软件介绍
SENIC是一种同时重建基因调控网络并从单细胞RNA-seq数据中鉴定stable cell states的工具。基于共表达和DNA模基序 （motif）分析推断基因调控网络 ，然后在每个细胞中分析网络活性以鉴定细胞状态
https://www.nature.com/articles/nmeth.4463
参考帮助文档：https://rawcdn.githack.com/aertslab/SCENIC/0a4c96ed8d930edd8868f07428090f9dae264705/inst/doc/SCENIC_Running.html#optional_steps:
输入：SCENIC需要输入的是单细胞RNA-seq表达矩阵—— 每列对应于样品（细胞），每行对应一个基因。基因ID应该是gene-symbol并存储为rownames （尤其是基因名字部分是为了与RcisTarget数据库兼容）；表达数据是Gene的reads count。根据作者的测试，提供原始的或Normalized UMI count，无论是否log转换，或使用TPM值，结果相差不大。
软件的安装
if (!requireNamespace("BiocManager", quietly = TRUE)) install.packages("BiocManager")
BiocManager::install(c("AUCell", "RcisTarget"))
BiocManager::install(c("GENIE3"))
BiocManager::install(c("zoo", "mixtools", "rbokeh"))
BiocManager::install(c("DT", "NMF", "pheatmap", "R2HTML", "Rtsne"))
BiocManager::install(c("doMC", "doRNG"))
BiocManager::install(c("SingleCellExperiment"))
if (!requireNamespace("devtools", quietly = TRUE)) install.packages("devtools")
devtools::install_github("aertslab/SCopeLoomR", build_vignettes = TRUE)
if (!requireNamespace("devtools", quietly = TRUE)) install.packages("devtools")
devtools::install_github("aertslab/SCENIC")
packageVersion("SCENIC")
下载评分数据库
需要下载RcisTarget的物种特定数据库（https://resources.aertslab.org/cistarget/）
For Human,Mouse,Fly
mm_url="https://resources.aertslab.org/cistarget/databases/mus_musculus/mm9/refseq_r45/mc9nr/gene_based/mm9-500bp-upstream-7species.mc9nr.feather"
mm_url2="https://resources.aertslab.org/cistarget/databases/mus_musculus/mm9/refseq_r45/mc9nr/gene_based/mm9-tss-centered-10kb-7species.mc9nr.feather"
hg_url="https://resources.aertslab.org/cistarget/databases/homo_sapiens/hg19/refseq_r45/mc9nr/gene_based/hg19-500bp-upstream-7species.mc9nr.feather"
hg_url2="https://resources.aertslab.org/cistarget/databases/homo_sapiens/hg19/refseq_r45/mc9nr/gene_based/hg19-tss-centered-10kb-7species.mc9nr.feather"
fly_url="https://resources.aertslab.org/cistarget/databases/drosophila_melanogaster/dm6/flybase_r6.02/mc8nr/gene_based/dm6-5kb-upstream-full-tx-11species.mc8nr.feather"
wget -c $mm_url
wget -c $mm_url2
wget -c $hg_url
wget -c $hg_url2
wget -c $fly_url
不同数据格式的读入
对于loom文件
download.file("http://loom.linnarssonlab.org/clone/Previously%20Published/Cortex.loom", "Cortex.loom")
loomPath <- "Cortex.loom"
10x的输出文件
singleCellMatrix <- Seurat::Read10X(data.dir="data/pbmc3k/filtered_gene_bc_matrices/hg19/")
cellInfo <- data.frame(seuratCluster=Idents(seuratObject))
R objects (e.g. Seurat, SingleCellExperiment)
sce <- load_as_sce(dataPath) # any SingleCellExperiment object
exprMat <- counts(sce)
cellInfo <- colData(sce)
简单的SENIC工作流程
setwd("/media/sdb/project/20200223/SCENIC_MouseBrain")
loomPath <- system.file(package="SCENIC", "examples/mouseBrain_toy.loom")
