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DE.benchmark.Reproduciblity.Rmd
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---
title: "Reproduciblity technical replicates"
output: html_notebook
---
```{r}
library(SingleCellExperiment);library(tidyverse);library(reticulate);library(tidyverse)
library(ggplot2);library(scmap);library(fmsb);library(ggsci);library(scibet)
library(Seurat);library(M3Drop);library(ROCR);library(cluster);library(parallel);library(ROGUE)
```
```{r}
pro_10x <- function(.x){
matr <- Read10X(.x)
gene <- readr::read_rds("/home/pauling/projects/02_data/09_Gene/coding_gene.rds.gz")
over_gene <- intersect(gene$gene_name, rownames(matr))
matr <- matr[over_gene,]
matr <- as.matrix(matr)
matr <- t(matr)
return(matr)
}
pro_10x_mouse <- function(.x){
matr <- Read10X(.x)
gene <- readr::read_rds("/home/pauling/projects/02_data/09_Gene/mouse/protein_coding.rds.gz")
over_gene <- intersect(gene$gene_name, rownames(matr))
matr <- matr[over_gene,]
matr <- as.matrix(matr)
matr <- t(matr)
return(matr)
}
m3d_fun <- function(expr){
expr <- as.matrix(expr)
expr <- t(expr)
norm <- M3DropConvertData(expr, is.counts=TRUE)
DEgenes <- M3Drop::M3DropFeatureSelection(norm, suppress.plot = T, mt_threshold = 2)
DEgenes <- DEgenes %>%
dplyr::arrange(p.value)
return(DEgenes)
}
HVG_fun <- function(expr){
expr <- t(expr)
hvg.res <- BrenneckeGetVariableGenes(expr, suppress.plot = T, fdr = 2)
hvg.res <- hvg.res %>% dplyr::arrange(p.value)
return(hvg.res)
}
Gini_fun <- function(expr){
calcul.gini = function(x, unbiased = TRUE, na.rm = FALSE){
if (!is.numeric(x)){
warning("'x' is not numeric; returning NA")
return(NA)
}
if (!na.rm && any(na.ind = is.na(x)))
stop("'x' contain NAs")
if (na.rm)
x = x[!na.ind]
n = length(x)
mu = mean(x)
N = if (unbiased) n * (n - 1) else n * n
ox = x[order(x)]
dsum = drop(crossprod(2 * 1:n - n - 1, ox))
dsum / (mu * N)
}
expr <- t(expr)
ExprM.RawCounts <- expr
minCellNum = 0
minGeneNum = 0
expressed_cutoff = 1
gini.bi = 0
log2.expr.cutoffl = 0
log2.expr.cutoffh = 30
Gini.pvalue_cutoff = 0.0001
Norm.Gini.cutoff = 1
span = 0.9
outlier_remove = 0.75
GeneList = 1
Gamma = 0.9
diff.cutoff = 1
lr.p_value_cutoff = 1e-5
CountsForNormalized = 100000
ExpressedinCell_per_gene=apply(ExprM.RawCounts,1,function(x) length(x[x > expressed_cutoff ]))
nonMir = grep("MIR|Mir", rownames(ExprM.RawCounts), invert = T) # because Mir gene is usually not accurate
Genelist = intersect(rownames(ExprM.RawCounts)[nonMir],rownames(ExprM.RawCounts)[ExpressedinCell_per_gene >= minCellNum])
ExpressedGene_per_cell=apply(ExprM.RawCounts[Genelist,],2,function(x) length(x[x>0]))
ExprM.RawCounts.filter = ExprM.RawCounts[Genelist,ExpressedGene_per_cell >= 0]
if(gini.bi==0){
gini = apply(as.data.frame(ExprM.RawCounts.filter), 1, function(x){calcul.gini(as.numeric(x)) } ) #theoretically, gini have very low chance to have a 1 value
GiniIndex = as.data.frame(cbind(1:dim(ExprM.RawCounts.filter)[1], gini))
} else {
GiniIndex1 <- as.data.frame(apply(ExprM.RawCounts.filter, 1, function(x){calcul.gini(as.numeric(x)) } ) )
GiniIndex2 <- as.data.frame(apply(ExprM.RawCounts.filter+0.00001, 1, function(x){calcul.gini(as.numeric(1/x)) } ) ) #bi directional
GiniIndex <- cbind(GiniIndex1, GiniIndex2)
colnames(GiniIndex)=c("gini1","gini2")
GiniIndex$gini2_sign = 0 - GiniIndex$gini2;
GiniIndex$gini = apply(GiniIndex, 1, max)
GiniIndex <- na.omit(GiniIndex)
GiniIndex$gini_sign = GiniIndex$gini
for(genei in 1:dim(GiniIndex)[1])
{
GiniIndex[genei, 5] = ifelse( GiniIndex[genei, 1] > GiniIndex[genei,2], "up-regulation", "down-regulation")
}
}
Maxs = apply(ExprM.RawCounts.filter,1,max)
Means = apply(ExprM.RawCounts.filter,1,mean)
log2.Maxs = log2(Maxs+0.1)
ExprM.Stat1 = as.data.frame(cbind(Maxs,GiniIndex$gini,log2.Maxs))
colnames(ExprM.Stat1) = c("Maxs","Gini","log2.Maxs")
ExprM.Stat1 = ExprM.Stat1[ExprM.Stat1$log2.Maxs>log2.expr.cutoffl & ExprM.Stat1$log2.Maxs<=log2.expr.cutoffh ,] # is this necessary?
