通過查閱文章找到marker gene后手動注釋分群,，如何檢驗分群正確與否,？

這個圖是通過Seurat標(biāo)準(zhǔn)流程降維后得到的muscle組織分群圖

然后查閱文章得到muscle組織不同細(xì)胞的markergene

genes_VEC = c("Fabp4", "Cdh5", "Cav1") #vascular endothelial cells

genes_FC = c("Ddr2","Tcf21", "Col3a1", "Col1a2", "Col1a1")  #fibroblasts

genes_AC = c("Nppa", "Myl7", "Sln") #"Nppa", "Myl7" not found #atrial cardiomyocytes

genes_EC = c("Npr3", "Pecam1")  #endocardial cells

genes_IC = c("C1qa", "H2-Eb1")  #immune cells

genes_SMSC= c("Myf5", "Myod1","Sox9","Acta2","Chodl")   #skeletal muscle satellite cell

genes_MSC= c("Pdgfra","Chad")  #mesenchymal stem cell

genes_endothelialC=c("Atxn1")

genes_TmB = c('Ptprc')

genes_macrophage=c("Itgam")

genes_T=c("Cd3g","Cd4")

genes_B=c("Cd19")

genes_others=c("Acan")

genes_all = c(genes_VEC, genes_FC, genes_AC,genes_EC,genes_IC,genes_SMSC,genes_MSC,genes_endothelialC,genes_TmB,genes_macrophage,genes_T,genes_B,genes_others)

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以下三幅圖片都是想表達不同的markergene的表達情況：

FeaturePlot(tiss, genes_all, pt.size = 1,ncol = 4)

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DotPlot(tiss, features = genes_all)

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VlnPlot(tiss, genes_all, ncol = 4)

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有了這三幅圖，我們可以對這些muscle細(xì)胞進行一個手動的注釋,，得到下面的細(xì)胞分群圖：

#annotaion

[email protected]$free_annotation <- plyr::mapvalues(from = c(1,2,5,7,8,10,11,3,6,9,0,4),

                                                  to = c("vascular endothelial cells", rep("fibroblasts",6), rep("immune cells", 3),rep("skeletal muscle satellite cells", 2)),

                                                  x = [email protected]$seurat_clusters)

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TSNEPlot(object = tiss, group.by = "free_annotation")

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但是這個分群圖正確與否應(yīng)該怎么判斷呢,？

檢驗分群正確與否方法

Findmarkers 函數(shù)

singleR

Seruat4.0

一、Findmarkers 函數(shù)

首先需要加載所需的R包和數(shù)據(jù)集

library(Seurat)  

tiss<- #數(shù)據(jù)集加載

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以下為自己數(shù)據(jù)示例

levels(tiss)

markers_df <- FindMarkers(object = tiss, ident.1 = 1, min.pct = 0.25)#一號群

markers_df  #Fabp4", "Cdh5", "Cav1"文章中提到的

print(x = head(markers_df))

markers_genes =  rownames(head(x = markers_df, n = 5))

VlnPlot(object = tiss, features =markers_genes,log =T )

FeaturePlot(object = tiss, features=markers_genes )

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這樣我們可以得到每一個分群的markergene,，可以大概看一下這些markergene與文獻中提到的有沒有重合的,。這個只能當(dāng)作一種檢驗方法吧。

二,、SingleR

singleR自帶7個數(shù)據(jù)庫文件,，需要聯(lián)網(wǎng)才能下載，其中5個是人類數(shù)據(jù),，2個是小鼠的數(shù)據(jù)：

BlueprintEncodeData Labels

HumanPrimaryCellAtlasData Labels

DatabaseImmuneCellExpressionData Labels

NovershternHematopoieticData Labels

MonacoImmuneData Labels

ImmGenData Labels

MouseRNAseqData Labels

本文以MouseRNAseqData 數(shù)據(jù)集為例：

首先加載參考數(shù)據(jù)集

# load ref

library("SingleR")

cg <- MouseRNAseqData()

cg

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以下為加載測試數(shù)據(jù)集的過程:

#load test

library("openxlsx")

library("ggplot2")

library("Matrix")

mypwd <- "/mnt/raid64/Mouse_iso_atlas/analysis/SingleCell/NGS_ONT_analysis/01.Seurat/"

library("Seurat", lib.loc = "/home/zhangdan/R/x86_64-pc-linux-gnu-library/4.0")

source(paste0(mypwd, "00_data_ingest/02_tissue_analysis_rmd/boilerplate.R"))

tissue_of_interest = 'muscle'

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process_tissue = function(tiss, scale){

  tiss <- NormalizeData(object = tiss, scale.factor = scale)

  tiss <- FindVariableFeatures(object = tiss, do.plot = TRUE, x.high.cutoff = Inf, y.cutoff = 0.5)

  tiss <- ScaleData(object = tiss)

  tiss <- RunPCA(object = tiss, do.print = FALSE)

}

load_tissue_singleron = function(tissue_of_interest){

singleron_metadata_filename = paste0(mypwd, "00_data_ingest/00_singleron_raw_data/metadata_singleron.csv")

singleron_metadata <- read.csv(singleron_metadata_filename, sep=",", header = TRUE)

tissue_metadata <- singleron_metadata[singleron_metadata$Tissue == tissue_of_interest, ]

subfolder = tissue_metadata$Sample

raw.data <- Read10X(data.dir = paste0(mypwd, "00_data_ingest/00_singleron_raw_data/singleron/", subfolder[1]))

# Create the Seurat object with all the data

tiss <- CreateSeuratObject(counts = raw.data, project = tissue_of_interest)

tiss[["percent.mt"]] <- PercentageFeatureSet(tiss, pattern = "^mt-")

tiss[["percent.ribo"]] <- PercentageFeatureSet(tiss, pattern = "^Rp[sl][[:digit:]]")

[email protected][,'free_annotation'] <- NA

tiss[["Tissue"]] <- tissue_metadata[1,]$Tissue

tiss[["Sex"]] <- tissue_metadata[1,]$Sex

tiss[["Strain"]] <- tissue_metadata[1,]$Strain

tiss[["mouse.id"]] <- tissue_metadata[1,]$mouse.id

VlnPlot(tiss, features = c("nFeature_RNA", "nCount_RNA", "percent.mt","percent.ribo"), ncol = 2)

#tiss <- subset(tiss, subset = nFeature_RNA > 200 & nFeature_RNA < 6000 & percent.mt < 5)

tiss <- process_tissue(tiss, 1e4)

return(tiss)

}

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tiss = load_tissue_singleron(tissue_of_interest)

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測試數(shù)據(jù)集通過SingleR比對到參考數(shù)據(jù)集上：

data <- tiss@assays$RNA@data

pred.hesc <- SingleR(test = data, ref = cg, assay.type.test=1,

    labels = cg$label.main)

pred.hesc

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#比對結(jié)果,，會顯示有哪些細(xì)胞及其細(xì)胞數(shù)量

# Summarizing the distribution:

table(pred.hesc$labels)

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plotScoreHeatmap(pred.hesc)

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[email protected]$lables <- pred.hesc$labels

DimPlot(tiss, group.by="lables", label = T)

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三、Seurat4.0

還在學(xué)習(xí)中 后續(xù)補上

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版權(quán)聲明：本文為CSDN博主「微光**」的原創(chuàng)文章,，遵循CC 4.0 BY-SA版權(quán)協(xié)議,，轉(zhuǎn)載請附上原文出處鏈接及本聲明。

原文鏈接：https://blog.csdn.net/zengwanqin/article/details/114655570