library(SCopeLoomR)
loom <- open_loom(loomPath)
exprMat <- get_dgem(loom)
cellInfo <- get_cellAnnotation(loom)
close_loom(loom)
#查看矩阵大小
#dim(exprMat)
library(SCENIC)
#scenicOptions <- initializeScenic(org="mgi", dbDir="cisTarget_databases", nCores=10)
scenicOptions <- initializeScenic(org="mgi", dbDir="/media/sdb/project/20200223/data", nCores=10)
saveRDS(scenicOptions, file="int/scenicOptions.Rds")
### Co-expression network
genesKept <- geneFiltering(exprMat, scenicOptions)
exprMat_filtered <- exprMat[genesKept, ]
runCorrelation(exprMat_filtered, scenicOptions)
exprMat_filtered_log <- log2(exprMat_filtered+1)
runGenie3(exprMat_filtered_log, scenicOptions)
### Build and score the GRN
exprMat_log <- log2(exprMat+1)
scenicOptions@settings$dbs <- scenicOptions@settings$dbs["10kb"] # Toy run settings
runSCENIC_1_coexNetwork2modules(scenicOptions)
runSCENIC_2_createRegulons(scenicOptions, coexMethod=c("top5perTarget")) # Toy run settings
runSCENIC_3_scoreCells(scenicOptions, exprMat_log)
# Export: 运行这个时可能报错
#export2scope(scenicOptions, exprMat)
# Binarize activity?
# aucellApp <- plotTsne_AUCellApp(scenicOptions, exprMat_log)
# savedSelections <- shiny::runApp(aucellApp)
# newThresholds <- savedSelections$thresholds
# scenicOptions@fileNames$int["aucell_thresholds",1] <- "int/newThresholds.Rds"
# saveRDS(newThresholds, file=getIntName(scenicOptions, "aucell_thresholds"))
# saveRDS(scenicOptions, file="int/scenicOptions.Rds")
runSCENIC_4_aucell_binarize(scenicOptions)
### Exploring output
# Check files in folder 'output'
# .loom file @ http://scope.aertslab.org
# output/Step2_MotifEnrichment_preview.html in detail/subset:
motifEnrichment_selfMotifs_wGenes <- loadInt(scenicOptions, "motifEnrichment_selfMotifs_wGenes")
tableSubset <- motifEnrichment_selfMotifs_wGenes[highlightedTFs=="Sox8"]
viewMotifs(tableSubset)
# output/Step2_regulonTargetsInfo.tsv in detail:
regulonTargetsInfo <- loadInt(scenicOptions, "regulonTargetsInfo")
tableSubset <- regulonTargetsInfo[TF=="Stat6" & highConfAnnot==TRUE]
viewMotifs(tableSubset)
运行SENIC
建立基因调控网络（Gene Regulation Network，GRN）：
- 基于共表达识别每个转录因子TF的潜在靶标。过滤表达矩阵并运行GENIE3或者GRNBoost，它们是利用表达矩阵推断基因调控网络的一种算法，能得到转录因子和潜在靶标的相关性网络；将目标从GENIE3或者GRNBoost格式转为共表达模块。
- 根据DNA模序分析（
motif）选择潜在的直接结合靶标（调节因子）（利用RcisTarget包：TF基序分析）
确定细胞状态及其调节因子：
3. 分析每个细胞中的网络活性（AUCell） 在细胞中评分调节子（计算AUC）
SCENIC完整流程
导入数据
loomPath <- system.file(package="SCENIC", "examples/mouseBrain_toy.loom")
library(SCopeLoomR)
loom <- open_loom(loomPath) #mode='r' 如果报错可以加上
exprMat <- get_dgem(loom)
cellInfo <- get_cellAnnotation(loom)
close_loom(loom)
Initialize settings 初始设置，导入评分数据库
library(SCENIC)
#先下载数据库，org用来选择物种，这里选择的是小鼠
scenicOptions <- initializeScenic(org="mgi", dbDir="cisTarget_databases", nCores=10)
