网站创建域名,wordpress首页title的调用,苏州百度推广排名,网站中的链接是一个一个html做的第六节#xff0c;我们使用结核病基因数据#xff0c;做了一个数据预处理的实操案例。例子中结核类型#xff0c;包括结核#xff0c;潜隐进展#xff0c;对照和潜隐#xff0c;四个类别。第七节延续上个数据#xff0c;进行了差异分析。 第八节对差异基因进行富集分析。… 第六节我们使用结核病基因数据做了一个数据预处理的实操案例。例子中结核类型包括结核潜隐进展对照和潜隐四个类别。第七节延续上个数据进行了差异分析。 第八节对差异基因进行富集分析。本节进行WGCNA分析。 更新版本GEO生信数据挖掘九肺结核数据-差异分析-WGCNA分析900行代码整理注释更新版本
目录
加载数据进行聚类
初次聚类观察
自己定义红线位置进行切割划分
载入性状数据
增加形状信息后再次聚类
网络构建
选取soft-thresholding powers
基于tom的差异的基因聚类绘制聚类树
根据聚类情况设置颜色
计算eigengenes
模块的自动合并
模块与临床形状的关系热图 关键数据
红色模块样本表达情况相关性大
产生了很多数据各个模块的和临床性状
后续挖掘核心基因时需要用到Cytoscape生成绘图所需要的数据 加载数据进行聚类
library(WGCNA)
#读取目录名称方便复制粘贴
dir()
#加载数据
load(DEG_TB_LTBI_step13.Rdata)#这里行为样品名列为基因名
################################样品聚类####################
datExpr t(dataset_TB_LTBI_DEG)
#初次聚类
sampleTree hclust(dist(datExpr), method average)
# Plot the sample tree: Open a graphic output window of size 20 by 15 inches
# The user should change the dimensions if the window is too large or too small.
sizeGrWindow(12,9)
#pdf(filesampleCluestering.pdf,width 12,height 9)
par(cex0.6)
par(marc(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)#结果图片自己导出PDF文件名1_sampleClustering.pdf### Plot a line to show the cut
abline(h 87, col red)##剪切高度不确定故无红线dev.off()
初次聚类观察 自己定义红线位置进行切割划分
本例发现右侧有些样本孤立适合被剔除设置红线87切割。
左侧也被切成两块需要做处理保留。 ### Determine cluster under the line
clust cutreeStatic(sampleTree, cutHeight 87, minSize 10)
table(clust)
#clust
#0 1 2
#5 57 40### clust 1 contains the samples we want to keep.
keepSamples (clust1|clust2)
datExpr0 datExpr[keepSamples, ]
dim(datExpr0) #[1] 97 2813
#保存数据
save(datExpr0,filedatExpr0_cluster_filter.Rdata)
载入性状数据
匹配样本名称性状数据与表达数据保证一致 #################### 载入性状数据###########################
#加载自己的性状数据
load(design_TB_LTBI.Rdata)
traitDatadesign
#Loading clinical trait data
#traitData read.table(trait_D.txt,row.names1,headerT,comment.char ,check.namesF)########trait file name can be changed######性状数据文件名根据实际修改如果工作路径不是实际性状数据路径需要添加正确的数据路径
dim(traitData)
#names(traitData)
# remove columns that hold information we do not need.
#allTraits traitData
dim(traitData)
names(traitData)# Form a data frame analogous to expression data that will hold the clinical traits.
fpkmSamples rownames(datExpr0)
traitSamples rownames(traitData)
#匹配样本名称性状数据与表达数据保证一致
traitRows match(fpkmSamples, traitSamples)
datTraits traitData[traitRows,]
rownames(datTraits)
collectGarbage()
增加形状信息后再次聚类 # Re-cluster samples
sampleTree2 hclust(dist(datExpr0), 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.#sizeGrWindow(20,20)
##pdf(file2_Sample dendrogram and trait heatmap.pdf,width12,height12)
plotDendroAndColors(sampleTree2, traitColors,groupLabels names(datTraits),main Sample dendrogram and trait heatmap)dev.off()
下方红色大致分成了两类效果不错。 网络构建
#############################network constr######################################### Allow multi-threading within WGCNA. At present this call is necessary.
