前往小程序,Get更优阅读体验!
立即前往
首页
学习
活动
专区
工具
TVP
发布
社区首页 >专栏 >单细胞代码解析-妇科癌症单细胞转录组及染色质可及性分析4

单细胞代码解析-妇科癌症单细胞转录组及染色质可及性分析4

原创
作者头像
小胡子刺猬的生信学习123
发布2022-08-21 13:03:37
5140
发布2022-08-21 13:03:37
举报

单细胞代码解析-妇科癌症单细胞转录组及染色质可及性分析1:https://cloud.tencent.com/developer/article/2055573

单细胞代码解析-妇科癌症单细胞转录组及染色质可及性分析2:https://cloud.tencent.com/developer/article/2072069

单细胞代码解析-妇科癌症单细胞转录组及染色质可及性分析3:https://cloud.tencent.com/developer/article/2078159

图片.png
图片.png
代码语言:javascript
复制
###########################################################
# Part 3: scRNA-seq processing after doublet removal
###########################################################
setwd(dir)

# Load the RNA dataset
counts.init <- Read10X_h5(filename = "./filtered_feature_bc_matrix.h5")
# Initialize the Seurat object with the raw (non-normalized data).
rna <- CreateSeuratObject(counts = counts.init, min.cells = 3)# genes not present in at least 3 cells are removed
rna

rna <- rna[,cells]
#这部分放入了以前鉴定得到的双细胞的结果;%in%:意思都是x的值是否在y里面
rna <- rna[,!(colnames(rna) %in% doublets)]
dim(rna)


#Normalize;标准换
rna <- NormalizeData(rna, normalization.method = "LogNormalize", scale.factor = 10000)

#Feature Selection;寻找高变基因
rna <- FindVariableFeatures(rna, selection.method = "vst", nfeatures = 2000)

#Scaling
all.genes <- rownames(rna)
rna <- ScaleData(rna, features = all.genes)
rna <- RunPCA(rna)


feats <- list(stromal,immune,fibroblast,endothelial,epithelial,smooth,plasma)
#利用seurat包的一个打分函数AddModuleScore,依据基因的平均表达水平进行分析
rna <- AddModuleScore(rna,features = feats,name = c("stromal.","immune.","fibroblast.","endothelial.",
                                                    "epithelial.","smooth.","plasma."),search = T)


# Add PC1 to metadata
##对前面提到的细胞类群进行相关性分析
rna@meta.data$PC1 <- rna@reductions$pca@cell.embeddings[,1]

count_cor_PC1 <- cor(rna$PC1,rna$nCount_RNA,method = "spearman")

stromal.cor <- cor(rna$PC1,rna$stromal.1,method = "spearman")
immune.cor <- cor(rna$PC1,rna$immune.2,method = "spearman")
fibroblast.cor <- cor(rna$PC1,rna$fibroblast.3,method = "spearman")
endothelial.cor <- cor(rna$PC1,rna$endothelial.4,method = "spearman")
epithelial.cor <- cor(rna$PC1,rna$epithelial.5,method = "spearman")
smooth.cor <- cor(rna$PC1,rna$smooth.6,method = "spearman")
plasma.cor <- cor(rna$PC1,rna$plasma.7,method = "spearman")


# Make JackStraw Plot
rna <- JackStraw(rna, num.replicate = 100,dims = 50)
rna <- ScoreJackStraw(rna,dims = 1:50)
JackStrawPlot(rna, dims = 1:50)+ggsave("JackStraw_postdoublet.png")
##下面是循环分析
# If PC1 is correalted with read depth, check to see if biological variation is corralted to PC1
#首先确定亚群分析的resolution确定,将resolution确定为0.7
#round:它四舍五入到给定的位数,如果没有提供四舍五入的位数,它会将数字四舍五入到最接近的整数
if (round(abs(count_cor_PC1),2) > 0.5){
  
  if( round(abs(stromal.cor),2) >= 0.5 |
      round(abs(immune.cor),2) >= 0.5 |
      round(abs(fibroblast.cor),2) >= 0.5 |
      round(abs(endothelial.cor),2) >= 0.5 |
      round(abs(epithelial.cor),2) >= 0.5 |
      round(abs(smooth.cor),2) >= 0.5 |
      round(abs(plasma.cor),2) >= 0.5){
    
    rna <- FindNeighbors(rna,dims = 1:50)
    rna <- FindClusters(rna,resolution = 0.7)
    rna <- RunUMAP(rna,dims = 1:50)
    Idents(rna) <- "RNA_snn_res.0.7"
    
