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R/operations.R
In genArise: Microarray Analysis tool
Defines functions
spotUnique
alter.unique
meanUnique
global.norm
grid.norm
bg.correct
Documented in
alter.unique
bg.correct
global.norm
grid.norm
meanUnique
spotUnique
require(locfit)
bg.correct <- function(mySpot){
spot <- as.list(attr(mySpot, "spotData"))
spot$Cy3 <- spot$Cy3 - spot$BgCy3
spot$Cy5 <- spot$Cy5 - spot$BgCy5
spotName <- attr(mySpot, "name")
if(is.null(spot$Symdesc)){
SD <- list(Cy3 = spot$Cy3, Cy5 = spot$Cy5, BgCy3 = spot$BgCy3, BgCy5 = spot$BgCy5, Id = spot$Id)
}
else{
SD <- list(Cy3 = spot$Cy3, Cy5 = spot$Cy5, BgCy3 = spot$BgCy3, BgCy5 = spot$BgCy5, Id = spot$Id, Symdesc = spot$Symdesc)
}
new("Spot", name = spotName, spotData= SD)
}
# Filtering
# Intensity-based filtering of array elements
filter.spot <- function (mySpot){
spot <- attr(mySpot, "spotData")
cys <- (!is.na(spot$Cy3)) & (!is.na(spot$Cy5)) & (!is.na(spot$BgCy3)) & (!is.na(spot$BgCy5)) & (!is.na(spot$Id))
spot$Cy3 <- spot$Cy3[cys]
spot$Cy5 <- spot$Cy5[cys]
spot$BgCy3 <- spot$BgCy3[cys]
spot$BgCy5 <- spot$BgCy5[cys]
spot$Id <- spot$Id[cys]
condition <- spot$Cy3 > 2 * sd(spot$BgCy3) & spot$Cy5 > 2 * sd(spot$BgCy5)
spot$Cy3 <- spot$Cy3[condition]
spot$Cy5 <- spot$Cy5[condition]
spot$BgCy3 <- spot$BgCy3[condition]
spot$BgCy5 <- spot$BgCy5[condition]
spot$Id <- spot$Id[condition]
if(!is.null(spot$Symdesc)){
spot$Symdesc <- spot$Symdesc[cys]
spot$Symdesc <- spot$Symdesc[condition]
newSpot <- list(Cy3 = spot$Cy3 , Cy5= spot$Cy5 , BgCy3 = spot$BgCy3 , BgCy5 = spot$BgCy5, Id = spot$Id, Symdesc = spot$Symdesc)
}
else{
newSpot <- list(Cy3 = spot$Cy3 , Cy5= spot$Cy5 , BgCy3 = spot$BgCy3 , BgCy5 = spot$BgCy5, Id = spot$Id)
}
spotName <- attr(mySpot, "name")
new("Spot", name = spotName, spotData= newSpot)
}
# Normalisation
# Normalisation by grid
grid.norm <- function(mySpot, nr, nc){
spot <- attr(mySpot, "spotData")
x <- spot$Cy3
y <- spot$Cy5
k <-(nc * nr)-1
j <- 1
Cy3 <- Cy5 <- BgCy3 <- BgCy5<- Id <- Symdesc <- list()
bloque <- 1
n <- length (x)
while(j<(n+1)){
a <- unlist(sapply(j : (j+k),function(x) spot$Cy3[x]))
b <- unlist(sapply(j : (j+k),function(x) spot$Cy5[x]))
c <- unlist(sapply(j : (j+k),function(x) spot$BgCy3[x]))
d <- unlist(sapply(j : (j+k),function(x) spot$BgCy5[x]))
e <- unlist(sapply(j : (j+k),function(x) spot$Id[x]))
if(!is.null(spot$Symdesc)){
f <- unlist(sapply(j : (j+k),function(x) spot$Symdesc[x]))
index.temp <- a > 0 & b > 0 & (!is.na(a)) & (!is.na(b)) & (!is.na(c)) & (!is.na(d)) & (!is.na(e)) & (!is.na(f))
f <- f[index.temp]
}
else{
