我选好了细胞拿去推断
你再给我过滤一遍 吃掉了我一部分细胞。。。
还不能设置参数取消过滤
坑爹的R包千篇一律，
被迫debug的少年万中无一。
我把你过滤的源码注释掉即可。
这样既可以保证所有细胞被推断。
copykat <- function (rawmat = rawdata, id.type = "S", cell.line = "no",
ngene.chr = 0, LOW.DR = 0.05, UP.DR = 0.1, win.size = 25,
norm.cell.names = "", KS.cut = 0.1, sam.name = "", distance = "euclidean",
n.cores = 1)
{
start_time <- Sys.time()
set.seed(1)
sample.name <- paste(sam.name, "_copykat_", sep = "")
print("running copykat v1.0.4")
print("step1: read and filter data ...")
print(paste(nrow(rawmat), " genes, ", ncol(rawmat), " cells in raw data",
sep = ""))
# genes.raw <- apply(rawmat, 2, function(x) (sum(x > 0)))
# if (sum(genes.raw > 200) == 0)
# stop("none cells have more than 200 genes")
# if (sum(genes.raw < 100) > 1) {
# rawmat <- rawmat[, -which(genes.raw < 200)]
# print(paste("filtered out ", sum(genes.raw <= 200),
# " cells with less than 200 genes; remaining ", ncol(rawmat),
# " cells", sep = ""))
# }
der <- apply(rawmat, 1, function(x) (sum(x > 0)))/ncol(rawmat)
if (sum(der > LOW.DR) >= 1) {
rawmat <- rawmat[which(der > LOW.DR), ]
print(paste(nrow(rawmat), " genes past LOW.DR filtering",
sep = ""))
}
WNS1 <- "data quality is ok"
if (nrow(rawmat) < 7000) {
WNS1 <- "low data quality"
UP.DR <- LOW.DR
print("WARNING: low data quality; assigned LOW.DR to UP.DR...")
}
print("step 2: annotations gene coordinates ...")
anno.mat <- annotateGenes.hg20(mat = rawmat, ID.type = id.type)
anno.mat <- anno.mat[order(anno.mat$abspos, decreasing = FALSE),
]
HLAs <- anno.mat$hgnc_symbol[grep("^HLA-", anno.mat$hgnc_symbol)]
toRev <- which(anno.mat$hgnc_symbol %in% c(as.vector(cyclegenes[[1]]),
HLAs))
# if (length(toRev) > 0) {
# anno.mat <- anno.mat[-toRev, ]
# }
# ToRemov2 <- NULL
# for (i in 8:ncol(anno.mat)) {
# cell <- cbind(anno.mat$chromosome_name, anno.mat[, i])
# cell <- cell[cell[, 2] != 0, ]
# if (length(as.numeric(cell)) < 5) {
# rm <- colnames(anno.mat)[i]
# ToRemov2 <- c(ToRemov2, rm)
# }
# else if (length(rle(cell[, 1])$length) < 23 | min(rle(cell[,
# 1])$length) < ngene.chr) {
# rm <- colnames(anno.mat)[i]
# ToRemov2 <- c(ToRemov2, rm)
# }
# i <- i + 1
# }
# if (length(ToRemov2) == (ncol(anno.mat) - 7))
# stop("all cells are filtered")
# if (length(ToRemov2) > 0) {
# anno.mat <- anno.mat[, -which(colnames(anno.mat) %in%
# ToRemov2)]
# }
rawmat3 <- data.matrix(anno.mat[, 8:ncol(anno.mat)])
norm.mat <- log(sqrt(rawmat3) + sqrt(rawmat3 + 1))
norm.mat <- apply(norm.mat, 2, function(x) (x <- x - mean(x)))
colnames(norm.mat) <- colnames(rawmat3)
print("step 3: smoothing data with dlm ...")
dlm.sm <- function(c) {
model <- dlm::dlmModPoly(order = 1, dV = 0.16, dW = 0.001)
x <- dlm::dlmSmooth(norm.mat[, c], model)$s
x <- x[2:length(x)]
x <- x - mean(x)
}
test.mc <- parallel::mclapply(1:ncol(norm.mat), dlm.sm,
mc.cores = n.cores)
norm.mat.smooth <- matrix(unlist(test.mc), ncol = ncol(norm.mat),
byrow = FALSE)
colnames(norm.mat.smooth) <- colnames(norm.mat)
print("step 4: measuring baselines ...")