    
    # Verify with inferCNV: is PC1 correlated with CNV events/Malignancy?
    #########################################################################
    # inferCNV: does PC1 also correlated with CNV/malignancy status?
    #InferCNV是一个由broad研究所开发的,利用单细胞转录组数据分析肿瘤细胞拷贝数变异(CNV)的工具。基本思路是在整个基因组范围内,通过计算肿瘤细胞关于参考的“正常”细胞的相对表达,利用滑窗思想对邻近的基因相对表达,计算拷贝数。
    library(infercnv)
    library(stringr)
    library(Seurat)
    counts_matrix = GetAssayData(rna, slot="counts")
    
    # Identify immune clusters
    #######################################################
    # Find immune cells by relative enrichment of ESTIMATE immune signature
    #描述性统计 —— R 'psych' 表型数据的描述性统计,是对表型数据进行的基础分析,包括最大值、最小是、均值、方差、极差等
    library(psych)
    test <- VlnPlot(rna,features = "immune.2")
    #describeBy:分组计算
    data <- describeBy(test$data$immune.2, test$data$ident, mat = TRUE)
    data.immune <- dplyr::filter(data,median > 0.1)
    
    test <- VlnPlot(rna,features = "plasma.7")
    data <- describeBy(test$data$plasma.7, test$data$ident, mat = TRUE)
    data.plasma <- dplyr::filter(data,median > 0.1)
    
    immune.clusters <- intersect(data.immune$group1,levels(Idents(rna)))
    plasma.clusters <- intersect(data.plasma$group1,levels(Idents(rna)))
    
    immune.clusters <- unique(append(immune.clusters,plasma.clusters))
    
    for (i in 1:length(immune.clusters)){
      j <- which(levels(Idents(rna)) == immune.clusters[i])
      levels(Idents(rna))[j] <- paste0("immune.",immune.clusters[i])
    }
    rna@meta.data$postdoublet.idents <- Idents(rna)
    idents <- data.frame(rownames(rna@meta.data),rna@meta.data$postdoublet.idents)
    
    
    colnames(idents) <- c("V1","V2")
    saveRDS(rna,"./rna_postdoublet_preinferCNV.rds")
    # Make inferCNV inputs
    
    rownames(idents) <- NULL
    colnames(idents) <- NULL
    write.table(idents,"./sample_annotation_file_inferCNV.txt",sep = "\t",row.names = FALSE)
    #R语言使用read.delim函数读取带分隔符的文本文件 
    ##构建注释文件
    idents <- read.delim("./sample_annotation_file_inferCNV.txt",header = F)
    gtf <- read.delim(GRCH38.annotations,header = F)
    library(EnsDb.Hsapiens.v86)
    convert.symbol = function(Data){
      ensembls <- Data$V1
      ensembls <- gsub("\\.[0-9]*$", "", ensembls)
      geneIDs1 <- ensembldb::select(EnsDb.Hsapiens.v86, keys= ensembls, keytype = "GENEID", columns = "SYMBOL")
      #cbind.fill:可填充缺失值并适用于任意数据类型
      Data <- cbind.fill(Data, geneIDs1, fill = NA)
      #去除NA
      Data <- na.omit(Data)
      Data$feature <- Data$SYMBOL
      Data.new <- data.frame(Data$SYMBOL,Data$V2,Data$V3,Data$V4)
      Data.new$Data.V2 <- paste("chr",Data.new$Data.V2,sep = "")
      Data.new$Data.SYMBOL <- make.unique(Data.new$Data.SYMBOL)
      return(Data.new)
    }
    ##convert.symbol:基因id的转换
    gtf <- convert.symbol(gtf)
    head(gtf)
    
    write.table(gtf,"./Homo_sapiens.GRCh38.86.symbol.txt",sep = "\t",row.names = FALSE,col.names = FALSE)
    
