examples/code/lung_Treutlein2014/2_Treutlein2014_runBioTP.R
In xyang2uchicago/BioTIP: BioTIP: An R package for characterization of Biological Tipping-Point
#### README ############
## There are 4 sections in this code
## Section 1) apply BioTIP to different clusters of 131 cells by different methods
## also apply BioTIP to different clusters of 196 cells by cell collection time (age), marker-based clusters, and unsupervised clusters
## section 2) quantify the robustness
## section 3) reporting table, compare the results of BioTIP to the resutls of QuanTC
## section 4) stability of BioTIP's identification
## section 5) TSNE plot for 3.2k HGV, 131 cells along the AT2 trajectory
## last update 2/8/2022
## by Holly Yang ##############
setwd('F:/projects/BioTIP/result/GSE52583/BioTIP_GSE52583_robustness/')
# the following funciton has been updated on github, but before updating the package to Bioconductor, you need to call it locally to test
source(file='F:/projects/scRNA/source/GSE87038_gastrulation/ForJennifer/optimize.sd_selection.R')
source(file='F:/projects/scRNA/source/GSE87038_gastrulation/GSE87038_git_copy/BioTIP.wrap.R')
source(file='F:/projects/scRNA/source/GSE87038_gastrulation/GSE87038_git_copy/Stability_score.R')
# normalized data were downloaded and reanalyzed
# refer to GSE87038_E8.25_normalize.R
library(BioTIP)
library(dplyr)
library(SingleCellExperiment)
library(ggplot2)
library(gridExtra) # required by grid.arrange()
library(ggpubr) # required by p-values on bar
###################################################
## load the R object ##
## focusing on 131 cells along the AT2 trajectory
###################################################
j="AT2.sce.RData"
if(j=="AT2.sce.RData") load('AT2.sce.RData') else load('sce.RData') #!!!!!!!!!!!!!!!
# format assayNames to run BioTIP
assayNames(sce) <- 'logcounts'
sce
#class: SingleCellExperiment
# dim: 3198 131
# metadata(0):
# assays(1): logcounts
# rownames(3198): 0610007C21Rik 0610007L01Rik ... Zyx l7Rn6
# rowData names(11): ensembl_gene_id gene_short_name ...
# num_cells_expressed use_for_ordering
# colnames(131): E18_1_C08 E18_1_C09 ... E16_1_C94 E16_1_C95
# colData names(17): age cellName ... C_SNNGraph_k8 C_Soft
# reducedDimNames(2): PCA TSNE
# altExpNames(0):
# logmat <- as.matrix(assays(sce)$logcounts)
# M <- cor.shrink(logmat, Y = NULL, MARGIN = 1, shrink = TRUE)
# if(j=="AT2.sce.RData") save(M, file="AT2_ShrinkM.RData", compress=TRUE) else save(M, file="ShrinkM.RData", compress=TRUE)
# dim(M) #3198 3198
# rm(logmat)
if(j=="AT2.sce.RData") load(file="AT2_ShrinkM.RData") else load(file="ShrinkM.RData")
dim(M) #3198 3198
####################################################################
## section 1) running BioTIP on different cell clustering outputs ##
####################################################################
{
######### 1.1) setting parameters, ######################
localHVG.preselect.cut = 0.1 # A positive numeric value setting how many propotion of global HVG to be selected per cell cluster
getNetwork.cut.fdr = 0.2 # to construct RW network and extract co-expressed gene moduels
getTopMCI.gene.minsize = 30 # min number of genes in an identified CTS
getTopMCI.n.states = 2 # A number setting the number of states to check the significance of DNB score (i.e. MCI)
# This parameter can be enlarge when inputting more clusters, usually the expected number of transition states +1
MCIbottom = 2 # A number setting the threshold to prioritize the initially selected CTS candidates.
# In our experiment, a number between 2 and 4 generated expected resutls.
############### 1.2) applying BioTIP to all new clustering outputs ##################
x <- grep("C_", colnames(colData(sce)))
colnames(colData(sce))[x]
# [1] "C_by_marker" "C_Monocle.k4" "C_consensus_ks3" "C_consensus_ks5"
# [5] "C_consensus_ks7" "C_Leiden_0.4" "C_Leiden_0.8" "C_Leiden_1.2"
# [9] "C_SNNGraph_k10" "C_SNNGraph_k8" "C_Soft"
set.seed(102020)
for(i in 1:length(x)){
samplesL <- split(rownames(colData(sce)), f = colData(sce)[x[i]])
(tmp = lengths(samplesL))
#outputpath = paste0(colnames(colData(sce))[x[i]],'/BioTIP_top',localHVG.preselect.cut,'FDR',getNetwork.cut.fdr,"_")
#load(file=paste0(outputpath,"optimized_local.HVG_selection.RData"))
res <- BioTIP.wrap(sce, samplesL, subDir=colnames(colData(sce))[x[i]],
getTopMCI.n.states=getTopMCI.n.states,
getTopMCI.gene.minsize=getTopMCI.gene.minsize,
MCIbottom=MCIbottom,
localHVG.preselect.cut=localHVG.preselect.cut, #logmat.local.HVG.testres=testres,
getNetwork.cut.fdr=getNetwork.cut.fdr,
M=M,
permutation.method='both',
empirical.IC.p.cut=0.1, ## when permutating both gene and sampels toestimate the Ic.shrink's significance,
## we can loose this creterial to get more meanfuling results
verbose=TRUE, plot=TRUE)
save(res, file=paste0(colnames(colData(sce))[x[i]],'/BioTIP.res.RData'))
}
## 1.3) Additional run for soft thresholding without TC
samplesL <- split(rownames(colData(sce)), f = colData(sce)$C_Soft)
(tmp = lengths(samplesL))
# C1 C2 TC
# 31 43 57
samplesL = samplesL[1:(length(samplesL)-1)]
# outputpath = paste0('C_Soft.wo.TC/BioTIP_top',localHVG.preselect.cut,'FDR',getNetwork.cut.fdr,"_")
# load(file=paste0(outputpath,"optimized_local.HVG_selection.RData"))
res <- BioTIP.wrap(sce, samplesL, subDir='C_Soft.wo.TC',
getTopMCI.n.states=getTopMCI.n.states,
getTopMCI.gene.minsize=getTopMCI.gene.minsize,
MCIbottom=MCIbottom,
localHVG.preselect.cut=localHVG.preselect.cut, #logmat.local.HVG.testres=testres,
getNetwork.cut.fdr=getNetwork.cut.fdr,
M=M,
permutation.method='both',
verbose=TRUE, plot=TRUE)
save(res, file='C_Soft.wo.TC/BioTIP.res.RData')
## 1.4) additional run for time-course
samplesL <- split(rownames(colData(sce)), f = colData(sce)$age)
(tmp = lengths(samplesL))
#14.5 16.5 18.5 Adult
#45 27 15 44
# outputpath = paste0('C_CollectionTime_allcells/BioTIP_top',localHVG.preselect.cut,'FDR',getNetwork.cut.fdr,"_")
# load(file=paste0(outputpath,"optimized_local.HVG_selection.RData"))
res <- BioTIP.wrap(sce, samplesL, subDir='C_CollectionTime',
getTopMCI.n.states=getTopMCI.n.states,
getTopMCI.gene.minsize=getTopMCI.gene.minsize,
MCIbottom=MCIbottom,
localHVG.preselect.cut=localHVG.preselect.cut, #logmat.local.HVG.testres=testres,
getNetwork.cut.fdr=getNetwork.cut.fdr,
M=M,
permutation.method='both',
verbose=TRUE, plot=TRUE)
save(res, file='C_CollectionTime/BioTIP.res.RData')
## 1.5) additional run for all collected time-course cells
load('sce.RData')
sce
# class: SingleCellExperiment
# dim: 3198 196
samplesL <- split(rownames(colData(sce)), f = colData(sce)$age)
(tmp = lengths(samplesL))
# 14.5 16.5 18.5 Adult
# 45 27 80 44
# format assayNames to run BioTIP
assayNames(sce) <- 'logcounts'
# outputpath = paste0('C_CollectionTime_allcells/BioTIP_top',localHVG.preselect.cut,'FDR',getNetwork.cut.fdr,"_")
# load(file=paste0(outputpath,"optimized_local.HVG_selection.RData"))
res <- BioTIP.wrap(sce, samplesL, subDir='C_CollectionTime_allcells',
getTopMCI.n.states=getTopMCI.n.states,
getTopMCI.gene.minsize=getTopMCI.gene.minsize,
MCIbottom=MCIbottom,
ocalHVG.preselect.cut=localHVG.preselect.cut, #logmat.local.HVG.testres=testres,
getNetwork.cut.fdr=getNetwork.cut.fdr,
M=M,
permutation.method='both',
verbose=TRUE, plot=TRUE)
save(res, file='C_CollectionTime_allcells/BioTIP.res.RData')
## 1.6) additionally run for all collected cells, clustered by gene markers
samplesL <- split(rownames(colData(sce)), f = colData(sce)$C_by_marker)
(tmp = lengths(samplesL))
# Ambiguous AT1 AT2 Unknown
# 6 63 77 50
# outputpath = paste0('C_by_marker_allcells/BioTIP_top',localHVG.preselect.cut,'FDR',getNetwork.cut.fdr,"_")
# load(file=paste0(outputpath,"optimized_local.HVG_selection.RData"))
res <- BioTIP.wrap(sce, samplesL, subDir='C_by_marker_allcells',
getTopMCI.n.states=getTopMCI.n.states,
getTopMCI.gene.minsize=getTopMCI.gene.minsize,
MCIbottom=MCIbottom,
localHVG.preselect.cut=localHVG.preselect.cut, # logmat.local.HVG.testres=testres,
getNetwork.cut.fdr=getNetwork.cut.fdr,
M=M,
permutation.method='both',
verbose=TRUE, plot=TRUE)
save(res, file='C_by_marker_allcells/BioTIP.res.RData')
## 1.7) copy QuanTC-identified transition genes
# BEGIN DO not repeat read and save QuanTC-predicted CTS genes ##
CTS <- list()
tmp <- read.table('../QuanTC_Output/AT2/transition_gene_name1.2.txt',
header=FALSE)#E14.5 in soft-thresholing clustering
CTS[['C1']] <- tmp[,1]
tmp <- read.table('../QuanTC_Output/AT2/transition_gene_name2.2.txt',
header=FALSE)#E16.5 in soft-thresholing clustering
CTS[['C3']] <- tmp[,1]
save(CTS, file='QuanTC_run/CTS.RData')
# END DO not repeat read and save QuanTC-predicted CTS genes ##
###################################################################
}
####################################################################
## section 2) quantify the robustness ##
####################################################################
i= "AT2.sce.RData"
if(j=="AT2.sce.RData") load('AT2.sce.RData') else load('sce.RData') #!!!!!!!!!!!!!!!
