synthetic_correctBias_nearsamples <- function(tumor, counts, bin.size = 100000, rm.centromere = TRUE, K = 5,
targetAnnotateBins = NULL, saveplot = TRUE, centromereBins = NULL, chrX = FALSE,
plot = TRUE, result.dir = NULL, prefix = NULL, reads.threshold = 50){
options(scipen = 50)
sampleData <- tumor
if(substr(sampleData[1, 1], 1, 3) == "chr") {
sampleData[, 1] <- gsub("chr", "", sampleData[, 1])
}
sampleData[, 1] <- gsub("X", "23", sampleData[, 1])
sampleData[, 1] <- gsub("Y", "24", sampleData[, 1])
if(nrow(counts) != nrow(sampleData)){
stop("Input data and \"counts\" size don't match!")
}
all.value <- NULL
for(i in 1:ncol(counts)){
normal <- counts[, i]
sampleData[sampleData[, "reads"] < reads.threshold, "reads"] <- 0
normal[normal < reads.threshold] <- 0
ratio <- sampleData[, "reads"]/normal
ratio <- ratio[is.finite(ratio) & ratio != 0 ]
ratio <- ratio/median(ratio, na.rm = T)
log.ratio <- log2(ratio[!is.na(ratio)]+0.001)
diff.sumsquare <- abs(diff(log.ratio))^2
variance <- mean(diff.sumsquare[diff.sumsquare < quantile(diff.sumsquare, 0.9)])
all.value <- c(all.value, variance)
}
minIs <- order(all.value)[1:K]
if(K > 1){
normal <- apply(counts[, minIs], 1, median)
} else {
normal <- counts[, minIs]
}
normal[normal < reads.threshold] <- 0
ratio <- sampleData[, "reads"]/normal
ratio <- ratio/median(ratio[is.finite(ratio) & ratio != 0], na.rm = T)
ratio[is.infinite(ratio) | is.nan(ratio)] <- NA
ratio.res <- data.frame(sampleData[, c(1:3)], ratio)
if(rm.centromere == TRUE) {
if(is.null(centromereBins)){
if(!bin.size %in% c(10000, 25000, 50000, 100000)){
stop(paste0("SynthEx doesn't have centromere bins pre-calculated for bin size of", bin.size, "; Please use createCentromereBins()
to generate the required file or consider to use another bin size."))
} else {
data(CentromereAnnotations)
ss <- paste0("centromere <- CentromereAnnotations$bin", bin.size)
eval(parse(text=ss))
}
} else {
centromere <- read.delim(centromereBins, header = F, as.is = T)
}
centromere[, 2] <- centromere[, 2] + 1
centromere.IDs <- paste0(centromere[, 1], ":", centromere[, 2])
ratio.IDs <- paste0(ratio.res[, "chr"], ":", ratio.res[, "start"])
ratio.res <- ratio.res[! ratio.IDs%in%centromere.IDs, ]
}
ratio <- ratio.res[, "ratio"]
ratio.IDs <- paste0(ratio.res[, "chr"], ":", ratio.res[, "start"])
if(is.null(targetAnnotateBins)){
if(! bin.size %in% c(10000, 25000, 50000, 100000)){
stop(paste0("SynthEx doesn't have centromere bins pre-calculated for bin size of", bin.size, "; Please use createTargetBins()
to generate the required file or consider to use another bin size."))