# Any error here may be ignored but you may want to update WGCNA if you see one.
# Caution: skip this line if you run RStudio or other third-party R environments.
# See note above.
enableWGCNAThreads()# Choose a set of soft-thresholding powers
powers c(1:15)# Call the network topology analysis function
sft pickSoftThreshold(datExpr0, powerVector powers, verbose 5)# Plot the results:
sizeGrWindow(15, 9)
#pdf(file3_Scale independence.pdf,width9,height5)
#pdf(fileRplot03.pdf,width9,height5)
par(mfrow c(1,2))
cex1 0.9
# Scale-free topology fit index as a function of the soft-thresholding power
plot(sft$fitIndices[,1], -sign(sft$fitIndices[,3])*sft$fitIndices[,2],xlabSoft Threshold (power),ylabScale Free Topology Model Fit,signed R^2,typen,main paste(Scale independence));
text(sft$fitIndices[,1], -sign(sft$fitIndices[,3])*sft$fitIndices[,2],labelspowers,cexcex1,colred);
# this line corresponds to using an R^2 cut-off of h
abline(h0.90,colred)
# Mean connectivity as a function of the soft-thresholding power
plot(sft$fitIndices[,1], sft$fitIndices[,5],xlabSoft Threshold (power),ylabMean Connectivity, typen,main paste(Mean connectivity))
text(sft$fitIndices[,1], sft$fitIndices[,5], labelspowers, cexcex1,colred)
dev.off()选取soft-thresholding powers
测试阈值注意观察突破红线的附近时取值下方代码时候的是自适应的方法选取 soft-thresholding powers ######chose the softPower
#datExpr0 datExpr0[,-1]
softPower sft$powerEstimate
adjacency adjacency(datExpr0, power softPower)##### Turn adjacency into topological overlap
TOM TOMsimilarity(adjacency);
dissTOM 1-TOM# Call the hierarchical clustering function
geneTree hclust(as.dist(dissTOM), method average);
# Plot the resulting clustering tree (dendrogram)#sizeGrWindow(12,9)
pdf(file4_Gene clustering on TOM-based dissimilarity.pdf,width12,height9)
plot(geneTree, xlab, sub, main Gene clustering on TOM-based dissimilarity,labels FALSE, hang 0.04)
dev.off()
基于tom的差异的基因聚类绘制聚类树 根据聚类情况设置颜色 # We like large modules, so we set the minimum module size relatively high:
minModuleSize 30
# Module identification using dynamic tree cut:
dynamicMods cutreeDynamic(dendro geneTree, distM dissTOM,deepSplit 2, pamRespectsDendro FALSE,minClusterSize minModuleSize);
table(dynamicMods)# Convert numeric lables into colors
dynamicColors labels2colors(dynamicMods)
table(dynamicColors)
# Plot the dendrogram and colors underneath
#sizeGrWindow(8,6)
pdf(file5_Dynamic Tree Cut.pdf,width8,height6)
plotDendroAndColors(geneTree, dynamicColors, Dynamic Tree Cut,dendroLabels FALSE, hang 0.03,addGuide TRUE, guideHang 0.05,main Gene dendrogram and module colors)
dev.off() 计算eigengenes # Calculate eigengenes
MEList moduleEigengenes(datExpr0, colors dynamicColors)
MEs MEList$eigengenes
# Calculate dissimilarity of module eigengenes
MEDiss 1-cor(MEs);
# Cluster module eigengenes
METree hclust(as.dist(MEDiss), method average)
# Plot the result
#sizeGrWindow(7, 6)
pdf(file6_Clustering of module eigengenes.pdf,width7,height6)
plot(METree, main Clustering of module eigengenes,xlab , sub )
MEDissThres 0.25######剪切高度可修改
# Plot the cut line into the dendrogram
abline(hMEDissThres, col red)
dev.off() 模块的自动合并 # Call an automatic merging function
merge mergeCloseModules(datExpr0, dynamicColors, cutHeight MEDissThres, verbose 3)
# The merged module colors
mergedColors merge$colors
# Eigengenes of the new merged modules:
mergedMEs merge$newMEs#sizeGrWindow(12, 9)