# format assayNames to run BioTIP
assayNames(sce) <- 'logcounts'
{
## 2. 1) Summarize the CT identifications
{
running.methods <- list.dirs()
running.methods <- running.methods[grepl('/C_',running.methods)]
methods <- lapply(running.methods, function(x) unlist(strsplit(x, "/C_"))[2]) %>% unlist()
x <- which(is.na(methods))
if(length(x)>0){
running.methods = running.methods[-x]
methods = methods[-x]
}
CT <- list()
for(i in 1:length(running.methods)){
load(file=paste0(running.methods[i],'/BioTIP.res.RData'))
if(length(res)>1) {
CT[[i]] <- res$significant[which(res$significant)] %>% names() %>% unique()
} else CT[[i]] <- NA
}
names(CT) <- paste0("C_", methods)
}
lapply(CT, toString) %>% unlist()
# C_by_marker C_by_marker_allcells C_CollectionTime
# "AT2" "" ""
# C_CollectionTime_allcells C_consensus_ks3 C_consensus_ks5
# "18.5" "1" "3"
# C_consensus_ks7 C_Leiden_0.4 C_Leiden_0.8
# "" "2" "NA"
# C_Leiden_1.2 C_Monocle.k4 C_SNNGraph_k10
# "NA" "2" ""
# C_SNNGraph_k8 C_Soft C_Soft.wo.TC
# "1" "" ""
## manually add the QuanTC's prediction about CT states,
CT$QuanTC_run <- c('C3','C1') # the soft-thresholding cluster IDs
## 2.2) Annotate cell identities by comparing to expected CT cluster ##
{
## load the QuanTC-identified transition cells
tmp <- read.table('../QuanTC_Output/AT2/C_TC.txt')
tmp <- tmp[,1]
table(tmp)
# 1 2 3 4 5
# 36 43 13 2 37
colData(sce)$QuanTC_run = tmp
## replace the QuanTC.cluster IDs
tmp <- data.frame(colData(sce))
tmp <- tmp %>% mutate(QuanTC_run = replace(QuanTC_run, QuanTC_run == 1, 'C1'))
tmp <- tmp %>% mutate(QuanTC_run = replace(QuanTC_run, QuanTC_run == 2, 'C2'))
tmp <- tmp %>% mutate(QuanTC_run = replace(QuanTC_run, QuanTC_run == 3, 'C3'))
tmp <- tmp %>% mutate(QuanTC_run = replace(QuanTC_run, QuanTC_run == 4, 'C4'))
tmp <- tmp %>% mutate(QuanTC_run = replace(QuanTC_run, QuanTC_run == 5, 'TC'))
colData(sce)$QuanTC_run <- tmp$QuanTC_run
colData(sce)$age_cellType <- paste(colData(sce)$age, colData(sce)$putative_cell_type, sep="_")
table(colData(sce)$QuanTC_run, colData(sce)$age_cellType)
# 14.5_NA 16.5_NA 18.5_AT2 18.5_BP 18.5_ciliated 18.5_Clara Adult_NA
# C1 36 0 0 0 0 0 0
# C2 0 0 0 0 0 0 43
# C3 0 13 0 0 0 0 0
# C4 0 0 1 1 0 0 0
# TC 9 14 10 0 1 2 1
colnames(colData(sce))
#
x <- grep("C_", colnames(colData(sce)))
colnames(colData(sce))[x]
best.matched.clusterID <- data.frame()
query = c('16.5_NA','18.5_AT2') # the age_cellType for bifurcation which are named
# as early HEP, later HEP, EP, HP, respectively
# among the studied clusters C3, C13, C10, C6, C15, C7
for(i in x){
tmp <- table(sce$age_cellType, colData(sce)[,i])
n1 <- nrow(tmp)
n2 <- ncol(tmp)
Jaccard_cell = tmp*0
for(a in 1:n1){
for(b in 1:n2){
Jaccard_cell[a,b] <- tmp[a,b]/(sum(tmp[a,])+sum(tmp[,b])-tmp[a,b])
}
}
best.match <- colnames(Jaccard_cell)[apply(Jaccard_cell, 1, which.max)]
names(best.match) <- rownames(Jaccard_cell)
best.matched.clusterID <- rbind(best.matched.clusterID,
best.match[query])
}
rownames(best.matched.clusterID) <- colnames(colData(sce))[x]
colnames(best.matched.clusterID) <- c('E16.5', 'E18.5_AT2')
## additionally recalculate without the TC among QuanTC's clusters
i= grep('Soft_k4', colnames(colData(sce))) # 7
tmp <- table(sce$age_cellType, colData(sce)[,i])
tmp = tmp[,1:(ncol(tmp)-1)] # masking TC
n1 <- nrow(tmp)
n2 <- ncol(tmp)
F1 = tmp*0
for(a in 1:n1){
for(b in 1:n2){
F1[a,b] <- tmp[a,b]/(sum(tmp[a,])+sum(tmp[,b])-tmp[a,b])
}
}
best.match <- colnames(F1)[apply(F1, 1, which.max)]
names(best.match) <- rownames(F1)
best.matched.clusterID <- rbind(best.matched.clusterID,
'C_Soft.wo.TC'= best.match[query])
### add the best matches for the additional BioTIP runs and the original QuanTC run
setdiff(names(CT), rownames(best.matched.clusterID))
# [1] "C_by_marker_allcells" "C_CollectionTime"
# [3] "C_CollectionTime_allcells"
# Now, manually add the best-match infor for these additional BioTIP runs
n <- nrow(best.matched.clusterID) # 14
best.matched.clusterID <- rbind(best.matched.clusterID,
best.matched.clusterID['C_by_marker',],
c('16.5','18.5'), c('16.5','18.5'))
rownames(best.matched.clusterID)[(n+1):(n+3)] <- setdiff(names(CT), rownames(best.matched.clusterID))
}
save(best.matched.clusterID, file='best.matched.clusterID.RData')
best.matched.clusterID
# E16.5 E18.5_AT2
# C_by_marker Unknown AT2
# C_Monocle.k4 2 1
# C_consensus_ks3 1 3
# C_consensus_ks5 3 1
# C_consensus_ks7 7 2
# C_Leiden_0.4 2 1
# C_Leiden_0.8 1 5
# C_Leiden_1.2 5 6
# C_SNNGraph_k10 4 1
# C_SNNGraph_k8 1 4
# C_Soft TC TC
# QuanTC_run C3 TC
# C_Soft.wo.TC C3 C4
# C_by_marker_allcells Unknown AT2
# C_CollectionTime 16.5 18.5
# C_CollectionTime_allcells 16.5 18.5
## 2.3) calculate Jaccard-similarity score for identifing the 4 potential bufurcations ##
## among the tested cells of the original cluster IDs C3, C13, C10, C6, C15, C7 ##
## between each predictions and that of our original run of BioTIP with the SNNGraph clusters ##
## after finding the reference cluster ID (i.e. best-matched IDs) for the QuanTC clusters ##
## And maually adding the 'best matches' for additional BioTIP/QuanTC runs of this datasets ##
{
jaccard.CT <- array()
for(i in 1:length(CT)){
x <- names(CT)[i]
n=1 ## 2 potntial bifurcation clusters !!!!!!!!!!!!
reference <- as.matrix(best.matched.clusterID[x,1:n])
# # adjust the reference cluster ID for shoft-thresholding clustering
# if(grepl('C_Soft', x) & !grepl('wo.TC', x) & !grepl('QuanTC_QuanTC', x)) {
# reference <- c(reference, best.matched.clusterID[paste0(x,'.wo.TC'),1:n]) %>% unlist()
# }
jaccard.CT[i] <- jaccard.sim(unique(reference), CT[[x]])
}
names(jaccard.CT) <- names(CT)
}
round(jaccard.CT, 2)
# C_by_marker C_by_marker_allcells C_CollectionTime
# 0 0 0
# C_CollectionTime_allcells C_consensus_ks3 C_consensus_ks5
# 0 1 1
# C_consensus_ks7 C_Leiden_0.4 C_Leiden_0.8
# 0 1 0
# C_Leiden_1.2 C_Monocle.k4 C_SNNGraph_k10
# 0 1 0
# C_SNNGraph_k8 C_Soft C_Soft.wo.TC
# 1 0 0
# QuanTC_run
# 0.5
## 2.4 we generate a proxy 'golden standard' of CTS genes,
## which were identifed by at least 3 out of five identfications for E16.5
###################################################################################
{
# select <- c('by_marker', 'Leiden_0.4','QuanTC_run','CollectionTime_allcells') # for E18.5
n.select <- 3 # floor(length(select) * 0.8))
select <- c( 'consensus_ks3', 'consensus_ks5', 'SNNGraph_k8', 'Leiden_0.4', 'QuanTC_run')
GS.CTS = NULL
for(i in 1:length(select)){
if(select[i] == 'QuanTC_run') {
load(file=paste0(select[i],'/CTS.RData'))
set2 <- CTS[[2]] # CTS[[1]] for E18.5
GS.CTS = c(GS.CTS, unlist(set2[x]))
rm(CTS)
} else {
load(file=paste0('C_',select[i],'/BioTIP.res.RData'))
set2 <- res$CTS.candidate[which(res$significant)]
best.match <- best.matched.clusterID[paste0('C_',select[i]),'E16.5'] #E18.5_AT2']
x <- grep(best.match, names(set2))
GS.CTS = c(GS.CTS, unlist(set2[x]))
}
}
x <- table(GS.CTS)
GS.CTS <- names(x)[which(x>= n.select)]
GS.CTS
# "9130023H24Rik" "Capsl" "Gm6320" "Nme5" "Rsph1"
# [6] "Tmem107"
(n.GS <- length(GS.CTS)) # 6
}
write.table(GS.CTS, file=paste0('GS.CTS.E16.5_at.least.',n.select,'identificaitons_fr.',length(select),'predicitons.txt'),
row.names=F, col.names=F)
## 2.5) evaluate the F1 score for the identified CTS genes at E16.5
## for any commonly identifed CTs from two identifications ##
################################################################
CTS.reference <- GS.CTS # inferred reference for this study !!