index.temp <- a > 0 & b > 0 & (!is.na(a)) & (!is.na(b)) & (!is.na(c)) & (!is.na(d)) & (!is.na(e))
}
a <- a[index.temp]
b <- b[index.temp]
c <- c[index.temp]
d <- d[index.temp]
e <- e[index.temp]
if(length(a) > 0){
R <- log(b,2) - log(a,2)
I <- log(a,10) + log( b, 10)
zero.index <- R == 0
R <- R[!zero.index]
I <- I[!zero.index]
a <- a[!zero.index]
b <- b[!zero.index]
c <- c[!zero.index]
d <- d[!zero.index]
e <- e[!zero.index]
if(!is.null(spot$Symdesc)){
f <- f[!zero.index]
Symdesc <- c(Symdesc, f, recursive = TRUE)
}
}
tmp <- fitted(locfit(R~I,alpha = 0.1, deg = 1, ev=rbox(cut = 0.9)))
Cy5 <- c(Cy5, b * (1/2^tmp), recursive = TRUE)
Cy3 <- c(Cy3, a, recursive = TRUE)
BgCy3 <- c(BgCy3, c, recursive = TRUE)
BgCy5 <- c(BgCy5, d, recursive = TRUE)
Id <- c(Id, e, recursive = TRUE)
j <- j + k + 1
}
Cy3 <- as.integer(Cy3)
Cy5 <- as.integer(Cy5)
BgCy5 <- as.integer(BgCy5)
BgCy3 <- as.integer(BgCy3)
if(is.null(spot$Symdesc)){
SD <- list(Cy3 = Cy3, Cy5 = Cy5, BgCy3 = BgCy3, BgCy5 = BgCy5, Id = Id)
}
else{
SD <- list(Cy3 = Cy3, Cy5 = Cy5, BgCy3 = BgCy3, BgCy5 = BgCy5, Id = Id, Symdesc = Symdesc)
}
new ("Spot", name = attr(mySpot, "name"), spotData = SD)
}
global.norm <- function(mySpot){
spot <- attr(mySpot, "spotData")
Cy3 <- Cy5 <- BgCy3 <- BgCy5<- Id <- Symdesc <- list()
index.temp <- spot$Cy3 > 0 & spot$Cy5 > 0
a <- spot$Cy3[index.temp]
b <- spot$Cy5[index.temp]
c <- spot$BgCy3[index.temp]
d <- spot$BgCy5[index.temp]
e <- spot$Id[index.temp]
if(!is.null(spot$Symdesc)){
f <- spot$Symdesc[index.temp]
}
if(length(a) > 0){
R <- log(b,2) - log(a,2)
I <- log(a,10) + log( b, 10)
zero.index <- R == 0
R <- R[!zero.index]
I <- I[!zero.index]
a <- a[!zero.index]
b <- b[!zero.index]
c <- c[!zero.index]
d <- d[!zero.index]
e <- e[!zero.index]
if(!is.null(spot$Symdesc)){
f <- f[!zero.index]
Symdesc <- f
}
tmp <- fitted(locfit(R~I,alpha = 0.1, deg = 1, ev=rbox(cut = 0.9) ))
Cy5 <- b * (1/2^tmp)
Cy3 <- a
BgCy3 <- c
BgCy5 <- d
Id <- e
}
Cy3 <- as.integer(Cy3)
Cy5 <- as.integer(Cy5)
BgCy5 <- as.integer(BgCy5)
BgCy3 <- as.integer(BgCy3)
if(is.null(spot$Symdesc)){
SD <- list(Cy3 = Cy3, Cy5 = Cy5, BgCy3 = BgCy3, BgCy5 = BgCy5, Id = Id)
}
else{
SD <- list(Cy3 = Cy3, Cy5 = Cy5, BgCy3 = BgCy3, BgCy5 = BgCy5, Id = Id, Symdesc = Symdesc)
}
new ("Spot", name = attr(mySpot, "name"), spotData = SD)
}
# Duplicate Analysis
# Mean Replicate filtering
meanUnique <- function(mySpot){
spot <- attr(mySpot, "spotData")
idHash <- new.env(hash=TRUE)
n <- length(spot$Cy3)
spot$Id <- toupper(spot$Id)
for(i in 1:n){
if(spot$Id[i] == ""){
spot$Id[i] <- "EMPTY"
}
if(exists( spot$Id[i], envir = idHash)){