} else {
data(TargetAnnotations)
ss <- paste0("target <- TargetAnnotations$bin", bin.size)
eval(parse(text=ss))
}
} else {
target <- read.delim(targetAnnotateBins, header = F, as.is = T)
}
if(substr(target[1, 4], 1, 3) == "chr") {
target[, 4] <- gsub("chr", "", target[, 4])
}
target[, 5] <- target[, 5]+1
target.IDs <- paste0(target[, 4], ":", target[, 5])
target.IDs <- target.IDs[!duplicated(target.IDs)]
#identify target and off target regions
target.status <- ifelse(ratio.IDs %in% target.IDs, "target", "non-target")
all.dis.target.left <- NULL
all.dis.target.right <- NULL
all.dis.non.target.left <- NULL
all.dis.non.target.right <- NULL
all.dis.within.target.left <- NULL
all.dis.within.target.right <- NULL
allchrs <- 1:22
for(i in allchrs){
chr.ratio <- ratio[grep(paste0(i, ":"), ratio.IDs)]
chr.names <- ratio.IDs[grep(paste0(i, ":"), ratio.IDs)]
chr.pos <- unlist(strsplit(chr.names, ":"))[seq(2, 2*length(chr.names), by=2)]
chr.pos <- (as.numeric(chr.pos)-1)/bin.size
chr.target.status <- target.status[grep(paste0(i, ":"), ratio.IDs)]
chr.ratio <- chr.ratio[order(chr.pos)]
target.bins <- c(1:length(chr.ratio))[chr.target.status == "target"]
all.neighbor.bins <- sort(unique(c(target.bins - 1, target.bins, target.bins + 1)))
sub.chr.ratio <- chr.ratio[all.neighbor.bins]
#Calculate Target-NonTarget Ratios
select.target.bins <- target.bins[!target.bins %in% c(target.bins - 1) & !target.bins %in% c(target.bins + 1, 1, nrow(chr.ratio))]
select.target.bins.ratio <- chr.ratio[select.target.bins]
select.left.target.bins.ratio <- chr.ratio[select.target.bins - 1]
select.right.target.bins.ratio <- chr.ratio[select.target.bins + 1]
dis.target.left <- select.target.bins.ratio - select.left.target.bins.ratio
dis.target.right <- select.target.bins.ratio - select.right.target.bins.ratio
#Calculate NonTarget-NonTarget ratios
non.target.bins <- c(1:length(chr.ratio))[-c(target.bins, 1, nrow(chr.ratio))]
non.target.bins.ratio <- chr.ratio[non.target.bins]
non.left.target.bins.ratio <- chr.ratio[non.target.bins - 1]
non.right.target.bins.ratio <- chr.ratio[non.target.bins + 1]
dis.non.target.left <- non.target.bins.ratio - non.left.target.bins.ratio
dis.non.target.right <- non.target.bins.ratio- non.right.target.bins.ratio
#Calculate Target-Target Ratios
select.left.target.target.bins <- target.bins[target.bins %in% c(target.bins - 1)]
select.right.target.target.bins <- target.bins[target.bins %in% c(target.bins + 1)]
dis.target.target.left <- chr.ratio[select.left.target.target.bins] - chr.ratio[select.left.target.target.bins + 1]
dis.target.target.right <- chr.ratio[select.right.target.target.bins] - chr.ratio[select.right.target.target.bins - 1]
all.dis.target.left <- c(all.dis.target.left, dis.target.left)
all.dis.target.right <- c(all.dis.target.right, dis.target.right)
all.dis.non.target.left <- c(all.dis.non.target.left, dis.non.target.left)
all.dis.non.target.right <- c(all.dis.non.target.right, dis.non.target.right)
all.dis.within.target.left <- c(all.dis.within.target.left, dis.target.target.left)
all.dis.within.target.right <- c(all.dis.within.target.right, dis.target.target.right)
}
all.dis.target <- c(all.dis.target.left, all.dis.target.right)
all.dis.non.target <- c(all.dis.non.target.left, all.dis.non.target.right)