{
# load the CTS prediction with different clustering methods
running.methods <- list.dirs()
running.methods <- running.methods[grepl("./C_", running.methods)]
running.methods
# [1] "./C_by_marker" "./C_by_marker_allcells" "./C_CollectionTime"
# [4] "./C_CollectionTime_allcells" "./C_consensus_ks3" "./C_consensus_ks5"
# [7] "./C_consensus_ks7" "./C_Leiden_0.4" "./C_Leiden_0.8"
# [10] "./C_Leiden_1.2" "./C_Monocle.k4" "./C_SNNGraph_k10"
# [13] "./C_SNNGraph_k8" "./C_Soft" "./C_Soft.wo.TC"
methods <- lapply(running.methods, function(x) unlist(strsplit(x, "./C_"))[2]) %>% unlist()
x <- which(is.na(methods))
if(length(x)>0) {
methods <- methods[-x]
running.methods <- running.methods[-x]
}
## manually adding the QuanTC's predictions
running.methods <- c(running.methods, './QuanTC_run')
methods <- c(methods,'QuanTC_run')
F1.res <- get.multiple.F1.score(match.col.name='E16.5',# MCIbottom,
CTS.reference = GS.CTS,
QuanTC.genes.list.id = 2,
running.methods=running.methods, methods=methods,
sce = sce,
best.matched.clusterID=best.matched.clusterID)
F1.CTS <- F1.res$F1.scores$E16.5
F1.CTS.ctl <- F1.res$F1.ctl$E16.5
round(F1.CTS,3)
# round(F1.CTS,3)
# by_marker by_marker_allcells CollectionTime
# 0.000 0.000 0.000
# CollectionTime_allcells consensus_ks3 consensus_ks5
# 0.000 0.000 0.162
# consensus_ks7 Leiden_0.4 Leiden_0.8
# 0.000 0.136 0.000
# Leiden_1.2 Monocle.k4 SNNGraph_k10
# 0.000 0.000 0.000
# SNNGraph_k8 Soft Soft.wo.TC
# 0.176 0.000 0.000
# QuanTC_run
# 0.000
## calculating F1 scores for the gene module with the highest DNB score
# which is C2_38g of the Leiden_0.4 run
load('C_Leiden_0.4/BioTIP.res.RData')
lengths(res$CTS.candidate)
# 2 1 2 1
# 38 67 44 39
intersect(GS.CTS, res$CTS.candidate[[1]])
#character(0)
F1.DNB <- F_score.CTS(list('2'=GS.CTS), res$CTS.candidate[1], weight=TRUE)$F1
F1.ctl.DNB <- F_score.CTS(list('2'=GS.CTS),
list('2'=sample(rownames(sce),38)),
weight=TRUE)$F1
nn = length(F1.CTS) # 16
Normalized.F1 <- Normalize.F1(c(F1.CTS.ctl, F1.CTS, F1.ctl.DNB, F1.DNB))
Norm.F1.CTS.ctl <- Normalized.F1[1:nn]
Norm.F1.CTS <- Normalized.F1[(nn+1):(2*nn)]
Norm.F1.ctl.DNB <- Normalized.F1[(2*nn+1)]
Norm.F1.DNB <- Normalized.F1[(2*nn+2)]
}
###########################################################
## Section 3) reproting table, compare BioTIP to QuanTC ##
###########################################################
{
tb <- data.frame(Jaccard.CT = round(jaccard.CT,3),
F1.CTS.ctl = round(F1.CTS.ctl,3),
F1.CTS = round(F1.CTS,3),
Norm.F1.CTS.ctl = round(Norm.F1.CTS.ctl,3),
Norm.F1.CTS = round(Norm.F1.CTS,3),
F1.ctl.DNB = round(F1.ctl.DNB,3),
F1.DNB = round(F1.DNB,3),
Norm.F1.ctl.DNB = round(Norm.F1.ctl.DNB,3),
Norm.F1.DNB = round(Norm.F1.DNB,3)
)
nrow(tb) #[1] 16
# reorder to show
myorder <- c('C_by_marker', 'C_CollectionTime',
paste0('C_consensus_ks',c(3,5,7)),
paste0('C_Leiden_',c(0.4, 0.8, 1.2)),
'C_SNNGraph_k8','C_SNNGraph_k10',
'QuanTC_run'
)
tb$detected.CT <- lapply(CT, toString) %>% unlist()
(mytable <- tb[myorder,
c('detected.CT','Jaccard.CT',
'F1.CTS.ctl', 'F1.CTS', 'F1.ctl.DNB','F1.DNB',
'Norm.F1.CTS.ctl', 'Norm.F1.CTS', 'Norm.F1.ctl.DNB','Norm.F1.DNB')])
# detected.CT Jaccard.CT F1.CTS F1.nor
# C_by_marker AT2 0 0.000 0.322
# C_CollectionTime 0 0.000 0.322
# C_consensus_ks3 1 1 0.000 0.322
# C_consensus_ks5 3 1 0.162 0.980
# C_consensus_ks7 0 0.000 0.322
# C_Leiden_0.4 2 1 0.136 0.952
# C_Leiden_0.8 NA 0 0.000 0.322
# C_Leiden_1.2 NA 0 0.000 0.322
# C_SNNGraph_k8 1 1 0.176 0.989
# C_SNNGraph_k10 0 0.000 0.322
# QuanTC_run C3 1 0.000 0.322
## in fact, we only estimated for DNB using Leiden_0.4 clustering
mytable[which(rownames(mytable) !='C_Leiden_0.4'),
c('Norm.F1.ctl.DNB', 'Norm.F1.DNB')] <- NA
write.table(mytable,
file='jaccard.CT_F1.CTS.txt',
sep='\t')
###############
## PLOT #######
###############
## generate bar plot #######################
df <- read.table('jaccard.CT_F1.CTS.txt', header=T, sep='\t')
df <- df[, -which(colnames(df) %in% c('Norm.F1.ctl.DNB', 'Norm.F1.DNB'))]
tmp <- rownames(df)
tmp = gsub('C_', '', tmp)
df$method=tmp
df$method=factor(df$method, levels=df$method)
p1 <- ggbarplot(df, "method", "Jaccard.CT", orientation = "horiz") +
geom_col(aes(fill = Jaccard.CT)) +
scale_fill_gradient2(low = "white",high = "green") +
theme_bw(base_size=10) + # use a black-and-white theme with set font size
theme(legend.position = "none") + coord_flip()
p2 <- ggbarplot(df, "method", "Norm.F1.CTS", orientation = "horiz") +
geom_col(aes(fill = Norm.F1.CTS)) +
scale_fill_gradient2(low = "white",high = "blue") +
theme_bw(base_size=10) + # use a black-and-white theme with set font size
theme(legend.position = "none") + coord_flip()
pdf(file='bar_robustness.pdf', width=6, height=4)
gridExtra::grid.arrange(
p1, p2#, p3
,ncol=2)
dev.off()
}
###########################################################
## Section 4) stability using the Leiden_0.4 clusters ##
###########################################################
{
if(j=="AT2.sce.RData") load(file="AT2_ShrinkM.RData") else load(file="ShrinkM.RData")
dim(M) #3198 3198
colnames(colData(sce))
# [1] "age" "cellName" "GEO.access" "putative_cell_type"
# [5] "C_by_marker" "C_Monocle.k4" "Pseudotime" "Size_Factor"
# [9] "C_consensus_ks3" "C_consensus_ks5" "C_consensus_ks7" "C_Leiden_0.4"
# [13] "C_Leiden_0.8" "C_Leiden_1.2" "C_SNNGraph_k10" "C_SNNGraph_k8"
# [17] "C_Soft"
sce$label <- sce$C_Leiden_0.4 # selected clustering method
rowData(sce) <- NULL # to allow run sample()
######### 4.1) setting parameters, the same as section 1 ######################
## but try multiple getTopMCI.gene.minsize
getTopMCI.gene.minsize <- c(30,20,10)
######### 4.2) run BioTIP on downsized data ##################
n.scability <- 20
downsize <- 0.95
n1 <- nrow(sce)
n2 <- ncol(sce)
for(m in 1:length(getTopMCI.gene.minsize)){
for(i in 1:length(MCIbottom)){
set.seed(102020)
## get n.scability sets of predictions
res <- list()
flag = counts = 0
repeat{
cat(counts, '\t')
counts = counts+1 # control the maxmum trials for searching two sets of signficant CTSs, given the current parameter settings
sce.dnsize <- sce[sample(n1, floor(n1*downsize)), sample(n2, floor(n2*downsize))]
samplesL <- split(rownames(colData(sce.dnsize)), f = sce.dnsize$label)
this.run <- BioTIP.wrap(sce.dnsize, samplesL, subDir='stability', globa.HVG.select=FALSE,
getTopMCI.n.states=getTopMCI.n.states, getTopMCI.gene.minsize=getTopMCI.gene.minsize,
n.getMaxMCImember = 2, MCIbottom=MCIbottom,
local.HVG.optimize =TRUE, localHVG.preselect.cut=localHVG.preselect.cut, localHVG.runs=100, #logmat.local.HVG.testres=testres,
getNetwork.cut.fdr=getNetwork.cut.fdr,
n.permutation = 100, M=M,
permutation.method='both',
verbose=FALSE, plot=FALSE)
if(!is.null(this.run) ) {
if(length(which(this.run$significant))>0) { # control at least 1 significant CT to be identified !!!