idData <- get( spot$Id[i], envir = idHash)
if(!is.null(spot$Symdesc)){
assign(spot$Id[i], list(spotSymdesc = c(idData$spotSymdesc, spot$Symdesc[i], recursive = TRUE), spotCy3 = c( idData$spotCy3, spot$Cy3[i],recursive=TRUE) ,
spotCy5 = c( idData$spotCy5, spot$Cy5[i],recursive=TRUE),spotBgCy3 = c( idData$spotBgCy3, spot$BgCy3[i],recursive=TRUE),
spotBgCy5 = c( idData$spotBgCy5, spot$BgCy5[i],recursive=TRUE)), envir = idHash)
}
else{
assign(spot$Id[i], list(spotCy3 = c( idData$spotCy3, spot$Cy3[i],recursive=TRUE) ,
spotCy5 = c( idData$spotCy5, spot$Cy5[i],recursive=TRUE),spotBgCy3 = c( idData$spotBgCy3, spot$BgCy3[i],recursive=TRUE),
spotBgCy5 = c( idData$spotBgCy5, spot$BgCy5[i],recursive=TRUE)), envir = idHash)
}
}
else{
if(!is.null(spot$Symdesc)){
assign(spot$Id[i], list(spotSymdesc = spot$Symdesc[i], spotCy3 = spot$Cy3[i] ,spotCy5 = spot$Cy5[i],spotBgCy3 = spot$BgCy3[i],
spotBgCy5 = spot$BgCy5[i]), envir = idHash)
}
else{
assign(spot$Id[i], list(spotCy3 = spot$Cy3[i] ,spotCy5 = spot$Cy5[i],spotBgCy3 = spot$BgCy3[i],
spotBgCy5 = spot$BgCy5[i]), envir = idHash)
}
}
}
compara <- function(lista.cy3, lista.cy5){
resultado <- TRUE
R.list <- log2(lista.cy5)-log2(lista.cy3)
n <- length(R.list)
if(n > 1){
for( i in 1:(length(R.list)-1)) {
if((R.list[i] < 0 & R.list[i+1] > 0) | (R.list[i] > 0 & R.list[i+1] < 0)){
return( FALSE )
}
if(abs(R.list[i]) > (abs(R.list[i+1]) * 1.2)){
resultado <- FALSE
}
}
}
resultado
}
ids <- ls(envir = idHash)
replicates <- function( id ){
mainList <- get(id, envir = idHash)
if(compara(as.numeric(mainList$spotCy3), as.numeric(mainList$spotCy5)))
if(!is.null(spot$Symdesc))
list(Id = id, Symdesc = unique(mainList$spotSymdesc), Cy3 = mean(mainList$spotCy3), Cy5 = mean(mainList$spotCy5), BgCy3 = mainList$spotBgCy3[length(mainList$spotBgCy3)], BgCy5 = mainList$spotBgCy5[length(mainList$spotBgCy3)])
else
list(Id = id, Cy3 = mean(mainList$spotCy3), Cy5 = mean(mainList$spotCy5), BgCy3 = mainList$spotBgCy3[length(mainList$spotBgCy3)], BgCy5 = mainList$spotBgCy5[length(mainList$spotBgCy3)])
}
restCy3 <- restCy5 <- restBgCy3 <- restBgCy5 <- restId <- restSymdesc <- list()
for(i in 1:length(ids)){
rest <- replicates(ids[i])
restCy3 <- c(restCy3,rest$Cy3,recursive=TRUE)
restCy5 <- c(restCy5,rest$Cy5,recursive=TRUE)
restBgCy3 <- c(restBgCy3,rest$BgCy3,recursive=TRUE)
restBgCy5 <- c(restBgCy5,rest$BgCy5,recursive=TRUE)
restId <- c(restId,rest$Id,recursive=TRUE)
if(!is.null(spot$Symdesc))
restSymdesc <- c(restSymdesc,rest$Symdesc,recursive=TRUE)
}
if(!is.null(spot$Symdesc))