all.dis.within.target <- c(all.dis.within.target.left, all.dis.within.target.right)
d1 <- density(all.dis.target, na.rm = T)
d2 <- density(all.dis.non.target, na.rm = T)
d3 <- density(all.dis.within.target.left, na.rm = T)
all.dis.others <- c(all.dis.non.target, all.dis.within.target)
target.data <- all.dis.target[!is.nan(all.dis.target) & !is.infinite(all.dis.target)]
nontarget.data <- all.dis.others[!is.nan(all.dis.others) & !is.infinite(all.dis.others)]
target.sd <- sd(target.data, na.rm = T)
non.target.sd <- sd(nontarget.data, na.rm = T)
target.max <- mean(target.data, na.rm = T)
target.bias.statistics <- round(c(target.max, mean(nontarget.data, na.rm = T), target.sd, non.target.sd), 3)
names(target.bias.statistics) <- c("TargetMean", "NonTargetMean", "TargetSd", "NonTargetSd")
if(plot == TRUE) {
if(abs(target.max) > 1){
print("Caution: The ratios between bins at the boundaries of target and non-target regions deviate a lot from zero, indicating the ratios from the target and non-target regions are very different")
if(saveplot == TRUE){
if(!is.null(prefix)){
pdf(paste0(result.dir, "/", prefix, "-DensityPlot--CautionSample.pdf"))
} else {
pdf(paste0(result.dir, "/DensityPlot--CautionSample.pdf"))
}
}
plot(d1, col = "red", xlim = c(-3, 3), ylim = c(0, max(d1$y, d2$y, d3$y)), main = prefix, xlab = "Distance", lwd = 2)
lines(d2, lwd = 2)
lines(d3, col = "blue", lwd = 2)
legend("topright", c("Target-Non", "Non-Non", "Target-Target"), lty = 1, lwd = 2, col = c("red", "black", "blue"))
if(saveplot == TRUE){
dev.off()
}
} else{
if(saveplot == TRUE){
if(!is.null(prefix)){
pdf(paste0(result.dir, "/", prefix, "-DensityPlot.pdf"))
} else {
pdf(paste0(result.dir, "/DensityPlot.pdf"))
}
}
plot(d1, col = "red", xlim = c(-3, 3), ylim = c(0, max(d1$y, d2$y, d3$y)), main = prefix, xlab = "Successive Difference", lwd = 2)
lines(d2, lwd = 2)
lines(d3, col = "blue", lwd = 2)
legend("topright", c("Target-Non", "Non-Non", "Target-Target"), lty = 1, lwd = 2, col = c("red", "black", "blue"))
if(saveplot == TRUE){
dev.off()
}
}
}
#Adjustment factor
bias <- target.max
ratio <- ifelse(!is.na(ratio) & !is.nan(ratio) & ratio.IDs %in% target.IDs, ratio - bias, ratio)
ratio <- ifelse(!is.na(ratio) & !is.nan(ratio) & ratio < 0, 0, ratio)
ratio[is.infinite(ratio) | is.nan(ratio)] <- NA
ratio <- round(ratio/median(ratio, na.rm = T), 3)
ratio.res[, "ratio"] <- ratio
if(!is.null(prefix)){
log2ratio <- ratio.res
log2ratio[, "start"] <- log2ratio[, "start"] - 1
log2ratio[, "ratio"] <- log2(ratio.res[, "ratio"]+0.0001)
write.table(log2ratio, paste0(result.dir, "/", prefix, "_Ratio.bed"), sep = "\t", quote = FALSE, col.names = TRUE, row.names = FALSE)
} else {
log2ratio <- ratio.res
log2ratio[, "start"] <- log2ratio[, "start"] - 1
log2ratio[, "ratio"] <- log2(ratio.res[, "ratio"]+0.0001)
write.table(log2ratio, paste0(result.dir, "/Ratio.bed"), sep = "\t", quote = FALSE, col.names = TRUE, row.names = FALSE)
}
if(chrX == FALSE){
ratio.res <- ratio.res[ratio.res[, "chr"] != 23 & ratio.res[, "chr"] != 24, ]
}
res <- list(target.bias.statistics, ratio.res, TRUE, minIs)
names(res) <- c("TargetBiasStatistics", "Ratio", "Synthetic", "Usednormal")
class(res) <- "RatioCorrectBiasInTargets"
return(res)
}
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