# scenicOptions@inputDatasetInfo$cellInfo <- "int/cellInfo.Rds"
saveRDS(scenicOptions, file="int/scenicOptions.Rds")
共表达网络
根据已有的表达矩阵推断潜在的转录因子靶标，使用GENIE3或GRNBoost。首先需要进行基因的过滤。
genesKept <- geneFiltering(exprMat, scenicOptions=scenicOptions,
minCountsPerGene=3*.01*ncol(exprMat),
minSamples=ncol(exprMat)*.01)
过滤表达矩阵，保留只有过滤后的基因
exprMat_filtered <- exprMat[genesKept, ]
计算相关性，这一步时间会比较长
runCorrelation(exprMat_filtered, scenicOptions)
exprMat_filtered_log <- log2(exprMat_filtered+1)
runGenie3(exprMat_filtered_log, scenicOptions)
Build and score the GRN 构建并给基因调控网络（GRN）打分
exprMat_log <- log2(exprMat+1)
scenicOptions@settings$dbs <- scenicOptions@settings$dbs["10kb"] # Toy run settings
runSCENIC_1_coexNetwork2modules(scenicOptions)
runSCENIC_2_createRegulons(scenicOptions, coexMethod=c("top5perTarget")) # Toy run settings
runSCENIC_3_scoreCells(scenicOptions, exprMat_log)
输入表达矩阵
在本教程中，我们提供了一个示例，样本是小鼠大脑的200个细胞和862个基因：
loomPath <- system.file(package="SCENIC", "examples/mouseBrain_toy.loom")
打开loom文件并加载表达矩阵；
library(SCopeLoomR)
loom <- open_loom(loomPath, mode="r")
exprMat <- get_dgem(loom)
cellInfo <- get_cellAnnotation(loom)
close_loom(loom)
#dim(exprMat)
细胞信息/表型
# cellInfo$nGene <- colSums(exprMat>0)
head(cellInfo)
cellInfo <- data.frame(cellInfo)
cellTypeColumn <- "Class"
colnames(cellInfo)[which(colnames(cellInfo)==cellTypeColumn)] <- "CellType"
cbind(table(cellInfo$CellType))
saveRDS(cellInfo, file="int/cellInfo.Rds")
# Color to assign to the variables (same format as for NMF::aheatmap)
colVars <- list(CellType=c("microglia"="forestgreen",
"endothelial-mural"="darkorange",
"astrocytes_ependymal"="magenta4",
"oligodendrocytes"="hotpink",
"interneurons"="red3",
"pyramidal CA1"="skyblue",
"pyramida SS"="darkblue"
))
colVars$CellType <- colVars$CellType[intersect(names(colVars$CellType), cellInfo$CellType)]
saveRDS(colVars, file="int/colVars.Rds")
plot.new()
legend(0,1, fill=colVars$CellType, legend=names(colVars$CellType))
初始化SCENIC设置
为了在SCENIC的多个步骤中保持设置一致，SCENIC包中的大多数函数使用一个公共对象，该对象存储当前运行的选项并代替大多数函数的“参数”。比如下面的org，dbDir等，可以在开始就将物种rog（mgi—— mouse， hgnc —— human， dmel —— fly）和RcisTarge数据库位置分别读给对象org，dbDir，之后统一用函数initializeScenic得到对象scenicOptions。具体参数设置可以用?initializeScenichelp一下。
library(SCENIC)
org="mgi" # or hgnc, or
dmeldbDir="cisTarget_databases" # RcisTarget databases location
myDatasetTitle="SCENIC example on Mouse brain" # choose a name for your analysis
data(defaultDbNames)
dbs <- defaultDbNames[[org]]
scenicOptions <- initializeScenic(org=org, dbDir=dbDir, dbs=dbs, datasetTitle=myDatasetTitle, nCores=10)
# 如果有需要就修改这个地方
scenicOptions@inputDatasetInfo$cellInfo <- "int/cellInfo.Rds
"scenicOptions@inputDatasetInfo$colVars <- "int/colVars.Rds"