SD <- list(Cy3 = restCy3, Cy5 = restCy5, BgCy3 = restBgCy3, BgCy5 = restBgCy5, Id = restId, Symdesc = restSymdesc)
else
SD <- list(Cy3 = restCy3, Cy5 = restCy5, BgCy3 = restBgCy3, BgCy5 = restBgCy5, Id = restId)
spotName <- attr(mySpot, "name")
new("Spot", name = spotName, spotData = SD)
}
# Non-extreme values replicate filtering
alter.unique <- function(mySpot){
spot <- attr(mySpot, "spotData")
myHash <- new.env(hash=TRUE)
n <- length(spot$Cy3)
spot$Id <- toupper(spot$Id)
for(i in 1:n){
if(spot$Id[i] == ""){
spot$Id[i] <- "EMPTY"
}
if(exists( spot$Id[i], envir = myHash)){
idData <- get( spot$Id[i], envir = myHash)
if(!is.null(spot$Symdesc)){
assign(spot$Id[i], list(spotSymdesc = c(idData$spotSymdesc, spot$Symdesc[i], recursive = TRUE), spotCy3 = c( idData$spotCy3, spot$Cy3[i],recursive=TRUE) ,
spotCy5 = c( idData$spotCy5, spot$Cy5[i],recursive=TRUE),spotBgCy3 = c( idData$spotBgCy3, spot$BgCy3[i],recursive=TRUE),
spotBgCy5 = c( idData$spotBgCy5, spot$BgCy5[i],recursive=TRUE)), envir = myHash)
}
else{
assign(spot$Id[i], list(spotCy3 = c( idData$spotCy3, spot$Cy3[i]) ,
spotCy5 = c( idData$spotCy5, spot$Cy5[i]),spotBgCy3 = c( idData$spotBgCy3, spot$BgCy3[i]),
spotBgCy5 = c( idData$spotBgCy5, spot$BgCy5[i])), envir = myHash)
}
}
else{
if(!is.null(spot$Symdesc)){
assign(spot$Id[i], list(spotSymdesc = spot$Symdesc[i], spotCy3 = spot$Cy3[i] ,spotCy5 = spot$Cy5[i],spotBgCy3 = spot$BgCy3[i],
spotBgCy5 = spot$BgCy5[i]), envir = myHash)
}
else{
assign(spot$Id[i], list(spotCy3 = spot$Cy3[i] ,spotCy5 = spot$Cy5[i],spotBgCy3 = spot$BgCy3[i],
spotBgCy5 = spot$BgCy5[i]), envir = myHash)
}
}
}
ids <- ls(envir = myHash)
Cy3.list <- Cy5.list <- BgCy3.list <- BgCy5.list <- Id.list <- Symdesc.list <- list()
Cy3s <- Cy5s <- BgCy3s <- BgCy5s <- Ids <- Symdescs <- list()
replicates2 <- function(id){
mainList <- get(id, envir = myHash)
Cy3.list <- mainList$spotCy3
Cy5.list <- mainList$spotCy5
BgCy3.list <- mainList$spotBgCy3
BgCy5.list <- mainList$spotBgCy5
if(!is.null(spot$Symdesc))
Symdesc.list <- mainList$spotSymdesc
R.list <- log2(Cy5.list)-log2(Cy3.list)
x.prueba <- R.list[R.list < 0]
Cy3s <- Cy5s <- BgCy3s <- BgCy5s <- ids <- Symdescs <- list()
if(length(x.prueba) == 0 || length(x.prueba) == length(Cy3.list)){
if(length(x.prueba) == 0){ # all positive
bueno.index <- as.numeric(which(R.list == max(R.list)))
}
else{# all negative
bueno.index <- as.numeric(which(R.list == min(R.list)))
}
Cy3s <- Cy3.list[bueno.index]
Cy5s <- Cy5.list[bueno.index]
BgCy3s <- BgCy3.list[bueno.index]
BgCy5s <- BgCy5.list[bueno.index]
if(!is.null(spot$Symdesc))
Symdescs <- Symdesc.list[bueno.index]
ids <- id
if(!is.null(spot$Symdesc))