flag = flag +1
res[[flag]] <- this.run
#rm(this.run)
}
}
if(flag ==n.scability | counts == 100) {
break
}
} # end repeat
counts-flag # 1, the number of runs without significant
save(res, flag, counts, file=paste0('stability/BioTIP_res_20runs_MCIbottom',MCIbottom[i],
'_Modulesize',getTopMCI.gene.minsize[m],'.RData'))
}
}
## 4.2) evaluate the stability of CT detection
## using the robustly detected C2 as reference
###########################################################
df.CT =NULL
for(m in 1:length(getTopMCI.gene.minsize)){
CT.list <- list()
Freq <- matrix(0, nrow=nlevels(sce$label), ncol=length(MCIbottom))#!!!
rownames(Freq) <- levels(sce$label) #!!!
colnames(Freq) <- MCIbottom
for(i in 1:length(MCIbottom)){
load(file=paste0('stability/BioTIP_res_20runs_MCIbottom',MCIbottom[i],
'_Modulesize',getTopMCI.gene.minsize[m],'.RData'))
## count which cluster has been identified repeatily ###########
CT <- list()
# for(j in 1:n.stability)
for(j in 1:length(res)){ # in case n.stability>length(res), i.e. some run has no isgnificant prediction
CT[[j]] <- res[[j]]$significant[which(res[[j]]$significant)] %>% names() %>% unique()
}
tmp <- table(unlist(CT))
Freq[names(tmp),i] <- tmp
#Freq <- cbind(Freq, tmp)
CT.list[[i]] <- CT
}
# Freq <- Freq/length(res)
names(CT.list) <- MCIbottom
df <- data.frame(Frequency = as.numeric(Freq),
clusterID = rep(rownames(Freq), ncol(Freq)),
MCIbottom = lapply(as.character(MCIbottom ), function(x) rep(x,nrow(Freq))) %>% unlist()
)
head(df)
# Frequency clusterID MCIbottom
# 1 10 C13 2
# 2 1 C3 2
# 3 5 C5 2
# 4 2 C7 2
# 5 3 C8 2
# 6 12 C6 2
df$clusterID <- factor(df$clusterID, levels =levels(sce$label)) #!!!
df$moduleSize <- rep(getTopMCI.gene.minsize[m], nrow(df))
df.CT <- rbind(df.CT, df)
# save(CT.list, file= paste0('stability/CT.detection_variable.MCIbottom_Modulesize',getTopMCI.gene.minsize[m],'.Rdata'))
}
df.CT$Frequency = df.CT$Frequency/n.scability
save(CT.list, df.CT,
file= 'stability/CT.detection_variable.MCIbottom_Modulesize.Rdata')
## 4.3) plot for variable Modulesize, when MCIbottom==2 ##
#############################################################
load(file= 'stability/CT.detection_variable.MCIbottom_Modulesize.Rdata')
df.CT$moduleSize <- as.character(df.CT$moduleSize )
ggplot(data=subset(df.CT, MCIbottom==2),
aes(x=clusterID, y=Frequency, group=moduleSize)) +
geom_line(aes(color=moduleSize), size=1.5)+
geom_point(aes(color=moduleSize))
dev.copy2pdf(file='stability/CT.detection_variable.MCIbottom2_Modulesize.pdf')
## 4.4) evaluate the stability of CTS identification
## for the C2
###########################################################
load(file= 'stability/CT.detection_variable.MCIbottom_Modulesize.Rdata')
names(CT.list)
#[1] "2"
# load('original_run/CTS.RData')
# names(CTS)
# #[1] "9" "10" "9"
# C9.ref = CTS[which(names(CTS)=='9')]
# C10.ref = CTS['10']
GS.CTS <- read.table('GS.CTS.E16.5_at.least.3identificaitons_fr.5predicitons.txt')
C2.ref <- list('2'=GS.CTS[,1])
MCIbottom # 2
i=1
F1.C2 <- F1.C2.ctl <- list()
for(m in 1:length(getTopMCI.gene.minsize)){
load(file=paste0('stability/BioTIP_res_20runs_MCIbottom',MCIbottom[i],
'_Modulesize',getTopMCI.gene.minsize[m],'.RData'))
F1.CTS.C2 <- F1.bk.C2 <- array(dim=n.scability)
for(j in 1:n.scability){
# evaluate the F1 score for two CTS identificitons
set2 <- res[[j]]$CTS.candidate[which(res[[j]]$significant)]
F1.CTS.C2[j] <- F_score.CTS(C2.ref, set2, weight=TRUE)$F1
# negative control
n.module <- lengths(set2)
random.set2 <- lapply(n.module, function(x) sample(rownames(sce), x))
F1.bk.C2[j] <- F_score.CTS(C2.ref, random.set2, weight=TRUE)$F1
}
F1.C2[[m]] = F1.CTS.C2
F1.C2.ctl[[m]] = F1.bk.C2
}
names(F1.C2) <- names(F1.C2.ctl) <- getTopMCI.gene.minsize
save(F1.C2, F1.C2.ctl,
file='stability/F1.CTS_MCIbottom2.RData')
lapply(F1.C2, mean) %>% unlist()
# 10 20 30
# 0.12294563 0.12294563 0.08011003
lapply(F1.C2.ctl, mean) %>% unlist()
#0.0024092971 0.0039024701 0.0008333333
df <- data.frame(F1 = c(unlist(F1.C2.ctl),unlist(F1.C2)),
minModuleSize = c(rep(getTopMCI.gene.minsize, lengths(F1.C2.ctl)),
rep(getTopMCI.gene.minsize, lengths(F1.C2))),
CT = c(rep('C2', sum(lengths(F1.C2.ctl))+sum(lengths(F1.C2)))),
CTS = c(rep('ctl', sum(lengths(F1.C2.ctl))), rep('CTS', sum(lengths(F1.C2))))
)
df$minModuleSize <- as.character(df$minModuleSize)
df$CTS <- factor(df$CTS, levels = c('ctl','CTS'))
df$color.lab <- paste0(df$minModuleSize,df$CTS)
df$color.lab <- factor(df$color.lab, levels=c('10ctl','10CTS','20ctl','20CTS','30ctl','30CTS'))
# To ensure that easy and difficult CT state have equal influence on the final score,
# we normalized the scores at each CT state across the different MCIbottom setting.
df$Norm.F1 <- Normalize.F1(df$F1)
library(ggbeeswarm)
library(grid)
library(gridExtra)
library(ggpubr)
p1 <- ggplot(data=df, aes(x=CT, y=F1, color=color.lab)) +
geom_violin(position = position_dodge(width = 0.5)) +
geom_quasirandom(dodge.width = 0.5, varwidth = TRUE) +
xlab('predicted for cluster') + ylab('F1 score for the CTS') +
scale_color_manual(values=c("grey", "orange","grey", "green","grey","blue"))
p2 <- ggplot(data=df, aes(x=CT, y=Norm.F1, color=color.lab)) +
geom_violin(position = position_dodge(width = 0.5)) +
geom_quasirandom(dodge.width = 0.5, varwidth = TRUE) +
xlab('predicted for cluster') + ylab('Normalized F1 score for the CTS') +
scale_color_manual(values=c("grey", "orange","grey", "green","grey","blue"))
pdf(file='stability/CTS.F1_variable.minModuleSize.pdf')
gridExtra::grid.arrange(
p1, p2, top = "CTS stability", bottom = "different module size settings"
,ncol=1)
dev.off()
tmp <- compare_means(Norm.F1 ~ color.lab, data=df, group='CT', method='t.test')
subset(tmp, group1=='10ctl' & group2=='10CTS')
# # A tibble: 2 x 9
## A tibble: 1 x 9
# CT .y. group1 group2 p p.adj p.format p.signif method
# <chr> <chr> <chr> <chr> <dbl> <dbl> <chr> <chr> <chr>
# 1 C2 Norm.F1 10ctl 10CTS 0.00000240 0.000031 2.4e-06 **** T-test
subset(tmp, group1=='20ctl' & group2=='20CTS')
# A tibble: 1 x 9
# CT .y. group1 group2 p p.adj p.format p.signif method
# <chr> <chr> <chr> <chr> <dbl> <dbl> <chr> <chr> <chr>
# 1 C2 Norm.F1 20ctl 20CTS 0.00000285 0.000031 2.8e-06 **** T-test
subset(tmp, group1=='30ctl' & group2=='30CTS')
# A tibble: 1 x 9
# CT .y. group1 group2 p p.adj p.format p.signif method
# <chr> <chr> <chr> <chr> <dbl> <dbl> <chr> <chr> <chr>
# 1 C2 Norm.F1 30ctl 30CTS 0.000284 0.0026 0.00028 *** T-test
}
###################################################################
## section 5) TSNE plot for 3.2k HGV, 131 cells along the AT2 trajectory
###################################################################
{
fid <- "C_Leiden_0.4/"
load(file=paste0(fid,"BioTIP_top0.1FDR0.2_optimized_local.HVG_selection.RData"))
names(testres)
#[1] "1" "2" "3"
library(scater)
pdf(file='tsne_131cells_C_Leiden_0.4.pdf')
plotReducedDim(sce, dimred='TSNE', shape_by='age',
colour_by = "C_Leiden_0.4",
text_by='putative_cell_type',
text_size = 4, text_colour='black', point_size=2) +
ggtitle('putative_cell_type') #+ ylim(5,20)
plotReducedDim(sce, dimred='TSNE', shape_by='age',
colour_by = 'putative_cell_type',
text_by="C_Leiden_0.4",
text_size = 4, text_colour='black', point_size=2) +
ggtitle('putative_cell_type') #+ ylim(5,20)
dev.off()
}
xyang2uchicago/BioTIP documentation built on June 30, 2024, 10:14 p.m.