if (cell.line == "yes") {
print("running pure cell line mode")
relt <- baseline.synthetic(norm.mat = norm.mat.smooth,
min.cells = 10, n.cores = n.cores)
norm.mat.relat <- relt$expr.relat
CL <- relt$cl
WNS <- "run with cell line mode"
preN <- NULL
}
else if (length(norm.cell.names) > 1) {
NNN <- length(colnames(norm.mat.smooth)[which(colnames(norm.mat.smooth) %in%
norm.cell.names)])
print(paste(NNN, " known normal cells found in dataset",
sep = ""))
if (NNN == 0)
stop("known normal cells provided; however none existing in testing dataset")
print("run with known normal...")
basel <- apply(norm.mat.smooth[, which(colnames(norm.mat.smooth) %in%
norm.cell.names)], 1, median)
print("baseline is from known input")
d <- parallelDist::parDist(t(norm.mat.smooth), threads = n.cores,
method = "euclidean")
km <- 6
fit <- hclust(d, method = "ward.D2")
CL <- cutree(fit, km)
while (!all(table(CL) > 5)) {
km <- km - 1
CL <- cutree(fit, k = km)
if (km == 2) {
break
}
}
WNS <- "run with known normal"
preN <- norm.cell.names
norm.mat.relat <- norm.mat.smooth - basel
}
else {
basa <- baseline.norm.cl(norm.mat.smooth = norm.mat.smooth,
min.cells = 5, n.cores = n.cores)
basel <- basa$basel
WNS <- basa$WNS
preN <- basa$preN
CL <- basa$cl
if (WNS == "unclassified.prediction") {
Tc <- colnames(rawmat)[which(as.numeric(apply(rawmat[which(rownames(rawmat) %in%
c("PTPRC", "LYZ", "PECAM1")), ], 2, mean)) >
1)]
length(Tc)
preN <- intersect(Tc, colnames(norm.mat.smooth))
if (length(preN) > 5) {
print("start manual mode")
WNS <- paste("copykat failed in locating normal cells; manual adjust performed with ",
length(preN), " immune cells", sep = "")
print(WNS)
basel <- apply(norm.mat.smooth[, which(colnames(norm.mat.smooth) %in%
preN)], 1, mean)
}
else {
basa <- baseline.GMM(CNA.mat = norm.mat.smooth,
max.normal = 5, mu.cut = 0.05, Nfraq.cut = 0.99,
RE.before = basa, n.cores = n.cores)
basel <- basa$basel
WNS <- basa$WNS
preN <- basa$preN
}
}
norm.mat.relat <- norm.mat.smooth - basel
}
DR2 <- apply(rawmat3, 1, function(x) (sum(x > 0)))/ncol(rawmat3)
norm.mat.relat <- norm.mat.relat[which(DR2 >= UP.DR), ]
anno.mat2 <- anno.mat[which(DR2 >= UP.DR), ]
# ToRemov3 <- NULL
# for (i in 8:ncol(anno.mat2)) {
# cell <- cbind(anno.mat2$chromosome_name, anno.mat2[,
# i])
# cell <- cell[cell[, 2] != 0, ]
# if (length(as.numeric(cell)) < 5) {
# rm <- colnames(anno.mat2)[i]
# ToRemov3 <- c(ToRemov3, rm)
# }
# else if (length(rle(cell[, 1])$length) < 23 | min(rle(cell[,
# 1])$length) < ngene.chr) {
# rm <- colnames(anno.mat2)[i]
# ToRemov3 <- c(ToRemov3, rm)
# }
# i <- i + 1
# }
# if (length(ToRemov3) == ncol(norm.mat.relat))
# stop("all cells are filtered")
# if (length(ToRemov3) > 0) {
# norm.mat.relat <- norm.mat.relat[, -which(colnames(norm.mat.relat) %in%
# ToRemov3)]
# }
CL <- CL[which(names(CL) %in% colnames(norm.mat.relat))]
CL <- CL[order(match(names(CL), colnames(norm.mat.relat)))]
print("step 5: segmentation...")