list(Cy3 = Cy3s[1], Cy5 = Cy5s[1], BgCy3 = BgCy3s[1], BgCy5 = BgCy5s[1], Id = ids, Symdesc = Symdescs[1])
else
list(Cy3 = Cy3s[1], Cy5 = Cy5s[1], BgCy3 = BgCy3s[1], BgCy5 = BgCy5s[1], Id = ids)
}
}
restCy3 <- restCy5 <- restBgCy3 <- restBgCy5 <- restId <- restSymdesc <- list()
for(i in 1:length(ids)){
rest <- replicates2(ids[i])
restCy3 <- c(restCy3,rest$Cy3,recursive=TRUE)
restCy5 <- c(restCy5,rest$Cy5,recursive=TRUE)
restBgCy3 <- c(restBgCy3,rest$BgCy3,recursive=TRUE)
restBgCy5 <- c(restBgCy5,rest$BgCy5,recursive=TRUE)
restId <- c(restId,rest$Id,recursive=TRUE)
if(!is.null(spot$Symdesc)){
restSymdesc <- c(restSymdesc,rest$Symdesc,recursive=TRUE)
}
}
if(!is.null(spot$Symdesc))
SD <- list(Cy3 = restCy3, Cy5 = restCy5, BgCy3 = restBgCy3, BgCy5 = restBgCy5, Id = restId, Symdesc = restSymdesc)
else
SD <- list(Cy3 = restCy3, Cy5 = restCy5, BgCy3 = restBgCy3, BgCy5 = restBgCy5, Id = restId)
spotName <- attr(mySpot, "name")
new("Spot", name = spotName, spotData = SD)
}
# Remove replicated observations, geometric mean
spotUnique <- function(mySpot){
spot <- attr(mySpot, "spotData")
idHash <- new.env(hash=TRUE)
n <- length(spot$Cy3)
spot$Id <- toupper(spot$Id)
for(i in 1:n){
if(spot$Id[i] == ""){
spot$Id[i] <- "EMPTY"
}
if(exists( spot$Id[i], envir = idHash)){
idData <- get( spot$Id[i], envir = idHash)
if(!is.null(spot$Symdesc)){
assign(spot$Id[i], list(spotSymdesc = c(idData$spotSymdesc, spot$Symdesc[i], recursive = TRUE), spotCy3 = c( idData$spotCy3, spot$Cy3[i],recursive=TRUE) ,
spotCy5 = c( idData$spotCy5, spot$Cy5[i],recursive=TRUE),spotBgCy3 = c( idData$spotBgCy3, spot$BgCy3[i],recursive=TRUE),
spotBgCy5 = c( idData$spotBgCy5, spot$BgCy5[i],recursive=TRUE)), envir = idHash)
}
else{
assign(spot$Id[i], list(spotCy3 = c( idData$spotCy3, spot$Cy3[i],recursive=TRUE) ,
spotCy5 = c( idData$spotCy5, spot$Cy5[i],recursive=TRUE),spotBgCy3 = c( idData$spotBgCy3, spot$BgCy3[i],recursive=TRUE),
spotBgCy5 = c( idData$spotBgCy5, spot$BgCy5[i],recursive=TRUE)), envir = idHash)
}
}
else{
if(!is.null(spot$Symdesc)){
assign(spot$Id[i], list(spotSymdesc = spot$Symdesc[i], spotCy3 = spot$Cy3[i] ,spotCy5 = spot$Cy5[i],spotBgCy3 = spot$BgCy3[i],
spotBgCy5 = spot$BgCy5[i]), envir = idHash)
}
else{
assign(spot$Id[i], list(spotCy3 = spot$Cy3[i] ,spotCy5 = spot$Cy5[i],spotBgCy3 = spot$BgCy3[i],
spotBgCy5 = spot$BgCy5[i]), envir = idHash)
}
}
}
geometric.mean <- function(x){
prod(x^(1/length(x)))
}
ids <- ls(envir = idHash)
replicates3 <- function(id){
mainList <- get(id, envir = idHash)
Cy3.list <- mainList$spotCy3
Cy5.list <- mainList$spotCy5