R Package Documentation
Browse R Packages
We want your feedback!
Note that we can't provide technical support on individual packages. You should contact the package authors for that.
#### README ############
## There are 4 sections in this code
## Section 1) apply BioTIP to different clusters of 131 cells by different methods
## also apply BioTIP to different clusters of 196 cells by cell collection time (age), marker-based clusters, and unsupervised clusters
## section 2) quantify the robustness
## section 3) reporting table, compare the results of BioTIP to the resutls of QuanTC
## section 4) stability of BioTIP's identification
## section 5) TSNE plot for 3.2k HGV, 131 cells along the AT2 trajectory
## last update 2/8/2022
## by Holly Yang ##############
setwd('F:/projects/BioTIP/result/GSE52583/BioTIP_GSE52583_robustness/')
# the following funciton has been updated on github, but before updating the package to Bioconductor, you need to call it locally to test
source(file='F:/projects/scRNA/source/GSE87038_gastrulation/ForJennifer/optimize.sd_selection.R')
source(file='F:/projects/scRNA/source/GSE87038_gastrulation/GSE87038_git_copy/BioTIP.wrap.R')
source(file='F:/projects/scRNA/source/GSE87038_gastrulation/GSE87038_git_copy/Stability_score.R')
# normalized data were downloaded and reanalyzed
# refer to GSE87038_E8.25_normalize.R
library(BioTIP)
library(dplyr)
library(SingleCellExperiment)
library(ggplot2)
library(gridExtra) # required by grid.arrange()
library(ggpubr) # required by p-values on bar
###################################################
## load the R object ##
## focusing on 131 cells along the AT2 trajectory
###################################################
j="AT2.sce.RData"
if(j=="AT2.sce.RData") load('AT2.sce.RData') else load('sce.RData') #!!!!!!!!!!!!!!!
# format assayNames to run BioTIP
assayNames(sce) <- 'logcounts'
sce
#class: SingleCellExperiment
# dim: 3198 131
# metadata(0):
# assays(1): logcounts
# rownames(3198): 0610007C21Rik 0610007L01Rik ... Zyx l7Rn6
# rowData names(11): ensembl_gene_id gene_short_name ...
# num_cells_expressed use_for_ordering
# colnames(131): E18_1_C08 E18_1_C09 ... E16_1_C94 E16_1_C95
# colData names(17): age cellName ... C_SNNGraph_k8 C_Soft
# reducedDimNames(2): PCA TSNE
# altExpNames(0):
# logmat <- as.matrix(assays(sce)$logcounts)
# M <- cor.shrink(logmat, Y = NULL, MARGIN = 1, shrink = TRUE)
# if(j=="AT2.sce.RData") save(M, file="AT2_ShrinkM.RData", compress=TRUE) else save(M, file="ShrinkM.RData", compress=TRUE)
# dim(M) #3198 3198
# rm(logmat)
if(j=="AT2.sce.RData") load(file="AT2_ShrinkM.RData") else load(file="ShrinkM.RData")
dim(M) #3198 3198
####################################################################
## section 1) running BioTIP on different cell clustering outputs ##
####################################################################
{
######### 1.1) setting parameters, ######################
localHVG.preselect.cut = 0.1 # A positive numeric value setting how many propotion of global HVG to be selected per cell cluster
getNetwork.cut.fdr = 0.2 # to construct RW network and extract co-expressed gene moduels
getTopMCI.gene.minsize = 30 # min number of genes in an identified CTS
getTopMCI.n.states = 2 # A number setting the number of states to check the significance of DNB score (i.e. MCI)
# This parameter can be enlarge when inputting more clusters, usually the expected number of transition states +1
MCIbottom = 2 # A number setting the threshold to prioritize the initially selected CTS candidates.
# In our experiment, a number between 2 and 4 generated expected resutls.
############### 1.2) applying BioTIP to all new clustering outputs ##################
x <- grep("C_", colnames(colData(sce)))
colnames(colData(sce))[x]
# [1] "C_by_marker" "C_Monocle.k4" "C_consensus_ks3" "C_consensus_ks5"
# [5] "C_consensus_ks7" "C_Leiden_0.4" "C_Leiden_0.8" "C_Leiden_1.2"
# [9] "C_SNNGraph_k10" "C_SNNGraph_k8" "C_Soft"
set.seed(102020)
for(i in 1:length(x)){
samplesL <- split(rownames(colData(sce)), f = colData(sce)[x[i]])
(tmp = lengths(samplesL))
#outputpath = paste0(colnames(colData(sce))[x[i]],'/BioTIP_top',localHVG.preselect.cut,'FDR',getNetwork.cut.fdr,"_")
#load(file=paste0(outputpath,"optimized_local.HVG_selection.RData"))
res <- BioTIP.wrap(sce, samplesL, subDir=colnames(colData(sce))[x[i]],
getTopMCI.n.states=getTopMCI.n.states,
getTopMCI.gene.minsize=getTopMCI.gene.minsize,
MCIbottom=MCIbottom,
localHVG.preselect.cut=localHVG.preselect.cut, #logmat.local.HVG.testres=testres,
getNetwork.cut.fdr=getNetwork.cut.fdr,
M=M,
permutation.method='both',
empirical.IC.p.cut=0.1, ## when permutating both gene and sampels toestimate the Ic.shrink's significance,
## we can loose this creterial to get more meanfuling results
verbose=TRUE, plot=TRUE)
save(res, file=paste0(colnames(colData(sce))[x[i]],'/BioTIP.res.RData'))
}
## 1.3) Additional run for soft thresholding without TC
samplesL <- split(rownames(colData(sce)), f = colData(sce)$C_Soft)
(tmp = lengths(samplesL))
# C1 C2 TC
# 31 43 57
samplesL = samplesL[1:(length(samplesL)-1)]
# outputpath = paste0('C_Soft.wo.TC/BioTIP_top',localHVG.preselect.cut,'FDR',getNetwork.cut.fdr,"_")
# load(file=paste0(outputpath,"optimized_local.HVG_selection.RData"))
res <- BioTIP.wrap(sce, samplesL, subDir='C_Soft.wo.TC',
getTopMCI.n.states=getTopMCI.n.states,
getTopMCI.gene.minsize=getTopMCI.gene.minsize,
MCIbottom=MCIbottom,
localHVG.preselect.cut=localHVG.preselect.cut, #logmat.local.HVG.testres=testres,
getNetwork.cut.fdr=getNetwork.cut.fdr,
M=M,
permutation.method='both',
verbose=TRUE, plot=TRUE)
save(res, file='C_Soft.wo.TC/BioTIP.res.RData')
## 1.4) additional run for time-course
samplesL <- split(rownames(colData(sce)), f = colData(sce)$age)
(tmp = lengths(samplesL))
#14.5 16.5 18.5 Adult
#45 27 15 44
# outputpath = paste0('C_CollectionTime_allcells/BioTIP_top',localHVG.preselect.cut,'FDR',getNetwork.cut.fdr,"_")
# load(file=paste0(outputpath,"optimized_local.HVG_selection.RData"))
res <- BioTIP.wrap(sce, samplesL, subDir='C_CollectionTime',
getTopMCI.n.states=getTopMCI.n.states,
getTopMCI.gene.minsize=getTopMCI.gene.minsize,
MCIbottom=MCIbottom,
localHVG.preselect.cut=localHVG.preselect.cut, #logmat.local.HVG.testres=testres,
getNetwork.cut.fdr=getNetwork.cut.fdr,
M=M,
permutation.method='both',
verbose=TRUE, plot=TRUE)
save(res, file='C_CollectionTime/BioTIP.res.RData')
## 1.5) additional run for all collected time-course cells
load('sce.RData')
sce
# class: SingleCellExperiment
# dim: 3198 196
samplesL <- split(rownames(colData(sce)), f = colData(sce)$age)
(tmp = lengths(samplesL))
# 14.5 16.5 18.5 Adult
# 45 27 80 44
# format assayNames to run BioTIP
assayNames(sce) <- 'logcounts'
# outputpath = paste0('C_CollectionTime_allcells/BioTIP_top',localHVG.preselect.cut,'FDR',getNetwork.cut.fdr,"_")
# load(file=paste0(outputpath,"optimized_local.HVG_selection.RData"))
res <- BioTIP.wrap(sce, samplesL, subDir='C_CollectionTime_allcells',
getTopMCI.n.states=getTopMCI.n.states,
getTopMCI.gene.minsize=getTopMCI.gene.minsize,
MCIbottom=MCIbottom,
ocalHVG.preselect.cut=localHVG.preselect.cut, #logmat.local.HVG.testres=testres,
getNetwork.cut.fdr=getNetwork.cut.fdr,
M=M,
permutation.method='both',
verbose=TRUE, plot=TRUE)
save(res, file='C_CollectionTime_allcells/BioTIP.res.RData')
## 1.6) additionally run for all collected cells, clustered by gene markers
samplesL <- split(rownames(colData(sce)), f = colData(sce)$C_by_marker)
(tmp = lengths(samplesL))
# Ambiguous AT1 AT2 Unknown
# 6 63 77 50
# outputpath = paste0('C_by_marker_allcells/BioTIP_top',localHVG.preselect.cut,'FDR',getNetwork.cut.fdr,"_")
# load(file=paste0(outputpath,"optimized_local.HVG_selection.RData"))
res <- BioTIP.wrap(sce, samplesL, subDir='C_by_marker_allcells',
getTopMCI.n.states=getTopMCI.n.states,
getTopMCI.gene.minsize=getTopMCI.gene.minsize,
MCIbottom=MCIbottom,
localHVG.preselect.cut=localHVG.preselect.cut, # logmat.local.HVG.testres=testres,
getNetwork.cut.fdr=getNetwork.cut.fdr,
M=M,
permutation.method='both',
verbose=TRUE, plot=TRUE)
save(res, file='C_by_marker_allcells/BioTIP.res.RData')
## 1.7) copy QuanTC-identified transition genes
# BEGIN DO not repeat read and save QuanTC-predicted CTS genes ##
CTS <- list()
tmp <- read.table('../QuanTC_Output/AT2/transition_gene_name1.2.txt',
header=FALSE)#E14.5 in soft-thresholing clustering
CTS[['C1']] <- tmp[,1]
tmp <- read.table('../QuanTC_Output/AT2/transition_gene_name2.2.txt',
header=FALSE)#E16.5 in soft-thresholing clustering
CTS[['C3']] <- tmp[,1]
save(CTS, file='QuanTC_run/CTS.RData')
# END DO not repeat read and save QuanTC-predicted CTS genes ##
###################################################################
}
####################################################################
## section 2) quantify the robustness ##
####################################################################
i= "AT2.sce.RData"
if(j=="AT2.sce.RData") load('AT2.sce.RData') else load('sce.RData') #!!!!!!!!!!!!!!!