pdf(file7_merged dynamic.pdf, width 9, height 6)
plotDendroAndColors(geneTree, cbind(dynamicColors, mergedColors),c(Dynamic Tree Cut, Merged dynamic),dendroLabels FALSE, hang 0.03,addGuide TRUE, guideHang 0.05)
dev.off()# Rename to moduleColors
moduleColors mergedColors
# Construct numerical labels corresponding to the colors
colorOrder c(grey, standardColors(50))
moduleLabels match(moduleColors, colorOrder)-1
MEs mergedMEs# Save module colors and labels for use in subsequent parts
save(MEs, TOM, dissTOM, moduleLabels, moduleColors, geneTree, sft, file networkConstruction-stepByStep.RData)模块与临床形状的关系热图 关键数据
#############################relate modules to external clinical triats######################################
# Define numbers of genes and samples
nGenes ncol(datExpr0)
nSamples nrow(datExpr0)moduleTraitCor cor(MEs, datTraits, use p)
moduleTraitPvalue corPvalueStudent(moduleTraitCor, nSamples)#sizeGrWindow(10,6)
pdf(file8_Module-trait relationships.pdf,width10,height6)
# Will display correlations and their p-values
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 #修改性状类型 data.frame
labeledHeatmap(Matrix moduleTraitCor,xLabels names(data.frame(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))
dev.off()挑选相关性最高的具有统计学意义的p0.05,red模块最佳 红色模块样本表达情况相关性大 产生了很多数据各个模块的和临床性状
######## Define variable weight containing all column of datTraits###MM and GS# names (colors) of the modules
modNames substring(names(MEs), 3)geneModuleMembership as.data.frame(cor(datExpr0, 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)#names of those trait
traitNamesnames(data.frame(datTraits))
class(datTraits)geneTraitSignificance as.data.frame(cor(datExpr0, datTraits, use p))
GSPvalue as.data.frame(corPvalueStudent(as.matrix(geneTraitSignificance), nSamples))names(geneTraitSignificance) paste(GS., traitNames, sep)
names(GSPvalue) paste(p.GS., traitNames, sep)####plot MM vs GS for each trait vs each module##########example:royalblue and CK
modulered
column match(module, modNames)
moduleGenes moduleColorsmoduletraitTB
traitColumnmatch(trait,traitNames)sizeGrWindow(7, 7)#par(mfrow c(1,1))
verboseScatterplot(abs(geneModuleMembership[moduleGenes, column]),
abs(geneTraitSignificance[moduleGenes, traitColumn]),
xlab paste(Module Membership in, module, module),
ylab paste(Gene significance for ,trait),
main paste(Module membership vs. gene significance\n),
cex.main 1.2, cex.lab 1.2, cex.axis 1.2, col module)
######for (trait in traitNames){traitColumnmatch(trait,traitNames)for (module in modNames){column match(module, modNames)moduleGenes moduleColorsmoduleif (nrow(geneModuleMembership[moduleGenes,]) 1){####进行这部分计算必须每个模块内基因数量大于2由于前面设置了最小数量是30这里可以不做这个判断但是grey有可能会出现1个gene,它会导致代码运行的时候中断故设置这一步#sizeGrWindow(7, 7)pdf(filepaste(9_, trait, _, module,_Module membership vs gene significance.pdf,sep),width7,height7)par(mfrow c(1,1))verboseScatterplot(abs(geneModuleMembership[moduleGenes, column]),abs(geneTraitSignificance[moduleGenes, traitColumn]),xlab paste(Module Membership in, module, module),ylab paste(Gene significance for ,trait),main paste(Module membership vs. gene significance\n),cex.main 1.2, cex.lab 1.2, cex.axis 1.2, col module)dev.off()}}
}#####
names(datExpr0)
probes names(datExpr0)#################export GS and MM############### geneInfo0 data.frame(probes probes,moduleColor moduleColors)for (Tra in 1:ncol(geneTraitSignificance))