# Databases:
# scenicOptions@settings$dbs <- c("mm9-5kb-mc8nr"="mm9-tss-centered-5kb-10species.mc8nr.feather")
# scenicOptions@settings$db_mcVersion <- "v8"
# Save to use at a later time...
saveRDS(scenicOptions, file="int/scenicOptions.Rds")
共表达网络
SCENIC工作流程的第一步是根据表达数据推断潜在的转录因子靶标。为此，我们使用GENIE3或GRNBoost，输入文件是表达矩阵（过滤后的）和转录因子列表。GENIE3/GRBBoost的输出结果和相关矩阵将用于创建共表达模块（runSCENIC_1_coexNetwork2modules（））。
基因过滤/选择
- 按每个基因的reads总数进行过滤。该filter旨在去除最可能是噪音的基因。默认情况下，它（
minCountsPerGene）保留所有样品中至少带有6个UMI reads的基因（例如，如果在1％的细胞中以3的值表达，则基因将具有的总数）。 - 通过基因的细胞数来实现过滤（例如 UMI > 0 ，或log 2（TPM）> 1 ）。默认情况下(
minSamples)，保留下来的基因能在至少1％的细胞中检测得到。 - 最后，只保留
RcisTarget数据库中可用的基因。
# (Adjust minimum values according to your dataset)
genesKept <- geneFiltering(exprMat, scenicOptions=scenicOptions,
minCountsPerGene=3*.01*ncol(exprMat),
minSamples=ncol(exprMat)*.01)
在进行网络推断之前，检查是否有任何已知的相关基因被过滤掉（如果缺少任何相关基因，请仔细检查filter设置是否合适）：
interestingGenes <- c("Sox9", "Sox10", "Dlx5")
interestingGenes[which(!interestingGenes %in% genesKept)]
相关性
GENIE33或者GRNBoost可以检测正负关联。为了区分潜在的激活和抑制，我们将目标分为正相关和负相关目标（比如TF与潜在目标之间的Spearman相关性）。
runCorrelation(exprMat_filtered, scenicOptions)
运行GENIE3得到潜在转录因子TF
## If launched in a new session, you will need to reload...
# setwd("...")
# loomPath <- "..."
# loom <- open_loom(loomPath, mode="r")
# exprMat <- get_dgem(loom)
# close_loom(loom)
# genesKept <- loadInt(scenicOptions, "genesKept")
# exprMat_filtered <- exprMat[genesKept,]
# library(SCENIC)
# scenicOptions <- readRDS("int/scenicOptions.Rds")
# Optional: add log (if it is not logged/normalized already)
exprMat_filtered <- log2(exprMat_filtered+1)
# Run GENIE3
runGenie3(exprMat_filtered, scenicOptions)
构建并评分GRN（runSCENIC_ …）
必要时重新加载表达式矩阵：
loom <- open_loom(loomPath, mode="r")
exprMat <- get_dgem(loom)
close_loom(loom)
# Optional: log expression (for TF expression plot, it does not affect any other calculation)
logMat <- log2(exprMat+1)
dim(exprMat)
使用wrapper函数运行其余步骤：
library(SCENIC)
scenicOptions <- readRDS("int/scenicOptions.Rds")