# format assayNames to run BioTIP
assayNames(sce) <- 'logcounts'
{
## 2. 1) Summarize the CT identifications
{
running.methods <- list.dirs()
running.methods <- running.methods[grepl('/C_',running.methods)]
methods <- lapply(running.methods, function(x) unlist(strsplit(x, "/C_"))[2]) %>% unlist()
x <- which(is.na(methods))
if(length(x)>0){
running.methods = running.methods[-x]
methods = methods[-x]
}
CT <- list()
for(i in 1:length(running.methods)){
load(file=paste0(running.methods[i],'/BioTIP.res.RData'))
if(length(res)>1) {
CT[[i]] <- res$significant[which(res$significant)] %>% names() %>% unique()
} else CT[[i]] <- NA
}
names(CT) <- paste0("C_", methods)
}
lapply(CT, toString) %>% unlist()
# C_by_marker C_by_marker_allcells C_CollectionTime
# "AT2" "" ""
# C_CollectionTime_allcells C_consensus_ks3 C_consensus_ks5
# "18.5" "1" "3"
# C_consensus_ks7 C_Leiden_0.4 C_Leiden_0.8
# "" "2" "NA"
# C_Leiden_1.2 C_Monocle.k4 C_SNNGraph_k10
# "NA" "2" ""
# C_SNNGraph_k8 C_Soft C_Soft.wo.TC
# "1" "" ""
## manually add the QuanTC's prediction about CT states,
CT$QuanTC_run <- c('C3','C1') # the soft-thresholding cluster IDs
## 2.2) Annotate cell identities by comparing to expected CT cluster ##
{
## load the QuanTC-identified transition cells
tmp <- read.table('../QuanTC_Output/AT2/C_TC.txt')
tmp <- tmp[,1]
table(tmp)
# 1 2 3 4 5
# 36 43 13 2 37
colData(sce)$QuanTC_run = tmp
## replace the QuanTC.cluster IDs
tmp <- data.frame(colData(sce))
tmp <- tmp %>% mutate(QuanTC_run = replace(QuanTC_run, QuanTC_run == 1, 'C1'))
tmp <- tmp %>% mutate(QuanTC_run = replace(QuanTC_run, QuanTC_run == 2, 'C2'))
tmp <- tmp %>% mutate(QuanTC_run = replace(QuanTC_run, QuanTC_run == 3, 'C3'))
tmp <- tmp %>% mutate(QuanTC_run = replace(QuanTC_run, QuanTC_run == 4, 'C4'))
tmp <- tmp %>% mutate(QuanTC_run = replace(QuanTC_run, QuanTC_run == 5, 'TC'))
colData(sce)$QuanTC_run <- tmp$QuanTC_run
colData(sce)$age_cellType <- paste(colData(sce)$age, colData(sce)$putative_cell_type, sep="_")
table(colData(sce)$QuanTC_run, colData(sce)$age_cellType)
# 14.5_NA 16.5_NA 18.5_AT2 18.5_BP 18.5_ciliated 18.5_Clara Adult_NA
# C1 36 0 0 0 0 0 0
# C2 0 0 0 0 0 0 43
# C3 0 13 0 0 0 0 0
# C4 0 0 1 1 0 0 0
# TC 9 14 10 0 1 2 1
colnames(colData(sce))
#
x <- grep("C_", colnames(colData(sce)))
colnames(colData(sce))[x]
best.matched.clusterID <- data.frame()
query = c('16.5_NA','18.5_AT2') # the age_cellType for bifurcation which are named
# as early HEP, later HEP, EP, HP, respectively
# among the studied clusters C3, C13, C10, C6, C15, C7
for(i in x){
tmp <- table(sce$age_cellType, colData(sce)[,i])
n1 <- nrow(tmp)
n2 <- ncol(tmp)
Jaccard_cell = tmp*0
for(a in 1:n1){
for(b in 1:n2){
Jaccard_cell[a,b] <- tmp[a,b]/(sum(tmp[a,])+sum(tmp[,b])-tmp[a,b])
}
}
best.match <- colnames(Jaccard_cell)[apply(Jaccard_cell, 1, which.max)]
names(best.match) <- rownames(Jaccard_cell)
best.matched.clusterID <- rbind(best.matched.clusterID,
best.match[query])
}
rownames(best.matched.clusterID) <- colnames(colData(sce))[x]
colnames(best.matched.clusterID) <- c('E16.5', 'E18.5_AT2')
## additionally recalculate without the TC among QuanTC's clusters
i= grep('Soft_k4', colnames(colData(sce))) # 7
tmp <- table(sce$age_cellType, colData(sce)[,i])
tmp = tmp[,1:(ncol(tmp)-1)] # masking TC
n1 <- nrow(tmp)
n2 <- ncol(tmp)
F1 = tmp*0
for(a in 1:n1){
for(b in 1:n2){
F1[a,b] <- tmp[a,b]/(sum(tmp[a,])+sum(tmp[,b])-tmp[a,b])
}
}
best.match <- colnames(F1)[apply(F1, 1, which.max)]
names(best.match) <- rownames(F1)
best.matched.clusterID <- rbind(best.matched.clusterID,
'C_Soft.wo.TC'= best.match[query])
### add the best matches for the additional BioTIP runs and the original QuanTC run
setdiff(names(CT), rownames(best.matched.clusterID))
# [1] "C_by_marker_allcells" "C_CollectionTime"
# [3] "C_CollectionTime_allcells"
# Now, manually add the best-match infor for these additional BioTIP runs
n <- nrow(best.matched.clusterID) # 14
best.matched.clusterID <- rbind(best.matched.clusterID,
best.matched.clusterID['C_by_marker',],
c('16.5','18.5'), c('16.5','18.5'))
rownames(best.matched.clusterID)[(n+1):(n+3)] <- setdiff(names(CT), rownames(best.matched.clusterID))
}
save(best.matched.clusterID, file='best.matched.clusterID.RData')
best.matched.clusterID
# E16.5 E18.5_AT2
# C_by_marker Unknown AT2
# C_Monocle.k4 2 1
# C_consensus_ks3 1 3
# C_consensus_ks5 3 1
# C_consensus_ks7 7 2
# C_Leiden_0.4 2 1
# C_Leiden_0.8 1 5
# C_Leiden_1.2 5 6
# C_SNNGraph_k10 4 1
# C_SNNGraph_k8 1 4
# C_Soft TC TC
# QuanTC_run C3 TC
# C_Soft.wo.TC C3 C4
# C_by_marker_allcells Unknown AT2
# C_CollectionTime 16.5 18.5
# C_CollectionTime_allcells 16.5 18.5
## 2.3) calculate Jaccard-similarity score for identifing the 4 potential bufurcations ##
## among the tested cells of the original cluster IDs C3, C13, C10, C6, C15, C7 ##
## between each predictions and that of our original run of BioTIP with the SNNGraph clusters ##
## after finding the reference cluster ID (i.e. best-matched IDs) for the QuanTC clusters ##
## And maually adding the 'best matches' for additional BioTIP/QuanTC runs of this datasets ##
{
jaccard.CT <- array()
for(i in 1:length(CT)){
x <- names(CT)[i]
n=1 ## 2 potntial bifurcation clusters !!!!!!!!!!!!
reference <- as.matrix(best.matched.clusterID[x,1:n])
# # adjust the reference cluster ID for shoft-thresholding clustering
# if(grepl('C_Soft', x) & !grepl('wo.TC', x) & !grepl('QuanTC_QuanTC', x)) {
# reference <- c(reference, best.matched.clusterID[paste0(x,'.wo.TC'),1:n]) %>% unlist()
# }
jaccard.CT[i] <- jaccard.sim(unique(reference), CT[[x]])
}
names(jaccard.CT) <- names(CT)
}
round(jaccard.CT, 2)
# C_by_marker C_by_marker_allcells C_CollectionTime
# 0 0 0
# C_CollectionTime_allcells C_consensus_ks3 C_consensus_ks5
# 0 1 1
# C_consensus_ks7 C_Leiden_0.4 C_Leiden_0.8
# 0 1 0
# C_Leiden_1.2 C_Monocle.k4 C_SNNGraph_k10
# 0 1 0
# C_SNNGraph_k8 C_Soft C_Soft.wo.TC
# 1 0 0
# QuanTC_run
# 0.5
## 2.4 we generate a proxy 'golden standard' of CTS genes,
## which were identifed by at least 3 out of five identfications for E16.5
###################################################################################
{
# select <- c('by_marker', 'Leiden_0.4','QuanTC_run','CollectionTime_allcells') # for E18.5
n.select <- 3 # floor(length(select) * 0.8))
select <- c( 'consensus_ks3', 'consensus_ks5', 'SNNGraph_k8', 'Leiden_0.4', 'QuanTC_run')
GS.CTS = NULL
for(i in 1:length(select)){
if(select[i] == 'QuanTC_run') {
load(file=paste0(select[i],'/CTS.RData'))
set2 <- CTS[[2]] # CTS[[1]] for E18.5
GS.CTS = c(GS.CTS, unlist(set2[x]))
rm(CTS)
} else {
load(file=paste0('C_',select[i],'/BioTIP.res.RData'))
set2 <- res$CTS.candidate[which(res$significant)]
best.match <- best.matched.clusterID[paste0('C_',select[i]),'E16.5'] #E18.5_AT2']
x <- grep(best.match, names(set2))
GS.CTS = c(GS.CTS, unlist(set2[x]))
}
}
x <- table(GS.CTS)
GS.CTS <- names(x)[which(x>= n.select)]
GS.CTS
# "9130023H24Rik" "Capsl" "Gm6320" "Nme5" "Rsph1"
# [6] "Tmem107"
(n.GS <- length(GS.CTS)) # 6
}
write.table(GS.CTS, file=paste0('GS.CTS.E16.5_at.least.',n.select,'identificaitons_fr.',length(select),'predicitons.txt'),
row.names=F, col.names=F)
## 2.5) evaluate the F1 score for the identified CTS genes at E16.5
## for any commonly identifed CTs from two identifications ##
################################################################
CTS.reference <- GS.CTS # inferred reference for this study !!