BgCy3.list <- mainList$spotBgCy3
BgCy5.list <- mainList$spotBgCy5
Symdesc.list <- mainList$spotSymdesc
Cy3 <- Cy5 <- BgCy3 <- BgCy5 <- Id <- Symdesc <- list()
tmp1 <- geometric.mean(Cy3.list)
tmp2 <- geometric.mean(Cy5.list)
if(tmp1 != 0 && tmp2 != 0){
Cy3 <- c(Cy3, round(tmp1,1), recursive=TRUE)
Cy5 <- c(Cy5, round(tmp2,1), recursive=TRUE)
BgCy3 <- c(BgCy3, BgCy3.list[length(mainList$spotBgCy3)],recursive=TRUE)
BgCy5 <- c(BgCy5, BgCy5.list[length(mainList$spotBgCy5)],recursive=TRUE)
Id <- c(Id, id, recursive=TRUE)
Symdesc <- c(Symdesc, Symdesc.list[1], recursive=TRUE)
}
list(Cy3 = Cy3, Cy5 = Cy5, BgCy3 = BgCy3, BgCy5 = BgCy5, Id = Id, Symdesc = Symdesc)
}
restCy3 <- restCy5 <- restBgCy3 <- restBgCy5 <- restId <- restSymdesc <- list()
for(i in 1:length(ids)){
rest <- replicates3(ids[i])
restCy3 <- c(restCy3,rest$Cy3,recursive=TRUE)
restCy5 <- c(restCy5,rest$Cy5,recursive=TRUE)
restBgCy3 <- c(restBgCy3,rest$BgCy3,recursive=TRUE)
restBgCy5 <- c(restBgCy5,rest$BgCy5,recursive=TRUE)
restId <- c(restId,rest$Id,recursive=TRUE)
if(!is.null(spot$Symdesc))
restSymdesc <- c(restSymdesc, rest$Symdesc, recursive=TRUE)
}
if(!is.null(spot$Symdesc))
SD <- list(Cy3 = restCy3, Cy5 = restCy5, BgCy3 = restBgCy3, BgCy5 = restBgCy5, Id = restId, Symdesc = restSymdesc)
else
SD <- list(Cy3 = restCy3, Cy5 = restCy5, BgCy3 = restBgCy3, BgCy5 = restBgCy5, Id = restId)
spotName <- attr(mySpot, "name")
new("Spot", name = spotName, spotData = SD)
}
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Defines functions spotUnique alter.unique meanUnique global.norm grid.norm bg.correct
Documented in alter.unique bg.correct global.norm grid.norm meanUnique spotUnique
require(locfit)
bg.correct <- function(mySpot){
spot <- as.list(attr(mySpot, "spotData"))
spot$Cy3 <- spot$Cy3 - spot$BgCy3
spot$Cy5 <- spot$Cy5 - spot$BgCy5
spotName <- attr(mySpot, "name")
if(is.null(spot$Symdesc)){
SD <- list(Cy3 = spot$Cy3, Cy5 = spot$Cy5, BgCy3 = spot$BgCy3, BgCy5 = spot$BgCy5, Id = spot$Id)
}
else{
SD <- list(Cy3 = spot$Cy3, Cy5 = spot$Cy5, BgCy3 = spot$BgCy3, BgCy5 = spot$BgCy5, Id = spot$Id, Symdesc = spot$Symdesc)
}
new("Spot", name = spotName, spotData= SD)
}
# Filtering
# Intensity-based filtering of array elements
filter.spot <- function (mySpot){
spot <- attr(mySpot, "spotData")
cys <- (!is.na(spot$Cy3)) & (!is.na(spot$Cy5)) & (!is.na(spot$BgCy3)) & (!is.na(spot$BgCy5)) & (!is.na(spot$Id))
spot$Cy3 <- spot$Cy3[cys]
spot$Cy5 <- spot$Cy5[cys]
spot$BgCy3 <- spot$BgCy3[cys]
spot$BgCy5 <- spot$BgCy5[cys]
spot$Id <- spot$Id[cys]
condition <- spot$Cy3 > 2 * sd(spot$BgCy3) & spot$Cy5 > 2 * sd(spot$BgCy5)