{oldNames names(geneInfo0)geneInfo0 data.frame(geneInfo0, geneTraitSignificance[,Tra],GSPvalue[, Tra])names(geneInfo0) c(oldNames,names(geneTraitSignificance)[Tra],names(GSPvalue)[Tra])
}for (mod in 1:ncol(geneModuleMembership))
{oldNames names(geneInfo0)geneInfo0 data.frame(geneInfo0, geneModuleMembership[,mod],MMPvalue[, mod])names(geneInfo0) c(oldNames,names(geneModuleMembership)[mod],names(MMPvalue)[mod])
}
geneOrder order(geneInfo0$moduleColor)
geneInfo geneInfo0[geneOrder, ]write.table(geneInfo, file 10_GS_and_MM.xls,sep\t,row.namesF)
####################################################Visualizing the gene network#######################################################nGenes ncol(datExpr0)
nSamples nrow(datExpr0)# 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 functionsizeGrWindow(9,9) #这个耗电脑内存
pdf(file12_Network heatmap plot_all gene.pdf,width9, height9)
TOMplot(plotTOM, geneTree, moduleColors, main Network heatmap plot, all genes)
dev.off()nSelect 400
# For reproducibility, we set the random seed
set.seed(10)
select sample(nGenes, size nSelect)
selectTOM dissTOM[select, select]
# Theres 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
plotDiss selectTOM^7
diag(plotDiss) NApdf(file13_Network heatmap plot_selected genes.pdf,width9, height9)
TOMplot(plotDiss, selectTree, selectColors, main Network heatmap plot, selected genes)
dev.off()####################################################Visualizing the gene network of eigengenes#####################################################sizeGrWindow(5,7.5)
pdf(file14_Eigengene dendrogram and Eigengene adjacency heatmap.pdf, width5, height7.5)
par(cex 0.9)
plotEigengeneNetworks(MEs, , marDendro c(0,4,1,2), marHeatmap c(3,4,1,2), cex.lab 0.8, xLabelsAngle 90)
dev.off()#or devide into two parts
# Plot the dendrogram
#sizeGrWindow(6,6);
pdf(file15_Eigengene dendrogram_2.pdf,width6, height6)
par(cex 1.0)
plotEigengeneNetworks(MEs, Eigengene dendrogram, marDendro c(0,4,2,0), plotHeatmaps FALSE)
dev.off()pdf(file15_Eigengene adjacency heatmap_2.pdf,width6, height6)
# Plot the heatmap matrix (note: this plot will overwrite the dendrogram plot)
par(cex 1.0)
plotEigengeneNetworks(MEs, Eigengene adjacency heatmap, marHeatmap c(3,4,2,2), plotDendrograms FALSE, xLabelsAngle 90)
dev.off()
后续挖掘核心基因时需要用到Cytoscape生成绘图所需要的数据
###########################Exporting to Cytoscape all one by one ########################### Select each moduleError in exportNetworkToCytoscape(modTOM, edgeFile paste(CytoscapeInput-edges-, : Cannot determine node names: nodeNames is NULL and adjMat has no dimnames.datExpr0 格式需要dataframemodules module
for (mod in 1:nrow(table(moduleColors)))
{modules names(table(moduleColors))[mod]# Select module probesprobes names(data.frame(datExpr0)) # inModule (moduleColors modules)modProbes probes[inModule]modGenes modProbes# Select the corresponding Topological OverlapmodTOM TOM[inModule, inModule]dimnames(modTOM) list(modProbes, modProbes)# Export the network into edge and node list files Cytoscape can readcyt exportNetworkToCytoscape(modTOM,edgeFile paste(CytoscapeInput-edges-, modules , .txt, sep),nodeFile paste(CytoscapeInput-nodes-, modules, .txt, sep),weighted TRUE,threshold 0.02,nodeNames modProbes,altNodeNames modGenes,nodeAttr moduleColors[inModule])
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