{
# load the CTS prediction with different clustering methods
running.methods <- list.dirs()
running.methods <- running.methods[grepl("./C_", running.methods)]
running.methods
# [1] "./C_by_marker" "./C_by_marker_allcells" "./C_CollectionTime"
# [4] "./C_CollectionTime_allcells" "./C_consensus_ks3" "./C_consensus_ks5"
# [7] "./C_consensus_ks7" "./C_Leiden_0.4" "./C_Leiden_0.8"
# [10] "./C_Leiden_1.2" "./C_Monocle.k4" "./C_SNNGraph_k10"
# [13] "./C_SNNGraph_k8" "./C_Soft" "./C_Soft.wo.TC"
methods <- lapply(running.methods, function(x) unlist(strsplit(x, "./C_"))[2]) %>% unlist()
x <- which(is.na(methods))
if(length(x)>0) {
methods <- methods[-x]
running.methods <- running.methods[-x]
}
## manually adding the QuanTC's predictions
running.methods <- c(running.methods, './QuanTC_run')
methods <- c(methods,'QuanTC_run')
F1.res <- get.multiple.F1.score(match.col.name='E16.5',# MCIbottom,
CTS.reference = GS.CTS,
QuanTC.genes.list.id = 2,
running.methods=running.methods, methods=methods,
sce = sce,
best.matched.clusterID=best.matched.clusterID)
F1.CTS <- F1.res$F1.scores$E16.5
F1.CTS.ctl <- F1.res$F1.ctl$E16.5
round(F1.CTS,3)
# round(F1.CTS,3)
# by_marker by_marker_allcells CollectionTime
# 0.000 0.000 0.000
# CollectionTime_allcells consensus_ks3 consensus_ks5
# 0.000 0.000 0.162
# consensus_ks7 Leiden_0.4 Leiden_0.8
# 0.000 0.136 0.000
# Leiden_1.2 Monocle.k4 SNNGraph_k10
# 0.000 0.000 0.000
# SNNGraph_k8 Soft Soft.wo.TC
# 0.176 0.000 0.000
# QuanTC_run
# 0.000
## calculating F1 scores for the gene module with the highest DNB score
# which is C2_38g of the Leiden_0.4 run
load('C_Leiden_0.4/BioTIP.res.RData')
lengths(res$CTS.candidate)
# 2 1 2 1
# 38 67 44 39
intersect(GS.CTS, res$CTS.candidate[[1]])
#character(0)
F1.DNB <- F_score.CTS(list('2'=GS.CTS), res$CTS.candidate[1], weight=TRUE)$F1
F1.ctl.DNB <- F_score.CTS(list('2'=GS.CTS),
list('2'=sample(rownames(sce),38)),
weight=TRUE)$F1
nn = length(F1.CTS) # 16
Normalized.F1 <- Normalize.F1(c(F1.CTS.ctl, F1.CTS, F1.ctl.DNB, F1.DNB))
Norm.F1.CTS.ctl <- Normalized.F1[1:nn]
Norm.F1.CTS <- Normalized.F1[(nn+1):(2*nn)]
Norm.F1.ctl.DNB <- Normalized.F1[(2*nn+1)]
Norm.F1.DNB <- Normalized.F1[(2*nn+2)]
}
###########################################################
## Section 3) reproting table, compare BioTIP to QuanTC ##
###########################################################
{
tb <- data.frame(Jaccard.CT = round(jaccard.CT,3),
F1.CTS.ctl = round(F1.CTS.ctl,3),
F1.CTS = round(F1.CTS,3),
Norm.F1.CTS.ctl = round(Norm.F1.CTS.ctl,3),
Norm.F1.CTS = round(Norm.F1.CTS,3),
F1.ctl.DNB = round(F1.ctl.DNB,3),
F1.DNB = round(F1.DNB,3),
Norm.F1.ctl.DNB = round(Norm.F1.ctl.DNB,3),
Norm.F1.DNB = round(Norm.F1.DNB,3)
)
nrow(tb) #[1] 16
# reorder to show
myorder <- c('C_by_marker', 'C_CollectionTime',
paste0('C_consensus_ks',c(3,5,7)),
paste0('C_Leiden_',c(0.4, 0.8, 1.2)),
'C_SNNGraph_k8','C_SNNGraph_k10',
'QuanTC_run'
)
tb$detected.CT <- lapply(CT, toString) %>% unlist()
(mytable <- tb[myorder,
c('detected.CT','Jaccard.CT',
'F1.CTS.ctl', 'F1.CTS', 'F1.ctl.DNB','F1.DNB',
'Norm.F1.CTS.ctl', 'Norm.F1.CTS', 'Norm.F1.ctl.DNB','Norm.F1.DNB')])
# detected.CT Jaccard.CT F1.CTS F1.nor
# C_by_marker AT2 0 0.000 0.322
# C_CollectionTime 0 0.000 0.322
# C_consensus_ks3 1 1 0.000 0.322
# C_consensus_ks5 3 1 0.162 0.980
# C_consensus_ks7 0 0.000 0.322
# C_Leiden_0.4 2 1 0.136 0.952
# C_Leiden_0.8 NA 0 0.000 0.322
# C_Leiden_1.2 NA 0 0.000 0.322
# C_SNNGraph_k8 1 1 0.176 0.989
# C_SNNGraph_k10 0 0.000 0.322
# QuanTC_run C3 1 0.000 0.322
## in fact, we only estimated for DNB using Leiden_0.4 clustering
mytable[which(rownames(mytable) !='C_Leiden_0.4'),
c('Norm.F1.ctl.DNB', 'Norm.F1.DNB')] <- NA
write.table(mytable,
file='jaccard.CT_F1.CTS.txt',
sep='\t')
###############
## PLOT #######
###############
## generate bar plot #######################
df <- read.table('jaccard.CT_F1.CTS.txt', header=T, sep='\t')
df <- df[, -which(colnames(df) %in% c('Norm.F1.ctl.DNB', 'Norm.F1.DNB'))]
tmp <- rownames(df)
tmp = gsub('C_', '', tmp)
df$method=tmp
df$method=factor(df$method, levels=df$method)
p1 <- ggbarplot(df, "method", "Jaccard.CT", orientation = "horiz") +
geom_col(aes(fill = Jaccard.CT)) +
scale_fill_gradient2(low = "white",high = "green") +
theme_bw(base_size=10) + # use a black-and-white theme with set font size
theme(legend.position = "none") + coord_flip()
p2 <- ggbarplot(df, "method", "Norm.F1.CTS", orientation = "horiz") +
geom_col(aes(fill = Norm.F1.CTS)) +
scale_fill_gradient2(low = "white",high = "blue") +
theme_bw(base_size=10) + # use a black-and-white theme with set font size
theme(legend.position = "none") + coord_flip()
pdf(file='bar_robustness.pdf', width=6, height=4)
gridExtra::grid.arrange(
p1, p2#, p3
,ncol=2)
dev.off()
}
###########################################################
## Section 4) stability using the Leiden_0.4 clusters ##
###########################################################
{
if(j=="AT2.sce.RData") load(file="AT2_ShrinkM.RData") else load(file="ShrinkM.RData")
dim(M) #3198 3198
colnames(colData(sce))
# [1] "age" "cellName" "GEO.access" "putative_cell_type"
# [5] "C_by_marker" "C_Monocle.k4" "Pseudotime" "Size_Factor"
# [9] "C_consensus_ks3" "C_consensus_ks5" "C_consensus_ks7" "C_Leiden_0.4"
# [13] "C_Leiden_0.8" "C_Leiden_1.2" "C_SNNGraph_k10" "C_SNNGraph_k8"
# [17] "C_Soft"
sce$label <- sce$C_Leiden_0.4 # selected clustering method
rowData(sce) <- NULL # to allow run sample()
######### 4.1) setting parameters, the same as section 1 ######################
## but try multiple getTopMCI.gene.minsize
getTopMCI.gene.minsize <- c(30,20,10)
######### 4.2) run BioTIP on downsized data ##################
n.scability <- 20
downsize <- 0.95
n1 <- nrow(sce)
n2 <- ncol(sce)
for(m in 1:length(getTopMCI.gene.minsize)){
for(i in 1:length(MCIbottom)){
set.seed(102020)
## get n.scability sets of predictions
res <- list()
flag = counts = 0
repeat{
cat(counts, '\t')
counts = counts+1 # control the maxmum trials for searching two sets of signficant CTSs, given the current parameter settings
sce.dnsize <- sce[sample(n1, floor(n1*downsize)), sample(n2, floor(n2*downsize))]
samplesL <- split(rownames(colData(sce.dnsize)), f = sce.dnsize$label)
this.run <- BioTIP.wrap(sce.dnsize, samplesL, subDir='stability', globa.HVG.select=FALSE,
getTopMCI.n.states=getTopMCI.n.states, getTopMCI.gene.minsize=getTopMCI.gene.minsize,
n.getMaxMCImember = 2, MCIbottom=MCIbottom,
local.HVG.optimize =TRUE, localHVG.preselect.cut=localHVG.preselect.cut, localHVG.runs=100, #logmat.local.HVG.testres=testres,
getNetwork.cut.fdr=getNetwork.cut.fdr,
n.permutation = 100, M=M,
permutation.method='both',
verbose=FALSE, plot=FALSE)
if(!is.null(this.run) ) {
if(length(which(this.run$significant))>0) { # control at least 1 significant CT to be identified !!!