log2.Maxs = ExprM.Stat1$log2.Maxs
Gini = ExprM.Stat1$Gini
Maxs = ExprM.Stat1$Maxs
# .3 fitting in max-gini space
Gini.loess.fit = loess(Gini~log2.Maxs, span=span, degree=1)
Normlized.Gini.Score = Gini.loess.fit$residuals #residuals = Gini - Gini.fitted
Gini.fitted = Gini.loess.fit$fitted
ExprM.Stat1 = as.data.frame(cbind(ExprM.Stat1[,c("Maxs","Gini", "log2.Maxs")], Normlized.Gini.Score, Gini.fitted))
colnames(ExprM.Stat1) = c("Maxs","Gini","log2.Maxs", "Norm.Gini", "Gini.fitted")
### remove 25% of first round outlier genes, do second round loess
Gini.loess.fit.residual = residuals(Gini.loess.fit)
thresh.outlier = quantile(Gini.loess.fit.residual[Gini.loess.fit.residual>0], outlier_remove)
id.genes.loess.fit = which(Gini.loess.fit.residual < thresh.outlier)
id.outliers.loess.fit = which(Gini.loess.fit.residual >= thresh.outlier)
log2.Maxs.genes = log2.Maxs[id.genes.loess.fit]
log2.Maxs.outliers = log2.Maxs[id.outliers.loess.fit]
Gini.loess.fit.2 = loess(Gini[id.genes.loess.fit]~log2.Maxs[id.genes.loess.fit], span=span, degree = 1)
Gini.loess.fit.2.predict = predict(Gini.loess.fit.2)
Gini.loess.fit.2.x.y = cbind(log2.Maxs.genes,Gini.loess.fit.2.predict)
Gini.loess.fit.2.x.y.uniq = as.data.frame(unique(Gini.loess.fit.2.x.y))
Gini.loess.fit.2.x.y.uniq = Gini.loess.fit.2.x.y.uniq[order(Gini.loess.fit.2.x.y.uniq[,1]),]
log2.Maxs.genes.sorted = log2.Maxs.genes[order(log2.Maxs.genes)]
Gini.loess.fit.2.predict.sorted = Gini.loess.fit.2.predict[order(log2.Maxs.genes)]
#using Gini.loess.fit.2 as model, predict gini value for those outlier which are not used for build model.
