In PaulingLiu/ROGUE: ROGUE (Ratio of Global Unshifted Entropy)

library(SingleCellExperiment);library(tidyverse);library(reticulate);
library(ggplot2);library(scmap);library(fmsb);
library(ggsci);library(scibet);library(Seurat);library(M3Drop);
library(ROCR);library(cluster);library(parallel);library(RaceID);
library(SC3)

CelSeq 2

expr <- readr::read_csv("/home/pauling/projects/04_SEmodel/07_NC_revision/03.data/01.clustering.ari/02.celseq2.3cl/sc_celseq2.count.csv.gz")
gene.name <- expr$Gene
expr <- as.matrix(expr[,-1])
rownames(expr) <- gene.name

cda <- readr::read_csv("/home/pauling/projects/04_SEmodel/07_NC_revision/03.data/01.clustering.ari/02.celseq2.3cl/sc_celseq2.metadata.csv.gz")
cda <- cda %>% dplyr::select(sample, cell_line) %>% dplyr::rename(label = cell_line)

t_expr <- t(expr)
res1 <- SE_fun(t_expr, span = 0.2)
res2 <- m3d_fun(t_expr)
res3 <- Gini_fun(t_expr)
res4 <- HVG_fun(t_expr)
res5 <- sct_fun(t_expr)
res6 <- FanoFactor_fun(t_expr)
res7 <- raceid_fun(t_expr)

tibble(method = c("SE","M3Drop","Gini","HVG","SCT","Fano","RaceID"),
       data = list(res1,res2,res3,res4,res5,res6,res7)) -> diff.gene

out.path <- "/home/pauling/projects/04_SEmodel/07_NC_revision/01.res/02.clustering/02.3cellline.celseq2"
readr::write_rds(diff.gene,file.path(out.path, "01.diff.gene.rds"))

RaceID3 Clustering

raceid.clustering <- function(expr, var.gene, cda, method = ""){
  n.cluster <- length(unique(cda$label))
  GeneNumber <- c(100,500,1000,1500,2000)
  res <- c()
  for (i in 1:length(GeneNumber)) {
    N <- GeneNumber[i]
    sc <- SCseq(expr[var.gene$Gene[1:N],])
    sc <- filterdata(sc, mintotal = 1, minexpr = 0, minnumber = 0)
    sc <- compdist(sc, FSelect = F, metric="pearson")
    sc <- clustexp(sc, cln=n.cluster, sat=FALSE, bootnr = 20)
    ari <- mclust::adjustedRandIndex(sc@cluster$kpart, cda$label)
    tmp <- tibble(ARI = ari, method = method, Number = N)
    res <- rbind(res,tmp)
  }
  return(res)
}

race.res1 <- raceid.clustering(expr, diff.gene$data[[1]], cda, diff.gene$method[1])
race.res2 <- raceid.clustering(expr, diff.gene$data[[2]], cda, diff.gene$method[2])
race.res3 <- raceid.clustering(expr, diff.gene$data[[3]], cda, diff.gene$method[3])
race.res4 <- raceid.clustering(expr, diff.gene$data[[4]], cda, diff.gene$method[4])
race.res5 <- raceid.clustering(expr, diff.gene$data[[5]], cda, diff.gene$method[5])
race.res6 <- raceid.clustering(expr, diff.gene$data[[6]], cda, diff.gene$method[6])
race.res7 <- raceid.clustering(expr, diff.gene$data[[7]], cda, diff.gene$method[7])

race.res <- rbind(race.res1,race.res2,race.res3,race.res4,
      race.res5,race.res6,race.res7)

race.res %>% readr::write_rds(file.path(out.path,"02.race.res.rds"))

race.res %>%
  ggplot(aes(Number, ARI)) +
  geom_line(aes(colour = method), lwd = 0.8) +
  geom_point(aes(colour = method), size = 2.5) +
  theme_bw() +
  scale_colour_d3() +
  pauling.theme(angle = 0) -> p
p

fig.path <- "/home/pauling/projects/04_SEmodel/07_NC_revision/02.figures/02.clustering/01.3cellline.celseq2"
ggsave(plot = p, filename = "02.raceid3.pdf", path = fig.path, width = 6, height = 4)