spot$Cy3 <- spot$Cy3[condition]
spot$Cy5 <- spot$Cy5[condition]
spot$BgCy3 <- spot$BgCy3[condition]
spot$BgCy5 <- spot$BgCy5[condition]
spot$Id <- spot$Id[condition]
if(!is.null(spot$Symdesc)){
spot$Symdesc <- spot$Symdesc[cys]
spot$Symdesc <- spot$Symdesc[condition]
newSpot <- list(Cy3 = spot$Cy3 , Cy5= spot$Cy5 , BgCy3 = spot$BgCy3 , BgCy5 = spot$BgCy5, Id = spot$Id, Symdesc = spot$Symdesc)
}
else{
newSpot <- list(Cy3 = spot$Cy3 , Cy5= spot$Cy5 , BgCy3 = spot$BgCy3 , BgCy5 = spot$BgCy5, Id = spot$Id)
}
spotName <- attr(mySpot, "name")
new("Spot", name = spotName, spotData= newSpot)
}
# Normalisation
# Normalisation by grid
grid.norm <- function(mySpot, nr, nc){
spot <- attr(mySpot, "spotData")
x <- spot$Cy3
y <- spot$Cy5
k <-(nc * nr)-1
j <- 1
Cy3 <- Cy5 <- BgCy3 <- BgCy5<- Id <- Symdesc <- list()
bloque <- 1
n <- length (x)
while(j<(n+1)){
a <- unlist(sapply(j : (j+k),function(x) spot$Cy3[x]))
b <- unlist(sapply(j : (j+k),function(x) spot$Cy5[x]))
c <- unlist(sapply(j : (j+k),function(x) spot$BgCy3[x]))
d <- unlist(sapply(j : (j+k),function(x) spot$BgCy5[x]))
e <- unlist(sapply(j : (j+k),function(x) spot$Id[x]))
if(!is.null(spot$Symdesc)){
f <- unlist(sapply(j : (j+k),function(x) spot$Symdesc[x]))
index.temp <- a > 0 & b > 0 & (!is.na(a)) & (!is.na(b)) & (!is.na(c)) & (!is.na(d)) & (!is.na(e)) & (!is.na(f))
f <- f[index.temp]
}
else{
index.temp <- a > 0 & b > 0 & (!is.na(a)) & (!is.na(b)) & (!is.na(c)) & (!is.na(d)) & (!is.na(e))
}
a <- a[index.temp]
b <- b[index.temp]
c <- c[index.temp]
d <- d[index.temp]
e <- e[index.temp]
if(length(a) > 0){
R <- log(b,2) - log(a,2)
I <- log(a,10) + log( b, 10)
zero.index <- R == 0
R <- R[!zero.index]
I <- I[!zero.index]
a <- a[!zero.index]
b <- b[!zero.index]
c <- c[!zero.index]
d <- d[!zero.index]
e <- e[!zero.index]
if(!is.null(spot$Symdesc)){
f <- f[!zero.index]
Symdesc <- c(Symdesc, f, recursive = TRUE)
}
}
tmp <- fitted(locfit(R~I,alpha = 0.1, deg = 1, ev=rbox(cut = 0.9)))
Cy5 <- c(Cy5, b * (1/2^tmp), recursive = TRUE)
Cy3 <- c(Cy3, a, recursive = TRUE)
BgCy3 <- c(BgCy3, c, recursive = TRUE)
BgCy5 <- c(BgCy5, d, recursive = TRUE)
Id <- c(Id, e, recursive = TRUE)
j <- j + k + 1
}
Cy3 <- as.integer(Cy3)
Cy5 <- as.integer(Cy5)
BgCy5 <- as.integer(BgCy5)
BgCy3 <- as.integer(BgCy3)
if(is.null(spot$Symdesc)){
SD <- list(Cy3 = Cy3, Cy5 = Cy5, BgCy3 = BgCy3, BgCy5 = BgCy5, Id = Id)
}
else{
SD <- list(Cy3 = Cy3, Cy5 = Cy5, BgCy3 = BgCy3, BgCy5 = BgCy5, Id = Id, Symdesc = Symdesc)
}
new ("Spot", name = attr(mySpot, "name"), spotData = SD)
}
global.norm <- function(mySpot){
spot <- attr(mySpot, "spotData")