#for each max in outliers set, find the id of max value which is most close in fitted data set
loc.outliers = apply(matrix(log2.Maxs.outliers),1,function(x){
if(x<max(log2.Maxs.genes.sorted)){
return(which(log2.Maxs.genes.sorted>=x)[1])
}else{
return(which.max(log2.Maxs.genes.sorted))
}})
#check the results
outlier_max_in_fit <- cbind(log2.Maxs.outliers, loc.outliers, log2.Maxs.genes.sorted[loc.outliers])
#based on Gini.loess.fit.2, predict outliers which was not used for fitting
Gini.outliers.predict = apply(cbind(seq(length(log2.Maxs.outliers)),log2.Maxs.outliers),1,function(x){
id = x[1]
value = x[2]
if(value == log2.Maxs.genes.sorted[loc.outliers[id]]){
return(as.numeric(Gini.loess.fit.2.x.y.uniq[which(Gini.loess.fit.2.x.y.uniq$log2.Maxs.genes>=value)[1],2]))
}else{
if(loc.outliers[id]>1){
return(Gini.loess.fit.2.predict.sorted[loc.outliers[id]-1]+(Gini.loess.fit.2.predict.sorted[loc.outliers[id]]-Gini.loess.fit.2.predict.sorted[loc.outliers[id]-1])*(value-log2.Maxs.genes.sorted[loc.outliers[id]-1])/(log2.Maxs.genes.sorted[loc.outliers[id]]-log2.Maxs.genes.sorted[loc.outliers[id]-1]))
}else{
return(Gini.loess.fit.2.predict.sorted[2]-(Gini.loess.fit.2.predict.sorted[2]-Gini.loess.fit.2.predict.sorted[1])*(log2.Maxs.genes.sorted[2]-value)/(log2.Maxs.genes.sorted[2]-log2.Maxs.genes.sorted[1]))
}
}
})
#plot outliers predict results
outliers.precit.x.y.uniq = as.data.frame(unique(cbind(log2.Maxs.outliers, Gini.outliers.predict)))
#plot(outliers.precit.x.y.uniq)
#plot whole fit2
colnames(outliers.precit.x.y.uniq) = colnames(Gini.loess.fit.2.x.y.uniq)
Gini.loess.fit.2.full.x.y.uniq = rbind(Gini.loess.fit.2.x.y.uniq, outliers.precit.x.y.uniq)
#plot(Gini.loess.fit.2.full.x.y.uniq)
#calcualte Normlized.Gini.Score2
Normlized.Gini.Score2 = rep(0,length(Gini.loess.fit.residual))
Normlized.Gini.Score2[id.genes.loess.fit] = residuals(Gini.loess.fit.2)
Normlized.Gini.Score2[id.outliers.loess.fit] = Gini[id.outliers.loess.fit] - Gini.outliers.predict
Gini.fitted2 = Gini - Normlized.Gini.Score2
ExprM.Stat1 = as.data.frame(cbind(ExprM.Stat1[,c("Maxs","Gini", "log2.Maxs", "Gini.fitted", "Norm.Gini" )], Gini.fitted2, Normlized.Gini.Score2))
colnames(ExprM.Stat1) = c("Maxs","Gini","log2.Maxs", "Gini.fitted","Norm.Gini", "Gini.fitted2", "Norm.Gini2")
Gini.pvalue = pnorm(-abs(scale(ExprM.Stat1$Norm.Gini2, center=TRUE,scale=TRUE)))
ExprM.Stat2 = cbind(ExprM.Stat1, Gini.pvalue) #first time use ExprM.Stat2
colnames(ExprM.Stat2) = c("Maxs","Gini","log2.Maxs", "Gini.fitted","Norm.Gini", "Gini.fitted2", "Norm.Gini2", "p.value")
ExprM.Stat2 %>%
tibble::rownames_to_column(var = 'Gene') %>%
dplyr::arrange(p.value)
}
sct_fun <- function(expr){
expr <- t(expr)
colnames(expr) <- paste0("Cell",1:ncol(expr))
sce <- CreateSeuratObject(counts = expr)
sce <- Seurat::SCTransform(sce, verbose = FALSE, do.center = F, variable.features.n = nrow(sce))
sce@[email protected] %>%
tibble::rownames_to_column(var = "Gene") %>%
dplyr::arrange(desc(residual_variance)) -> sct.res
sct.res <- sct.res %>%
dplyr::mutate(
p.value = 1-pnorm(
sct.res$residual_variance,
mean = mean(sct.res$residual_variance),
sd = sd(sct.res$residual_variance)))
return(sct.res)
}
FanoFactor_fun <- function(expr){
#calculate Fano factor
Fano <- apply(expr,2,function(x) var(x)/mean(x))
Fano <- Fano %>%
as.data.frame() %>%
tibble::rownames_to_column(var = "Gene")
colnames(Fano) <- c("Gene","fano")
