale_fill_gradientn(colors=cc(100))

p4 <- ggplot() +
          geom_segment(data=segment(dendr_head), aes(x=x, y=y, xend=xend, yend=yend)) +
          scale_y_reverse(expand=c(0, 0)) +
          theme(axis.line.y=element_blank(),
                axis.ticks.x=element_blank(),
                axis.text.x=element_blank(),
                axis.title=element_blank(),
                panel.background=element_rect(fill="white"),
                panel.grid=element_blank())

layout_P <- "
    AAAABCCCCCC
    AAAABCCCCCC
    AAAABCCCCCC
    AAAABCCCCCC
    AAAABCCCCCC
    AAAABCCCCCC
    #####DDDDDD
    "

p0 + p1 + p2 + p4 +  plot_layout(design = layout_P)

DEG_heatmap

WGCNA - 實戰

https://karobben.github.io/2020/06/10/R/WGCNA/

Author

Karobben

Posted on

2020-06-10

Updated on

2024-01-11

Licensed under

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