results <- CNA.MCMC(clu = CL, fttmat = norm.mat.relat, bins = win.size,
cut.cor = KS.cut, n.cores = n.cores)
if (length(results$breaks) < 25) {
print("too few breakpoints detected; decreased KS.cut to 50%")
results <- CNA.MCMC(clu = CL, fttmat = norm.mat.relat,
bins = win.size, cut.cor = 0.5 * KS.cut, n.cores = n.cores)
}
if (length(results$breaks) < 25) {
print("too few breakpoints detected; decreased KS.cut to 75%")
results <- CNA.MCMC(clu = CL, fttmat = norm.mat.relat,
bins = win.size, cut.cor = 0.5 * 0.5 * KS.cut, n.cores = n.cores)
}
if (length(results$breaks) < 25)
stop("too few segments; try to decrease KS.cut; or improve data")
colnames(results$logCNA) <- colnames(norm.mat.relat)
results.com <- apply(results$logCNA, 2, function(x) (x <- x -
mean(x)))
RNA.copycat <- cbind(anno.mat2[, 1:7], results.com)
write.table(RNA.copycat, paste(sample.name, "CNA_raw_results_gene_by_cell.txt",
sep = ""), sep = "\t", row.names = FALSE, quote = F)
print("step 6: convert to genomic bins...")
Aj <- convert.all.bins.hg20(DNA.mat = DNA.hg20, RNA.mat = RNA.copycat,
n.cores = n.cores)
uber.mat.adj <- data.matrix(Aj$RNA.adj[, 4:ncol(Aj$RNA.adj)])
print("step 7: adjust baseline ...")
if (cell.line == "yes") {
mat.adj <- data.matrix(Aj$RNA.adj[, 4:ncol(Aj$RNA.adj)])
write.table(cbind(Aj$RNA.adj[, 1:3], mat.adj), paste(sample.name,
"CNA_results.txt", sep = ""), sep = "\t", row.names = FALSE,
quote = F)
if (distance == "euclidean") {
hcc <- hclust(parallelDist::parDist(t(mat.adj),
threads = n.cores, method = distance), method = "ward.D")
}
else {
hcc <- hclust(as.dist(1 - cor(mat.adj, method = distance)),
method = "ward.D")
}
saveRDS(hcc, file = paste(sample.name, "clustering_results.rds",
sep = ""))
print("step 8: ploting heatmap ...")
my_palette <- colorRampPalette(rev(RColorBrewer::brewer.pal(n = 3,
name = "RdBu")))(n = 999)
chr <- as.numeric(Aj$DNA.adj$chrom)%%2 + 1
rbPal1 <- colorRampPalette(c("black", "grey"))
CHR <- rbPal1(2)[as.numeric(chr)]
chr1 <- cbind(CHR, CHR)
if (ncol(mat.adj) < 3000) {
h <- 10
}
else {
h <- 15
}
col_breaks = c(seq(-1, -0.4, length = 50), seq(-0.4,
-0.2, length = 150), seq(-0.2, 0.2, length = 600),
seq(0.2, 0.4, length = 150), seq(0.4, 1, length = 50))
if (distance == "euclidean") {
jpeg(paste(sample.name, "heatmap.jpeg", sep = ""),
height = h * 250, width = 4000, res = 100)
heatmap.3(t(mat.adj), dendrogram = "r", distfun = function(x) parallelDist::parDist(x,
threads = n.cores, method = distance), hclustfun = function(x) hclust(x,
method = "ward.D"), ColSideColors = chr1, Colv = NA,
Rowv = TRUE, notecol = "black", col = my_palette,
breaks = col_breaks, key = TRUE, keysize = 1,
density.info = "none", trace = "none", cexRow = 0.1,
cexCol = 0.1, cex.main = 1, cex.lab = 0.1, symm = F,
symkey = F, symbreaks = T, cex = 1, main = paste(WNS1,
"; ", WNS, sep = ""), cex.main = 4, margins = c(10,
10))
dev.off()
}
else {
jpeg(paste(sample.name, "heatmap.jpeg", sep = ""),
height = h * 250, width = 4000, res = 100)
heatmap.3(t(mat.adj), dendrogram = "r", distfun = function(x) as.dist(1 -