Fano <- Fano %>% dplyr::filter(!is.na(fano)) %>% dplyr::arrange(desc(fano))
Fano <- Fano %>%
dplyr::mutate(
p.value = 1-pnorm(
Fano$fano,
mean = mean(Fano$fano),
sd = sd(Fano$fano)))
return(Fano)
}
raceid_fun <- function(x, mthr = -1){
uvar <- function(x,fit){
err <- coef(summary(fit))[, "Std. Error"]
2**(coef(fit)[1] + err[1] + log2(x)*(coef(fit)[2] + err[2]) + (coef(fit)[3] + err[3]) * log2(x)**2)
}
x <- t(x)
m <- apply(x, 1, mean)
v <- apply(x, 1, var)
ml <- log2(m)
vl <- log2(v)
f <- ml > -Inf & vl > -Inf
ml <- ml[f]
vl <- vl[f]
mm <- -8
repeat {
fit <- lm(vl ~ ml + I(ml^2))
if (coef(fit)[3] >= 0 | mm >= mthr) {
break
}
mm <- mm + 0.5
f <- ml > mm
ml <- ml[f]
vl <- vl[f]
}
vln <- log2(v) - log2(sapply(m, FUN = uvar, fit = fit))
tibble(
Gene = names(vln),
vln = vln
) %>%
dplyr::filter(!is.na(vln)) %>%
dplyr::arrange(desc(vln)) -> race.res
race.res <- race.res %>%
dplyr::mutate(
p.value = 1-pnorm(
race.res$vln,
mean = mean(race.res$vln),
sd = sd(race.res$vln)))
return(race.res)
}
coord_radar <- function (theta = "x", start = 0, direction = 1) {
theta <- match.arg(theta, c("x", "y"))
r <- if (theta == "x") "y" else "x"
ggproto(NULL, CoordPolar, theta = theta, r = r, start = start,
direction = sign(direction),
expand = F,
is_linear = function(coord) TRUE)
}
pauling.theme <- function(poi = "right", size = 12, title.size = 15){
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 = size),
legend.text = element_text(size = size),
axis.text.y = element_text(color="black"),
axis.text.x = element_text(color="black"))
}
```
```{r}
#GSE65525 indrop mESC
mda1 <- readr::read_rds("/home/pauling/projects/04_SEmodel/01_data/05_Gene_Selection_Benchmark/02_Reproducibility/mESC_replicates/mda.rds.gz")
```
#PBMC 1k
```{r}
path_1 <- "/home/pauling/projects/04_SEmodel/01_data/05_Gene_Selection_Benchmark/02_Reproducibility/02_pbmc/V3_matrix"
path_2 <- "/home/pauling/projects/04_SEmodel/01_data/05_Gene_Selection_Benchmark/02_Reproducibility/02_pbmc/V3_2_matrix/"
matr1 <- pro_10x(path_1)
matr2 <- pro_10x(path_2)
```
#brain
```{r}
path_1 <- "/home/pauling/projects/04_SEmodel/01_data/05_Gene_Selection_Benchmark/02_Reproducibility/03_brain/brain_1k/"
path_2 <- "/home/pauling/projects/04_SEmodel/01_data/05_Gene_Selection_Benchmark/02_Reproducibility/03_brain/brain_10k/"
brain.matr1 <- pro_10x_mouse(path_1)
brain.matr2 <- pro_10x_mouse(path_2)
```
#heart
```{r}
path_1 <- "/home/pauling/projects/04_SEmodel/01_data/05_Gene_Selection_Benchmark/02_Reproducibility/04_heart/heart_1k/"
path_2 <- "/home/pauling/projects/04_SEmodel/01_data/05_Gene_Selection_Benchmark/02_Reproducibility/04_heart/heart_10k/"
heart.matr1 <- pro_10x_mouse(path_1)
heart.matr2 <- pro_10x_mouse(path_2)
```
```{r}
tibble(
tissue = c("mESC","mESC","PBMC","PBMC","brian","brain","heart","heart"),
expr = list(mda1$matr[[1]], mda1$matr[[2]], matr1, matr2, brain.matr1, brain.matr2, heart.matr1, heart.matr2)
) -> cda
cda %>%
dplyr::mutate(
expr = purrr::map(
.x = expr,
.f = function(.x){
.x <- matr.filter(.x)
.x <- as.matrix(.x)
return(.x)
}
)
) -> cda
cda %>%
dplyr::mutate(
res = purrr::map(
.x = expr,
.f = function(.x){
Res1 <- SE_fun(t(.x), span = 0.1)
m1 <- m3d_fun(.x)
HVG1 <- HVG_fun(.x)
G1 <- Gini_fun(.x)
sct <- sct_fun(.x)
fano.f <- FanoFactor_fun(.x)
race.res <- raceid_fun(.x)
list(Res1, m1, HVG1, G1, sct, fano.f, race.res)
}
)
) -> cda