    #R 语言中的 length () 函数用于获取或设置向量 (列表)或其他对象的长度。
    #CreateInfercnvObject () 函数构建infercnv对象,此处必须设置gene_order_file参数,其输入是一个基因的染色体位置信息文件,以制表符分隔
    num.immune.clusters = length(immune.clusters)
    # create the infercnv object
    if ( num.immune.clusters == 1) {
      infercnv_obj = CreateInfercnvObject(raw_counts_matrix=as.matrix(counts_matrix),
                                          annotations_file="./sample_annotation_file_inferCNV.txt",
                                          gene_order_file="./Homo_sapiens.GRCh38.86.symbol.txt",
                                          ref_group_names=paste0("immune.",immune.clusters[1]))
      
    } else if (num.immune.clusters == 2) {
      infercnv_obj = CreateInfercnvObject(raw_counts_matrix=as.matrix(counts_matrix),
                                          annotations_file="./sample_annotation_file_inferCNV.txt",
                                          gene_order_file="./Homo_sapiens.GRCh38.86.symbol.txt",
                                          ref_group_names=c(paste0("immune.",immune.clusters[1]),
                                                            paste0("immune.",immune.clusters[2])))
      
    } else if ( num.immune.clusters == 3 ) {
      infercnv_obj = CreateInfercnvObject(raw_counts_matrix=as.matrix(counts_matrix),
                                          annotations_file="./sample_annotation_file_inferCNV.txt",
                                          gene_order_file="./Homo_sapiens.GRCh38.86.symbol.txt",
                                          ref_group_names=c(paste0("immune.",immune.clusters[1]),
                                                            paste0("immune.",immune.clusters[2]),
                                                            paste0("immune.",immune.clusters[3])))
    } else if (num.immune.clusters == 4) {
      infercnv_obj = CreateInfercnvObject(raw_counts_matrix=as.matrix(counts_matrix),
                                          annotations_file="./sample_annotation_file_inferCNV.txt",
                                          gene_order_file="./Homo_sapiens.GRCh38.86.symbol.txt",
                                          ref_group_names=c(paste0("immune.",immune.clusters[1]),
                                                            paste0("immune.",immune.clusters[2]),
                                                            paste0("immune.",immune.clusters[3]),
                                                            paste0("immune.",immune.clusters[4])))
    } else if (num.immune.clusters == 5) {
      infercnv_obj = CreateInfercnvObject(raw_counts_matrix=as.matrix(counts_matrix),
                                          annotations_file="./sample_annotation_file_inferCNV.txt",
                                          gene_order_file="./Homo_sapiens.GRCh38.86.symbol.txt",
                                          ref_group_names=c(paste0("immune.",immune.clusters[1]),
                                                            paste0("immune.",immune.clusters[2]),
                                                            paste0("immune.",immune.clusters[3]),
                                                            paste0("immune.",immune.clusters[4]),
                                                            paste0("immune.",immune.clusters[5])))
    } else if (num.immune.clusters == 6) {
      infercnv_obj = CreateInfercnvObject(raw_counts_matrix=as.matrix(counts_matrix),
                                          annotations_file="./sample_annotation_file_inferCNV.txt",
                                          gene_order_file="./Homo_sapiens.GRCh38.86.symbol.txt",
                                          ref_group_names=c(paste0("immune.",immune.clusters[1]),
                                                            paste0("immune.",immune.clusters[2]),
                                                            paste0("immune.",immune.clusters[3]),
                                                            paste0("immune.",immune.clusters[4]),
                                                            paste0("immune.",immune.clusters[5]),
                                                            paste0("immune.",immune.clusters[6])))
    }else if (num.immune.clusters == 7) {
      infercnv_obj = CreateInfercnvObject(raw_counts_matrix=as.matrix(counts_matrix),
                                          annotations_file="./sample_annotation_file_inferCNV.txt",
                                          gene_order_file="./Homo_sapiens.GRCh38.86.symbol.txt",
                                          ref_group_names=c(paste0("immune.",immune.clusters[1]),
                                                            paste0("immune.",immune.clusters[2]),
                                                            paste0("immune.",immune.clusters[3]),
                                                            paste0("immune.",immune.clusters[4]),
                                                            paste0("immune.",immune.clusters[5]),
                                                            paste0("immune.",immune.clusters[6]),