flag = flag +1
res[[flag]] <- this.run
#rm(this.run)
}
}
if(flag ==n.scability | counts == 100) {
break
}
} # end repeat
counts-flag # 1, the number of runs without significant
save(res, flag, counts, file=paste0('stability/BioTIP_res_20runs_MCIbottom',MCIbottom[i],
'_Modulesize',getTopMCI.gene.minsize[m],'.RData'))
}
}
## 4.2) evaluate the stability of CT detection
## using the robustly detected C2 as reference
###########################################################
df.CT =NULL
for(m in 1:length(getTopMCI.gene.minsize)){
CT.list <- list()
Freq <- matrix(0, nrow=nlevels(sce$label), ncol=length(MCIbottom))#!!!
rownames(Freq) <- levels(sce$label) #!!!
colnames(Freq) <- MCIbottom
for(i in 1:length(MCIbottom)){
load(file=paste0('stability/BioTIP_res_20runs_MCIbottom',MCIbottom[i],
'_Modulesize',getTopMCI.gene.minsize[m],'.RData'))
## count which cluster has been identified repeatily ###########
CT <- list()
# for(j in 1:n.stability)
for(j in 1:length(res)){ # in case n.stability>length(res), i.e. some run has no isgnificant prediction
CT[[j]] <- res[[j]]$significant[which(res[[j]]$significant)] %>% names() %>% unique()
}
tmp <- table(unlist(CT))
Freq[names(tmp),i] <- tmp
#Freq <- cbind(Freq, tmp)
CT.list[[i]] <- CT
}
# Freq <- Freq/length(res)
names(CT.list) <- MCIbottom
df <- data.frame(Frequency = as.numeric(Freq),
clusterID = rep(rownames(Freq), ncol(Freq)),
MCIbottom = lapply(as.character(MCIbottom ), function(x) rep(x,nrow(Freq))) %>% unlist()
)
head(df)
# Frequency clusterID MCIbottom
# 1 10 C13 2
# 2 1 C3 2
# 3 5 C5 2
# 4 2 C7 2
# 5 3 C8 2
# 6 12 C6 2
df$clusterID <- factor(df$clusterID, levels =levels(sce$label)) #!!!
df$moduleSize <- rep(getTopMCI.gene.minsize[m], nrow(df))
df.CT <- rbind(df.CT, df)
# save(CT.list, file= paste0('stability/CT.detection_variable.MCIbottom_Modulesize',getTopMCI.gene.minsize[m],'.Rdata'))
}
df.CT$Frequency = df.CT$Frequency/n.scability
save(CT.list, df.CT,
file= 'stability/CT.detection_variable.MCIbottom_Modulesize.Rdata')
## 4.3) plot for variable Modulesize, when MCIbottom==2 ##
#############################################################
load(file= 'stability/CT.detection_variable.MCIbottom_Modulesize.Rdata')
df.CT$moduleSize <- as.character(df.CT$moduleSize )
ggplot(data=subset(df.CT, MCIbottom==2),
aes(x=clusterID, y=Frequency, group=moduleSize)) +
geom_line(aes(color=moduleSize), size=1.5)+
geom_point(aes(color=moduleSize))
dev.copy2pdf(file='stability/CT.detection_variable.MCIbottom2_Modulesize.pdf')
## 4.4) evaluate the stability of CTS identification
## for the C2
###########################################################
load(file= 'stability/CT.detection_variable.MCIbottom_Modulesize.Rdata')
names(CT.list)
#[1] "2"
# load('original_run/CTS.RData')
# names(CTS)
# #[1] "9" "10" "9"
# C9.ref = CTS[which(names(CTS)=='9')]
# C10.ref = CTS['10']
GS.CTS <- read.table('GS.CTS.E16.5_at.least.3identificaitons_fr.5predicitons.txt')
C2.ref <- list('2'=GS.CTS[,1])
MCIbottom # 2
i=1
F1.C2 <- F1.C2.ctl <- list()
for(m in 1:length(getTopMCI.gene.minsize)){
load(file=paste0('stability/BioTIP_res_20runs_MCIbottom',MCIbottom[i],
'_Modulesize',getTopMCI.gene.minsize[m],'.RData'))
F1.CTS.C2 <- F1.bk.C2 <- array(dim=n.scability)
for(j in 1:n.scability){
# evaluate the F1 score for two CTS identificitons
set2 <- res[[j]]$CTS.candidate[which(res[[j]]$significant)]
F1.CTS.C2[j] <- F_score.CTS(C2.ref, set2, weight=TRUE)$F1
# negative control
n.module <- lengths(set2)
random.set2 <- lapply(n.module, function(x) sample(rownames(sce), x))
F1.bk.C2[j] <- F_score.CTS(C2.ref, random.set2, weight=TRUE)$F1
}
F1.C2[[m]] = F1.CTS.C2
F1.C2.ctl[[m]] = F1.bk.C2
}
names(F1.C2) <- names(F1.C2.ctl) <- getTopMCI.gene.minsize
save(F1.C2, F1.C2.ctl,
file='stability/F1.CTS_MCIbottom2.RData')
lapply(F1.C2, mean) %>% unlist()
# 10 20 30
# 0.12294563 0.12294563 0.08011003
lapply(F1.C2.ctl, mean) %>% unlist()
#0.0024092971 0.0039024701 0.0008333333
df <- data.frame(F1 = c(unlist(F1.C2.ctl),unlist(F1.C2)),
minModuleSize = c(rep(getTopMCI.gene.minsize, lengths(F1.C2.ctl)),
rep(getTopMCI.gene.minsize, lengths(F1.C2))),
CT = c(rep('C2', sum(lengths(F1.C2.ctl))+sum(lengths(F1.C2)))),
CTS = c(rep('ctl', sum(lengths(F1.C2.ctl))), rep('CTS', sum(lengths(F1.C2))))
)
df$minModuleSize <- as.character(df$minModuleSize)
df$CTS <- factor(df$CTS, levels = c('ctl','CTS'))
df$color.lab <- paste0(df$minModuleSize,df$CTS)
df$color.lab <- factor(df$color.lab, levels=c('10ctl','10CTS','20ctl','20CTS','30ctl','30CTS'))
# To ensure that easy and difficult CT state have equal influence on the final score,
# we normalized the scores at each CT state across the different MCIbottom setting.
df$Norm.F1 <- Normalize.F1(df$F1)
library(ggbeeswarm)
library(grid)
library(gridExtra)
library(ggpubr)
p1 <- ggplot(data=df, aes(x=CT, y=F1, color=color.lab)) +
geom_violin(position = position_dodge(width = 0.5)) +
geom_quasirandom(dodge.width = 0.5, varwidth = TRUE) +
xlab('predicted for cluster') + ylab('F1 score for the CTS') +
scale_color_manual(values=c("grey", "orange","grey", "green","grey","blue"))
p2 <- ggplot(data=df, aes(x=CT, y=Norm.F1, color=color.lab)) +
geom_violin(position = position_dodge(width = 0.5)) +
geom_quasirandom(dodge.width = 0.5, varwidth = TRUE) +
xlab('predicted for cluster') + ylab('Normalized F1 score for the CTS') +
scale_color_manual(values=c("grey", "orange","grey", "green","grey","blue"))
pdf(file='stability/CTS.F1_variable.minModuleSize.pdf')
gridExtra::grid.arrange(
p1, p2, top = "CTS stability", bottom = "different module size settings"
,ncol=1)
dev.off()
tmp <- compare_means(Norm.F1 ~ color.lab, data=df, group='CT', method='t.test')
subset(tmp, group1=='10ctl' & group2=='10CTS')
# # A tibble: 2 x 9
## A tibble: 1 x 9
# CT .y. group1 group2 p p.adj p.format p.signif method
# <chr> <chr> <chr> <chr> <dbl> <dbl> <chr> <chr> <chr>
# 1 C2 Norm.F1 10ctl 10CTS 0.00000240 0.000031 2.4e-06 **** T-test
subset(tmp, group1=='20ctl' & group2=='20CTS')
# A tibble: 1 x 9
# CT .y. group1 group2 p p.adj p.format p.signif method
# <chr> <chr> <chr> <chr> <dbl> <dbl> <chr> <chr> <chr>
# 1 C2 Norm.F1 20ctl 20CTS 0.00000285 0.000031 2.8e-06 **** T-test
subset(tmp, group1=='30ctl' & group2=='30CTS')
# A tibble: 1 x 9
# CT .y. group1 group2 p p.adj p.format p.signif method
# <chr> <chr> <chr> <chr> <dbl> <dbl> <chr> <chr> <chr>
# 1 C2 Norm.F1 30ctl 30CTS 0.000284 0.0026 0.00028 *** T-test
}
###################################################################
## section 5) TSNE plot for 3.2k HGV, 131 cells along the AT2 trajectory
###################################################################
{
fid <- "C_Leiden_0.4/"
load(file=paste0(fid,"BioTIP_top0.1FDR0.2_optimized_local.HVG_selection.RData"))
names(testres)
#[1] "1" "2" "3"
library(scater)
pdf(file='tsne_131cells_C_Leiden_0.4.pdf')
plotReducedDim(sce, dimred='TSNE', shape_by='age',
colour_by = "C_Leiden_0.4",
text_by='putative_cell_type',
text_size = 4, text_colour='black', point_size=2) +
ggtitle('putative_cell_type') #+ ylim(5,20)
plotReducedDim(sce, dimred='TSNE', shape_by='age',
colour_by = 'putative_cell_type',
text_by="C_Leiden_0.4",
text_size = 4, text_colour='black', point_size=2) +
ggtitle('putative_cell_type') #+ ylim(5,20)
dev.off()
}
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