SC3 Clustering

sce <- SingleCellExperiment(
    assays = list(
        counts = as.matrix(expr),
        logcounts = log2(as.matrix(expr) + 1)
    )
)
rowData(sce)$feature_symbol <- rownames(sce)

sc3.clustering <- function(sce, var.gene, cda, method = ""){
  GeneNumber <- c(100,500,1000,1500,2000)
  res <- c()
  for (i in 1:length(GeneNumber)) {
    N <- GeneNumber[i]
    sc <- sce[as.character(var.gene$Gene[1:N]),]
    sc <- sc3(sc, ks = 3, biology = F, gene_filter = F)
    ari <- mclust::adjustedRandIndex(sc$sc3_3_clusters, cda$label)
    tmp <- tibble(ARI = ari, method = method, Number = N)
    res <- rbind(res,tmp)
  }
  return(res)
}

sc3.res1 <- sc3.clustering(sce, diff.gene$data[[1]], cda, diff.gene$method[1])
sc3.res2 <- sc3.clustering(sce, diff.gene$data[[2]], cda, diff.gene$method[2])
sc3.res3 <- sc3.clustering(sce, diff.gene$data[[3]], cda, diff.gene$method[3])
sc3.res4 <- sc3.clustering(sce, diff.gene$data[[4]], cda, diff.gene$method[4])
sc3.res5 <- sc3.clustering(sce, diff.gene$data[[5]], cda, diff.gene$method[5])
sc3.res6 <- sc3.clustering(sce, diff.gene$data[[6]], cda, diff.gene$method[6])
sc3.res7 <- sc3.clustering(sce, diff.gene$data[[7]], cda, diff.gene$method[7])

sc3.res <- rbind(sc3.res1,sc3.res2,sc3.res3,sc3.res4,sc3.res5,sc3.res6,sc3.res7)

sc3.res %>% readr::write_rds(file.path(out.path,"02.sc3.res.rds"))

sc3.res %>%
  ggplot(aes(Number, ARI)) +
  geom_line(aes(colour = method), lwd = 0.8) +
  geom_point(aes(colour = method), size = 2.5) +
  theme_bw() +
  scale_colour_d3() +
  pauling.theme(angle = 0) +
  ylim(0.9,0.98) -> p
p
ggsave(plot = p, filename = "02.sc3.pdf", path = fig.path, width = 6, height = 4)

Seurat Clustering

Cal.ARI.silh <- function(.x, .y, method = ""){

  ari <- mclust::adjustedRandIndex(Idents(.x), .y$label)
  tibble(ARI = ari, method = method)

}

get.ari.sl <- function(.x, DiffGene, cda, resolution, gene.numbers, method, n.cluster){
  ari.sl.res <- c()
  for (i in 1:length(gene.numbers)) {
    sce.tmp <- RunPCA(.x, features = DiffGene$Gene[1:gene.numbers[i]])
    sce.tmp <- FindNeighbors(sce.tmp, dims = 1:20)
    sce.tmp <- FindClusters(sce.tmp, resolution = resolution)
    if(length(levels(Idents(sce.tmp))) == n.cluster){
      ari.sl <- Cal.ARI.silh(sce.tmp, cda, method) %>% 
        dplyr::mutate(Number = gene.numbers[i])

      ari.sl.res <- rbind(ari.sl.res,ari.sl)
    }
  }
  return(ari.sl.res)
}

sce <- CreateSeuratObject(counts = expr)
sce <- NormalizeData(sce, normalization.method = "LogNormalize", scale.factor = 10000)
all.genes <- rownames(sce)
sce <- ScaleData(sce, features = all.genes, do.center = F, do.scale = F)

sce.se <- RunPCA(sce, features = diff.gene$data[[1]]$Gene[1:30])
sce.se <- FindNeighbors(sce.se, dims = 1:20)
sce.se <- RunUMAP(sce.se, dims = 1:20)
sce.se <- FindClusters(sce.se, resolution = 0.2)
DimPlot(sce.se, reduction = "umap")