                                                            paste0("immune.",immune.clusters[7])))
    }else if (num.immune.clusters == 8) {
      infercnv_obj = CreateInfercnvObject(raw_counts_matrix=as.matrix(counts_matrix),
                                          annotations_file="./sample_annotation_file_inferCNV.txt",
                                          gene_order_file="./Homo_sapiens.GRCh38.86.symbol.txt",
                                          ref_group_names=c(paste0("immune.",immune.clusters[1]),
                                                            paste0("immune.",immune.clusters[2]),
                                                            paste0("immune.",immune.clusters[3]),
                                                            paste0("immune.",immune.clusters[4]),
                                                            paste0("immune.",immune.clusters[5]),
                                                            paste0("immune.",immune.clusters[6]),
                                                            paste0("immune.",immune.clusters[7]),
                                                            paste0("immune.",immune.clusters[8])))
    }else if (num.immune.clusters == 9) {
      infercnv_obj = CreateInfercnvObject(raw_counts_matrix=as.matrix(counts_matrix),
                                          annotations_file="./sample_annotation_file_inferCNV.txt",
                                          gene_order_file="./Homo_sapiens.GRCh38.86.symbol.txt",
                                          ref_group_names=c(paste0("immune.",immune.clusters[1]),
                                                            paste0("immune.",immune.clusters[2]),
                                                            paste0("immune.",immune.clusters[3]),
                                                            paste0("immune.",immune.clusters[4]),
                                                            paste0("immune.",immune.clusters[5]),
                                                            paste0("immune.",immune.clusters[6]),
                                                            paste0("immune.",immune.clusters[7]),
                                                            paste0("immune.",immune.clusters[8]),
                                                            paste0("immune.",immune.clusters[9])))
    }else if (num.immune.clusters == 10) {
      infercnv_obj = CreateInfercnvObject(raw_counts_matrix=as.matrix(counts_matrix),
                                          annotations_file="./sample_annotation_file_inferCNV.txt",
                                          gene_order_file="./Homo_sapiens.GRCh38.86.symbol.txt",
                                          ref_group_names=c(paste0("immune.",immune.clusters[1]),
                                                            paste0("immune.",immune.clusters[2]),
                                                            paste0("immune.",immune.clusters[3]),
                                                            paste0("immune.",immune.clusters[4]),
                                                            paste0("immune.",immune.clusters[5]),
                                                            paste0("immune.",immune.clusters[6]),
                                                            paste0("immune.",immune.clusters[7]),
                                                            paste0("immune.",immune.clusters[8]),
                                                            paste0("immune.",immune.clusters[9]),
                                                            paste0("immune.",immune.clusters[10])))
    }
    #根据cluster的数量,进行else if的处理,去输出判断拷贝数的结果
    # perform infercnv operations to reveal cnv signal
    #infercnv::run():两步法进行拷贝数分析,不需要一步一步进行尝试,参考来源:[https://www.jianshu.com/p/70f7a168fe62](https://www.jianshu.com/p/70f7a168fe62)
    infercnv_obj = infercnv::run(infercnv_obj,
                                 cutoff=0.1,  # use 1 for smart-seq, 0.1 for 10x-genomics
                                 out_dir="./output_dir_CNV_postdoublet_PassedPC1Checks",  # dir is auto-created for storing outputs
                                 cluster_by_groups=T,   # cluster
                                 denoise=T,scale_data = T,
                                 HMM=T,HMM_type = "i6",analysis_mode = "samples",min_cells_per_gene = 10,
                                 BayesMaxPNormal = 0.4, num_threads = 8
                                 
    )
    
  
    regions <- read.delim("./output_dir_CNV_postdoublet_PassedPC1Checks/HMM_CNV_predictions.HMMi6.hmm_mode-samples.Pnorm_0.4.pred_cnv_regions.dat")
    probs <- read.delim("./output_dir_CNV_postdoublet_PassedPC1Checks/BayesNetOutput.HMMi6.hmm_mode-samples/CNV_State_Probabilities.dat")
    #R 编程语言中的 as.data.frame() 函数用于将对象转换为数据框
    probs <- as.data.frame(t(probs[3,]))
    colnames(probs) <- "Prob.Normal"
    probs <- dplyr::filter(probs,Prob.Normal < 0.05)
    cnvs <- rownames(probs)
    #gsub ()可以用于字段的删减、增补、替换和切割,可以处理一个字段也可以处理由字段组成的向量
    cnvs <- gsub("\\.","-",cnvs)
    
    regions <- regions[regions$cnv_name %in% cnvs, ]
    