scenicOptions@settings$verbose <- TRUE
scenicOptions@settings$nCores <- 10
scenicOptions@settings$seed <- 123
# For a very quick run:
# coexMethod=c("top5perTarget")
scenicOptions@settings$dbs <- scenicOptions@settings$dbs["10kb"] # For toy run
# save...
runSCENIC_1_coexNetwork2modules(scenicOptions)
runSCENIC_2_createRegulons(scenicOptions, coexMethod=c("top5perTarget")) #** Only for toy run!! 只用于测试数据
runSCENIC_3_scoreCells(scenicOptions, logMat)
可选步骤
将network activity转换成ON/OFF（二进制）格式
nPcs <- c(5) # For toy dataset
# nPcs <- c(5,15,50)
scenicOptions@settings$seed <- 123 # same seed for all of them
#使用不同的参数运行t-SNE
fileNames <- tsneAUC(scenicOptions, aucType="AUC", nPcs=nPcs, perpl=c(5,15,50))
fileNames <- tsneAUC(scenicOptions, aucType="AUC", nPcs=nPcs, perpl=c(5,15,50), onlyHighConf=TRUE, filePrefix="int/tSNE_oHC")
# 画图 (individual files in int/):
fileNames <- paste0("int/",grep(".Rds", grep("tSNE_", list.files("int"), value=T), value=T))
par(mfrow=c(length(nPcs), 3))
fileNames <- paste0("int/",grep(".Rds", grep("tSNE_AUC", list.files("int"), value=T, perl = T), value=T))
plotTsne_compareSettings(fileNames, scenicOptions, showLegend=FALSE, varName="CellType", cex=.5)
# Using only "high-confidence" regulons (normally similar)
par(mfrow=c(3,3))
fileNames <- paste0("int/",grep(".Rds", grep("tSNE_oHC_AUC", list.files("int"), value=T, perl = T), value=T))
plotTsne_compareSettings(fileNames, scenicOptions, showLegend=FALSE, varName="CellType", cex=.5)
输出到 loom/SCope
SCENIC生成的结果既能在http://scope.aertslab.org查看，还能用函数export2scope()（需要SCopeLoomR包）保存成.loom文件。
# DGEM (Digital gene expression matrix)
# (non-normalized counts)
# exprMat <- get_dgem(open_loom(loomPath))
# dgem <- exprMat
# head(colnames(dgem)) #should contain the Cell ID/name
# Export:
scenicOptions@fileNames$output["loomFile",] <- "output/mouseBrain_SCENIC.loom"
export2scope(scenicOptions, exprMat)
加载.loom文件中的结果
SCopeLoomR中也有函数可以导入.loom文件中的内容，比如调节因子，AUC和封装内容（比如regulon activity的t-SNE和UMAP结果）。
library(SCopeLoomR)
scenicLoomPath <- getOutName(scenicOptions, "loomFile")
loom <- open_loom(scenicLoomPath)
# Read information from loom file:
regulons_incidMat <- get_regulons(loom)
regulons <- regulonsToGeneLists(regulons_incidMat)
regulonsAUC <- get_regulonsAuc(loom)
regulonsAucThresholds <- get_regulonThresholds(loom)
embeddings <- get_embeddings(loom)
解读结果
1. 细胞状态
AUCell提供跨细胞的调节子的活性,AUCell使用“Area under Curve 曲线下面积”（AUC）来计算输入基因集的关键子集是否在每个细胞的表达基因中富集。通过该调节子活性（连续或二进制AUC矩阵）来聚类细胞，我们可以看出是否存在倾向于具有相同调节子活性的细胞群，并揭示在多个细胞中反复发生的网络状态。这些状态等同于网络的吸引子状态。将这些聚类与不同的可视化方法相结合，我们可以探索细胞状态与特定调节子的关联。
将AUC和TF表达投射到t-SNE上
logMat <- exprMat # Better if it is logged/normalized
aucellApp <- plotTsne_AUCellApp(scenicOptions, logMat) # default t-SNE
savedSelections <- shiny::runApp(aucellApp)
print(tsneFileName(scenicOptions))
tSNE_scenic <- readRDS(tsneFileName(scenicOptions))
aucell_regulonAUC <- loadInt(scenicOptions, "aucell_regulonAUC")
# Show TF expression:
par(mfrow=c(2,3))
AUCell::AUCell_plotTSNE(tSNE_scenic$Y, exprMat, aucell_regulonAUC[onlyNonDuplicatedExtended(rownames(aucell_regulonAUC))[c("Dlx5", "Sox10", "Sox9","Irf1", "Stat6")],], plots="Expression")