GeneNumer <- c(100,500,1000,1500,2000)
se.ari.sl <- get.ari.sl(sce, diff.gene$data[[1]], cda, 0.2, GeneNumer, "SE", 3)

sce.M3Drop <- RunPCA(sce, features = diff.gene$data[[2]]$Gene[1:30])
sce.M3Drop <- FindNeighbors(sce.M3Drop, dims = 1:20)
sce.M3Drop <- FindClusters(sce.M3Drop, resolution = 0.8)
sce.M3Drop <- RunUMAP(sce.M3Drop, dims = 1:20)
DimPlot(sce.M3Drop)

se.ari.M3D <- get.ari.sl(sce, diff.gene$data[[2]], cda, 0.8, GeneNumer, "M3Drop", 3)

sce.gini <- RunPCA(sce, features = diff.gene$data[[3]]$Gene[1:30])
sce.gini <- FindNeighbors(sce.gini, dims = 1:20)
sce.gini <- FindClusters(sce.gini, resolution = 0.2)
sce.gini <- RunUMAP(sce.gini, dims = 1:20)
DimPlot(sce.gini, reduction = "umap")

se.ari.gini <- get.ari.sl(sce, diff.gene$data[[3]], cda, 0.2, GeneNumer, "Gini",3)

sce.hvg <- RunPCA(sce, features = diff.gene$data[[4]]$Gene[1:100])
sce.hvg <- FindNeighbors(sce.hvg, dims = 1:20)
sce.hvg <- RunUMAP(sce.hvg, dims = 1:20)
sce.hvg <- FindClusters(sce.hvg, resolution = 1)
DimPlot(sce.hvg, reduction = "umap")

se.ari.hvg <- get.ari.sl(sce, diff.gene$data[[4]], cda, 1, GeneNumer, "HVG",3)

sce.sct <- RunPCA(sce, features = diff.gene$data[[5]]$Gene[1:100])
sce.sct <- FindNeighbors(sce.sct, dims = 1:20)
sce.sct <- RunUMAP(sce.sct, dims = 1:20)
sce.sct <- FindClusters(sce.sct, resolution = 0.2)
DimPlot(sce.sct, reduction = "umap")

se.ari.sct <- get.ari.sl(sce, diff.gene$data[[5]], cda, 0.2, GeneNumer, "SCT", 3)

sce.fano <- RunPCA(sce, features = diff.gene$data[[6]]$Gene[1:100])
sce.fano <- FindNeighbors(sce.fano, dims = 1:20)
sce.fano <- FindClusters(sce.fano, resolution = 0.2)
sce.fano <- RunUMAP(sce.fano, dims = 1:20)
DimPlot(sce.fano, reduction = "umap")

se.ari.fano <- get.ari.sl(sce, diff.gene$data[[6]], cda, 0.2, GeneNumer, "Fano", 3)

sce.race <- RunPCA(sce, features = diff.gene$data[[7]]$Gene[1:100])
sce.race <- FindNeighbors(sce.race, dims = 1:20)
sce.race <- FindClusters(sce.race, resolution = 0.2)
sce.race <- RunUMAP(sce.race, dims = 1:20)
DimPlot(sce.race, reduction = "umap")

se.ari.race <- get.ari.sl(sce, diff.gene$data[[7]], cda, 0.2, GeneNumer, "RaceID",3)

pauling.theme <- function(poi = "right", size = 12, title.size = 15, angle){
  theme(
    legend.position = poi,
    axis.title = element_text(size = title.size,color="black"),
    axis.text = element_text(size = size,color="black"),
    legend.title = element_text(size = 12),
    legend.text = element_text(size = 12),
    axis.text.y = element_text(color="black"),
    axis.text.x = element_text(color="black", angle = angle, hjust = 1))
}

seurat.res <- rbind(se.ari.sl, se.ari.gini, se.ari.fano, se.ari.sct, 
                    se.ari.race, se.ari.M3D, se.ari.hvg)

seurat.res %>% readr::write_rds(file.path(out.path,"02.seurat.res.rds"))

seurat.res %>% 
  ggplot(aes(Number, ARI)) +
  geom_line(aes(colour = method), lwd = 0.8) +
  geom_point(aes(colour = method), size = 2.5) +
  theme_bw() +
  scale_colour_d3() +
  pauling.theme(angle = 0) -> p
p
ggsave(plot = p, filename = "02..seurat.pdf", path = fig.path, width = 6, height = 4)