    #sub R语言中的函数用于替换字符串中模式的第一个匹配项
    cnv.groups <- sub("\\..*", "", regions$cell_group_name)
      
    length(which(rownames(rna@reductions$pca@cell.embeddings) == rownames(rna@meta.data)))
    rna$PC1.loading <- rna@reductions$pca@cell.embeddings[,1]
    rna$cell.barcode <- rownames(rna@meta.data)
    #ifelse()中的条件判断中可以得到多个逻辑结果,有多少个逻辑结果,ifelse()的返回值就有多少个元素
    rna$CNV.Pos <- ifelse(as.character(rna$postdoublet.idents) %in% cnv.groups,1,0)
    
    cnv.freq <- data.frame(table(regions$cell_group_name))
    cnv.freq$Var1 <- sub("\\..*", "", cnv.freq$Var1)
    
    rna$Total_CNVs <- ifelse(as.character(rna$postdoublet.idents) %in% cnv.freq$Var1,cnv.freq$Freq,0)
    
    boxplot.cnv <- ggplot(rna@meta.data,aes(x= postdoublet.idents,y=PC1.loading,color = as.factor(CNV.Pos)))+geom_boxplot()
    boxplot.cnv+ggsave("postdoublet_CNV_PC1_boxplot.png")
    #describeBy:分组计算
    data <- describeBy(boxplot.cnv$data$PC1.loading, boxplot.cnv$data$postdoublet.idents, mat = TRUE)
    data$CNV <- ifelse(data$group1 %in% cnv.groups,1,0)
    
    wilcox <- wilcox.test(data = rna@meta.data,PC1.loading~CNV.Pos)
    ##根据pvalue进行判断不同的数据属性
    if (wilcox$p.value < 0.05){
      rna <- rna
      library(stringr)
      #str_remove:删除字符串中的匹配模式。
      levels(Idents(rna)) <- str_remove(levels(Idents(rna)),"immune.")
      saveRDS(rna,"./rna_postdoublet_PassedPC1Checks.rds")
    }else{
      all.genes <- rownames(rna)
      rna <- ScaleData(rna, features = all.genes,vars.to.regress = "nCount_RNA")
      rna <- FindNeighbors(rna,dims = 1:50)
      rna <- FindClusters(rna,resolution = 0.7)
      rna <- RunUMAP(rna,dims = 1:50)
      Idents(rna) <- "RNA_snn_res.0.7"
      
      library(infercnv)
      library(stringr)
      library(Seurat)
      counts_matrix = GetAssayData(rna, slot="counts")
   

总结

作者大大的代码也太长了吧,根本一篇都弄不完。上面分析的主要思路是前期通过对细胞类型鉴定,然后筛选出了双细胞结果,根据细胞类型进行下面的分析,这次加入了肿瘤变异之间的拷贝数分析,感觉自己的分析中也可以应用到这个内容。然后作者的代码里有一个提取注释文件的方法也很好,也是可以保留下来,以后提取注释文件用到的。还有作者经常用到的else if语句也很多,可以减少后面的不停调试的方法。

原创声明:本文系作者授权腾讯云开发者社区发表,未经许可,不得转载。

如有侵权,请联系 cloudcommunity@tencent.com 删除。

原创声明:本文系作者授权腾讯云开发者社区发表,未经许可,不得转载。

如有侵权,请联系 cloudcommunity@tencent.com 删除。

评论
登录后参与评论
0 条评论
热度
最新
推荐阅读
目录
  • 总结
相关产品与服务
命令行工具
腾讯云命令行工具 TCCLI 是管理腾讯云资源的统一工具。使用腾讯云命令行工具,您可以快速调用腾讯云 API 来管理您的腾讯云资源。此外,您还可以基于腾讯云的命令行工具来做自动化和脚本处理,以更多样的方式进行组合和重用。
领券
问题归档专栏文章快讯文章归档关键词归档开发者手册归档开发者手册 Section 归档