cor(t(x), method = distance)), hclustfun = function(x) hclust(x,
method = "ward.D"), ColSideColors = chr1, Colv = NA,
Rowv = TRUE, notecol = "black", col = my_palette,
breaks = col_breaks, key = TRUE, keysize = 1,
density.info = "none", trace = "none", cexRow = 0.1,
cexCol = 0.1, cex.main = 1, cex.lab = 0.1, symm = F,
symkey = F, symbreaks = T, cex = 1, main = paste(WNS1,
"; ", WNS, sep = ""), cex.main = 4, margins = c(10,
10))
dev.off()
}
end_time <- Sys.time()
print(end_time - start_time)
reslts <- list(cbind(Aj$RNA.adj[, 1:3], mat.adj), hcc)
names(reslts) <- c("CNAmat", "hclustering")
return(reslts)
}
else {
if (distance == "euclidean") {
hcc <- hclust(parallelDist::parDist(t(uber.mat.adj),
threads = n.cores, method = distance), method = "ward.D")
}
else {
hcc <- hclust(as.dist(1 - cor(uber.mat.adj, method = distance)),
method = "ward.D")
}
hc.umap <- cutree(hcc, 2)
names(hc.umap) <- colnames(results.com)
cl.ID <- NULL
for (i in 1:max(hc.umap)) {
cli <- names(hc.umap)[which(hc.umap == i)]
pid <- length(intersect(cli, preN))/length(cli)
cl.ID <- c(cl.ID, pid)
i <- i + 1
}
com.pred <- names(hc.umap)
com.pred[which(hc.umap == which(cl.ID == max(cl.ID)))] <- "diploid"
com.pred[which(hc.umap == which(cl.ID == min(cl.ID)))] <- "nondiploid"
names(com.pred) <- names(hc.umap)
results.com.rat <- uber.mat.adj - apply(uber.mat.adj[,
which(com.pred == "diploid")], 1, mean)
results.com.rat <- apply(results.com.rat, 2, function(x) (x <- x -
mean(x)))
results.com.rat.norm <- results.com.rat[, which(com.pred ==
"diploid")]
dim(results.com.rat.norm)
cf.h <- apply(results.com.rat.norm, 1, sd)
base <- apply(results.com.rat.norm, 1, mean)
adjN <- function(j) {
a <- results.com.rat[, j]
a[abs(a - base) <= 0.25 * cf.h] <- mean(a)
a
}
mc.adjN <- parallel::mclapply(1:ncol(results.com.rat),
adjN, mc.cores = n.cores)
adj.results <- matrix(unlist(mc.adjN), ncol = ncol(results.com.rat),
byrow = FALSE)
colnames(adj.results) <- colnames(results.com.rat)
rang <- 0.5 * (max(adj.results) - min(adj.results))
mat.adj <- adj.results/rang
print("step 8: final prediction ...")
if (distance == "euclidean") {
hcc <- hclust(parallelDist::parDist(t(mat.adj),
threads = n.cores, method = distance), method = "ward.D")
}
else {
hcc <- hclust(as.dist(1 - cor(mat.adj, method = distance)),
method = "ward.D")
}
hc.umap <- cutree(hcc, 2)
names(hc.umap) <- colnames(results.com)
saveRDS(hcc, file = paste(sample.name, "clustering_results.rds",
sep = ""))
cl.ID <- NULL
for (i in 1:max(hc.umap)) {
cli <- names(hc.umap)[which(hc.umap == i)]
pid <- length(intersect(cli, preN))/length(cli)
cl.ID <- c(cl.ID, pid)
i <- i + 1
}
com.preN <- names(hc.umap)
com.preN[which(hc.umap == which(cl.ID == max(cl.ID)))] <- "diploid"
com.preN[which(hc.umap == which(cl.ID == min(cl.ID)))] <- "aneuploid"
names(com.preN) <- names(hc.umap)
if (WNS == "unclassified.prediction") {
com.preN[which(com.preN == "diploid")] <- "c1:diploid:low.conf"
com.preN[which(com.preN == "nondiploid")] <- "c2:aneuploid:low.conf"
}
print("step 9: saving results...")
