肿瘤样本中的正常上皮鉴定-参考Nature Communications

单细胞系列教程目录索引,持续更新...

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参考文献：Single-cell RNA sequencing demonstrates the molecular and cellular reprogramming of metastatic lung adenocarcinoma

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方法简述

1. 将推定恶性细胞比例调整至20%以下
2. 过滤掉信息量较少的基因（默认：少于 10 个细胞表达、在 log2 尺度上的平均表达少于 0.1）
3. 转化为Z-score，将尺度限制为-3~3
4. 按染色体位置对基因进行排序，估计 CNV 信号（默认 = 100 个基因）
5. 用两个参数汇总CNV信号，对恶性细胞和非恶性细胞进行分类。
   • CNV 信号（MS，平方均值）：CNV信号估计均方。
   • CORR（与高 CNV 信号细胞的相关性）：每个细胞的 CNV 与前 5% 细胞的平均值的相关性。
   • 如果它们的 CNV 信号 (MS) > 0.02(X轴) 或 CNV 相关性 (CORR) > 0.2(Y轴)，则鉴定为恶性细胞。

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代码可通过原作者Github获取。这里以作者给出的example进行示例。

library(ggplot2)
library(pals)
library(plyr)
library(dplyr)
library(Seurat)
library(gplots)
library(RColorBrewer)
####################################################################################################
source("R/calInferredCNA_for_CEP.R") ## calculate inferredCNV value
source("R/makingTCIDEA_for_CEP.R") ## make TCIDEA object
source("R/TCIDEA_obj_for_CEP.R") ## initiate TCIDEA object
source("R/calCNVScore_for_CEP.R") ## calculate CNV score (MS, Corr) with CEP result

###################################################################################################
## Example data ###################################################################################
#肿瘤样本注释
cell_annotation_with_tumor <- readRDS(file = "example/cell_info_tumor_example.Rds") # celltype = annotation cell types in transcriptome data
#肿瘤样本log2表达矩阵
tumor_example <- readRDS(file = "example/log2TPM_tumor_example.Rds")
#正常细胞log2表达矩阵
normal_example <- readRDS(file = "example/log2TPM_normal_example.Rds")
#基因组位置信息
ref_genome_example <- readRDS(file = "example/refgenome_example.Rds")
#输出目录
output.dir = paste0(getwd(), "/", "result")
###################################################################################################
## PARAMETERS #####################################################################################
#设定推断恶性细胞最大比例
EP_cutoff = 20 ## count if > 20% of EP -> add
#ep细胞亚群的名称
target.celltypes = "EP" ## declare name of epithelial cells in metadata
label = "example" 
###################################################################################################
## 1. check proportion of epithelial cells in tumor tissues ##
prop <- as.data.frame(table(cell_annotation_with_tumor$celltype))
prop$Percent = prop$Freq / nrow(cell_annotation_with_tumor) * 100

##2. Check the proportion (adding normal cells or not)
if(prop[prop$Var1 %in% target.celltypes,]$Percent > EP_cutoff){
  
  list <- addNormalDataset(tumor.data = tumor_example, tumor.ident = cell_annotation_with_tumor, target.celltypes = target.celltypes,
                           normal.data = normal_example)
  addnormal_example <- as.matrix(list$data); addnormal_annotation <- list$ident
  
  runCEP(target.normalized = addnormal_example,  
         sample.info = addnormal_annotation, label = paste0(label,"_AddNormal"),
         annotationdata = ref_genome_example, target.celltypes = target.celltypes, output.dir = output.dir,
         min.cells = 10, MYwalk = 100) ## Sample list of EP proportion > EP_cutoff (20%)
}else{
  runCEP(target.normalized = tumor_example,  
         sample.info = cell_annotation_with_tumor, label = label,
         annotationdata = ref_genome_example,target.celltypes = target.celltypes, output.dir = output.dir,
         min.cells = 10, MYwalk = 100) ## Sample list of EP proportion <= EP_cutoff (20%)
}

############################################

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结果提取

result=readRDS(file = "result/example_AddNormal_after_calc_CNV_score.Rds")
identification=data.frame(Barcord=result$Index,cell_type=result$cell_index)
head(identification)

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后续自行整合至seurat注释进行后续分析

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参考来源：
https://github.com/SGI-LungCancer/SingleCell
参考文献：
Single-cell RNA sequencing demonstrates the molecular and cellular reprogramming of metastatic lung adenocarcinoma
鸣谢：
I thank Dr.Jianming Zeng(University of Macau), and all the members of his bioinformatics team, biotrainee, for generously sharing their experience and codes.

问题交流：
Email: [email protected]