cda <- readr::read_rds("/home/pauling/projects/04_SEmodel/05_data_phase2/01.gene.selection.benchmark/04.reproducibility/cda.rds.gz")
```
```{r}
tissue <- c("mESC","PBMC","brian","heart")
out.path <- "/home/pauling/projects/04_SEmodel/04_figures/04_DE.benchmark/01.DE.benchmark/03.Reproducibility"
for (i in 1:4) {
tibble(
ID = rep(c(1:7),4),
gene_num = c(rep(500,7),rep(1000,7),rep(1500,7),rep(2000,7)),
method = rep(c("SE","M3Drop","HVG","Gini","SCT","Fano","RaceID"),4)
) %>%
dplyr::mutate(
Reproducibility = purrr::map2_dbl(
.x = ID,
.y = gene_num,
.f = function(.x, .y){
if(.x == 3){
cda$res[[2*i-1]][[.x]] <- cda$res[[2*i-1]][[.x]] %>% dplyr::arrange(p.value)
cda$res[[2*i]][[.x]] <- cda$res[[2*i]][[.x]] %>% dplyr::arrange(p.value)
}
round(length(intersect(cda$res[[2*i-1]][[.x]]$Gene[1:.y],cda$res[[2*i]][[.x]]$Gene[1:.y]))/.y,2)*100
}
)
) -> res1
res1 %>%
ggplot(aes(factor(method, levels = c("SE","M3Drop","HVG","Gini","SCT","Fano","RaceID")), Reproducibility)) +
geom_polygon(aes(colour = factor(gene_num), group = factor(gene_num)), fill = NA, lwd = 0.8) +
geom_point(aes(colour = factor(gene_num), fill = factor(gene_num)), shape = 21, alpha = 0.5, size = 3) +
coord_radar() +
theme_minimal() +
theme(axis.text = element_text(size = 12, colour = "black"),
axis.title = element_text(size = 13, colour = "black"),
) +
labs(
x = ""
) +
ylim(0, NA) -> p
ggsave(plot = p, filename = paste0(tissue[i],".pdf"), path = out.path, width = 6, height = 4)
}
```
```{r, fig.width=6, fig.height=4}
tissue <- c("mESC","PBMC","brian","heart")
out.path <- "/home/pauling/projects/04_SEmodel/07_NC_revision/02.figures/04.reproducibility"
for (i in 1:4) {
tibble(
ID = rep(c(1:7),4),
gene_num = c(rep(500,7),rep(1000,7),rep(1500,7),rep(2000,7)),
method = rep(c("SE","M3Drop","HVG","Gini","SCT","Fano","RaceID"),4)
) %>%
dplyr::mutate(
Reproducibility = purrr::map2_dbl(
.x = ID,
.y = gene_num,
.f = function(.x, .y){
if(.x == 3){
cda$res[[2*i-1]][[.x]] <- cda$res[[2*i-1]][[.x]] %>% dplyr::arrange(p.value)
cda$res[[2*i]][[.x]] <- cda$res[[2*i]][[.x]] %>% dplyr::arrange(p.value)
}
round(length(intersect(cda$res[[2*i-1]][[.x]]$Gene[1:.y],cda$res[[2*i]][[.x]]$Gene[1:.y]))/.y,2)
}
)
) -> res1
res1 %>%
ggplot(aes(gene_num, Reproducibility)) +
geom_point(aes(colour = method), size = 2) +
geom_line(aes(colour = method), lwd = 0.6) +
theme_classic() +
pauling.theme(size = 12, title.size = 13) +
scale_color_d3() +
labs(
x = "Number of genes",
y = "Reproducibility"
) -> p
ggsave(plot = p, filename = paste0(tissue[i],".pdf"), path = out.path, width = 5, height = 3)
}
```
```{r, fig.width=5, fig.height=3}
i <- 3
tibble(
ID = rep(c(1:7),4),
gene_num = c(rep(500,7),rep(1000,7),rep(1500,7),rep(2000,7)),
method = rep(c("SE","M3Drop","HVG","Gini","SCT","Fano","RaceID"),4)
) %>%
dplyr::mutate(
Reproducibility = purrr::map2_dbl(
.x = ID,
.y = gene_num,
.f = function(.x, .y){
if(.x == 3){
cda$res[[2*i-1]][[.x]] <- cda$res[[2*i-1]][[.x]] %>% dplyr::arrange(p.value)
cda$res[[2*i]][[.x]] <- cda$res[[2*i]][[.x]] %>% dplyr::arrange(p.value)
}
round(length(intersect(cda$res[[2*i-1]][[.x]]$Gene[1:.y],cda$res[[2*i]][[.x]]$Gene[1:.y]))/.y,2)*100
}
)
) -> res1
res1 %>%
ggplot(aes(gene_num, Reproducibility)) +
geom_point(aes(colour = method), size = 2) +
geom_line(aes(colour = method), lwd = 0.6) +
theme_classic() +
pauling.theme(size = 12, title.size = 13) +
scale_color_d3() +
labs(
x = "Number of genes",
y = "Reproducibility (%)"
)
```