res <- cbind(names(com.preN), com.preN)
colnames(res) <- c("cell.names", "copykat.pred")
write.table(res, paste(sample.name, "prediction.txt",
sep = ""), sep = "\t", row.names = FALSE, quote = FALSE)
write.table(cbind(Aj$RNA.adj[, 1:3], mat.adj), paste(sample.name,
"CNA_results.txt", sep = ""), sep = "\t", row.names = FALSE,
quote = F)
print("step 10: ploting heatmap ...")
my_palette <- colorRampPalette(rev(RColorBrewer::brewer.pal(n = 3,
name = "RdBu")))(n = 999)
chr <- as.numeric(Aj$DNA.adj$chrom)%%2 + 1
rbPal1 <- colorRampPalette(c("black", "grey"))
CHR <- rbPal1(2)[as.numeric(chr)]
chr1 <- cbind(CHR, CHR)
rbPal5 <- colorRampPalette(RColorBrewer::brewer.pal(n = 8,
name = "Dark2")[2:1])
compreN_pred <- rbPal5(2)[as.numeric(factor(com.preN))]
cells <- rbind(compreN_pred, compreN_pred)
if (ncol(mat.adj) < 3000) {
h <- 10
}
else {
h <- 15
}
col_breaks = c(seq(-1, -0.4, length = 50), seq(-0.4,
-0.2, length = 150), seq(-0.2, 0.2, length = 600),
seq(0.2, 0.4, length = 150), seq(0.4, 1, length = 50))
if (distance == "euclidean") {
jpeg(paste(sample.name, "heatmap.jpeg", sep = ""),
height = h * 250, width = 4000, res = 100)
heatmap.3(t(mat.adj), dendrogram = "r", distfun = function(x) parallelDist::parDist(x,
threads = n.cores, method = distance), hclustfun = function(x) hclust(x,
method = "ward.D"), ColSideColors = chr1, RowSideColors = cells,
Colv = NA, Rowv = TRUE, notecol = "black", col = my_palette,
breaks = col_breaks, key = TRUE, keysize = 1,
density.info = "none", trace = "none", cexRow = 0.1,
cexCol = 0.1, cex.main = 1, cex.lab = 0.1, symm = F,
symkey = F, symbreaks = T, cex = 1, main = paste(WNS1,
"; ", WNS, sep = ""), cex.main = 4, margins = c(10,
10))
legend("topright", paste("pred.", names(table(com.preN)),
sep = ""), pch = 15, col = RColorBrewer::brewer.pal(n = 8,
name = "Dark2")[2:1], cex = 1)
dev.off()
}
else {
jpeg(paste(sample.name, "heatmap.jpeg", sep = ""),
height = h * 250, width = 4000, res = 100)
heatmap.3(t(mat.adj), dendrogram = "r", distfun = function(x) as.dist(1 -
cor(t(x), method = distance)), hclustfun = function(x) hclust(x,
method = "ward.D"), ColSideColors = chr1, RowSideColors = cells,
Colv = NA, Rowv = TRUE, notecol = "black", col = my_palette,
breaks = col_breaks, key = TRUE, keysize = 1,
density.info = "none", trace = "none", cexRow = 0.1,
cexCol = 0.1, cex.main = 1, cex.lab = 0.1, symm = F,
symkey = F, symbreaks = T, cex = 1, main = paste(WNS1,
"; ", WNS, sep = ""), cex.main = 4, margins = c(10,
10))
legend("topright", paste("pred.", names(table(com.preN)),
sep = ""), pch = 15, col = RColorBrewer::brewer.pal(n = 8,
name = "Dark2")[2:1], cex = 1)
dev.off()
}
end_time <- Sys.time()
print(end_time - start_time)
reslts <- list(res, cbind(Aj$RNA.adj[, 1:3], mat.adj),
hcc)
names(reslts) <- c("prediction", "CNAmat", "hclustering")
return(reslts)
}
}