# 保存AUC图片:
Cairo::CairoPDF("output/Step4_BinaryRegulonActivity_tSNE_colByAUC.pdf", width=20, height=15)
par(mfrow=c(4,6))
AUCell::AUCell_plotTSNE(tSNE_scenic$Y, cellsAUC=aucell_regulonAUC, plots="AUC")
dev.off()
library(KernSmooth)
library(RColorBrewer)
dens2d <- bkde2D(tSNE_scenic$Y, 1)$fhat
image(dens2d, col=brewer.pal(9, "YlOrBr"), axes=FALSE)
contour(dens2d, add=TRUE, nlevels=5, drawlabels=FALSE)
#par(bg = "black")
par(mfrow=c(1,2))
regulonNames <- c( "Dlx5","Sox10")
cellCol <- plotTsne_rgb(scenicOptions, regulonNames, aucType="AUC", aucMaxContrast=0.6)
text(0, 10, attr(cellCol,"red"), col="red", cex=.7, pos=4)
text(-20,-10, attr(cellCol,"green"), col="green3", cex=.7, pos=4)
regulonNames <- list(red=c("Sox10", "Sox8"),
green=c("Irf1"),
blue=c( "Tef"))
cellCol <- plotTsne_rgb(scenicOptions, regulonNames, aucType="Binary")
text(5, 15, attr(cellCol,"red"), col="red", cex=.7, pos=4)
text(5, 15-4, attr(cellCol,"green"), col="green3", cex=.7, pos=4)
text(5, 15-8, attr(cellCol,"blue"), col="blue", cex=.7, pos=4)
GRN：Regulon靶标和模序
regulons <- loadInt(scenicOptions, "regulons")
regulons[c("Dlx5", "Irf1")]
regulons <- loadInt(scenicOptions, "aucell_regulons")
head(cbind(onlyNonDuplicatedExtended(names(regulons))))
regulonTargetsInfo <- loadInt(scenicOptions, "regulonTargetsInfo")
tableSubset <- regulonTargetsInfo[TF=="Stat6" & highConfAnnot==TRUE]
viewMotifs(tableSubset)
2. 细胞群的调控因子
regulonAUC <- loadInt(scenicOptions, "aucell_regulonAUC")
regulonAUC <- regulonAUC[onlyNonDuplicatedExtended(rownames(regulonAUC)),]
regulonActivity_byCellType <- sapply(split(rownames(cellInfo), cellInfo$CellType),function(cells) rowMeans(getAUC(regulonAUC)[,cells]))
regulonActivity_byCellType_Scaled <- t(scale(t(regulonActivity_byCellType), center = T, scale=T))
pheatmap::pheatmap(regulonActivity_byCellType_Scaled, color=colorRampPalette(c("blue","white","red"))(100), breaks=seq(-3, 3, length.out = 100),treeheight_row=10, treeheight_col=10, border_color=NA)
# filename="regulonActivity_byCellType.pdf", width=10, height=20)
topRegulators <- reshape2::melt(regulonActivity_byCellType_Scaled)
colnames(topRegulators) <- c("Regulon", "CellType", "RelativeActivity")
topRegulators <- topRegulators[which(topRegulators$RelativeActivity>0),]
viewTable(topRegulators)
minPerc <- .7
binaryRegulonActivity <- loadInt(scenicOptions, "aucell_binary_nonDupl")
cellInfo_binarizedCells <- cellInfo[which(rownames(cellInfo)%in% colnames(binaryRegulonActivity)),, drop=FALSE]
regulonActivity_byCellType_Binarized <- sapply(split(rownames(cellInfo_binarizedCells), cellInfo_binarizedCells$CellType),function(cells) rowMeans(binaryRegulonActivity[,cells, drop=FALSE]))
binaryActPerc_subset <- regulonActivity_byCellType_Binarized[which(rowSums(regulonActivity_byCellType_Binarized>minPerc)>0),]
pheatmap::pheatmap(binaryActPerc_subset,color = colorRampPalette(c("white","pink","red"))(100), breaks=seq(0, 1, length.out = 100),treeheight_row=10, treeheight_col=10, border_color=NA)