Cy3 <- Cy5 <- BgCy3 <- BgCy5<- Id <- Symdesc <- list()
index.temp <- spot$Cy3 > 0 & spot$Cy5 > 0
a <- spot$Cy3[index.temp]
b <- spot$Cy5[index.temp]
c <- spot$BgCy3[index.temp]
d <- spot$BgCy5[index.temp]
e <- spot$Id[index.temp]
if(!is.null(spot$Symdesc)){
f <- spot$Symdesc[index.temp]
}
if(length(a) > 0){
R <- log(b,2) - log(a,2)
I <- log(a,10) + log( b, 10)
zero.index <- R == 0
R <- R[!zero.index]
I <- I[!zero.index]
a <- a[!zero.index]
b <- b[!zero.index]
c <- c[!zero.index]
d <- d[!zero.index]
e <- e[!zero.index]
if(!is.null(spot$Symdesc)){
f <- f[!zero.index]
Symdesc <- f
}
tmp <- fitted(locfit(R~I,alpha = 0.1, deg = 1, ev=rbox(cut = 0.9) ))
Cy5 <- b * (1/2^tmp)
Cy3 <- a
BgCy3 <- c
BgCy5 <- d
Id <- e
}
Cy3 <- as.integer(Cy3)
Cy5 <- as.integer(Cy5)
BgCy5 <- as.integer(BgCy5)
BgCy3 <- as.integer(BgCy3)
if(is.null(spot$Symdesc)){
SD <- list(Cy3 = Cy3, Cy5 = Cy5, BgCy3 = BgCy3, BgCy5 = BgCy5, Id = Id)
}
else{
SD <- list(Cy3 = Cy3, Cy5 = Cy5, BgCy3 = BgCy3, BgCy5 = BgCy5, Id = Id, Symdesc = Symdesc)
}
new ("Spot", name = attr(mySpot, "name"), spotData = SD)
}
# Duplicate Analysis
# Mean Replicate filtering
meanUnique <- function(mySpot){
spot <- attr(mySpot, "spotData")
idHash <- new.env(hash=TRUE)
n <- length(spot$Cy3)
spot$Id <- toupper(spot$Id)
for(i in 1:n){
if(spot$Id[i] == ""){
spot$Id[i] <- "EMPTY"
}
if(exists( spot$Id[i], envir = idHash)){
idData <- get( spot$Id[i], envir = idHash)
if(!is.null(spot$Symdesc)){
assign(spot$Id[i], list(spotSymdesc = c(idData$spotSymdesc, spot$Symdesc[i], recursive = TRUE), spotCy3 = c( idData$spotCy3, spot$Cy3[i],recursive=TRUE) ,
spotCy5 = c( idData$spotCy5, spot$Cy5[i],recursive=TRUE),spotBgCy3 = c( idData$spotBgCy3, spot$BgCy3[i],recursive=TRUE),
spotBgCy5 = c( idData$spotBgCy5, spot$BgCy5[i],recursive=TRUE)), envir = idHash)
}
else{
assign(spot$Id[i], list(spotCy3 = c( idData$spotCy3, spot$Cy3[i],recursive=TRUE) ,
spotCy5 = c( idData$spotCy5, spot$Cy5[i],recursive=TRUE),spotBgCy3 = c( idData$spotBgCy3, spot$BgCy3[i],recursive=TRUE),
spotBgCy5 = c( idData$spotBgCy5, spot$BgCy5[i],recursive=TRUE)), envir = idHash)
}
}
else{
if(!is.null(spot$Symdesc)){
assign(spot$Id[i], list(spotSymdesc = spot$Symdesc[i], spotCy3 = spot$Cy3[i] ,spotCy5 = spot$Cy5[i],spotBgCy3 = spot$BgCy3[i],
spotBgCy5 = spot$BgCy5[i]), envir = idHash)
}
else{
assign(spot$Id[i], list(spotCy3 = spot$Cy3[i] ,spotCy5 = spot$Cy5[i],spotBgCy3 = spot$BgCy3[i],
spotBgCy5 = spot$BgCy5[i]), envir = idHash)
}
}
}
compara <- function(lista.cy3, lista.cy5){
resultado <- TRUE
R.list <- log2(lista.cy5)-log2(lista.cy3)
n <- length(R.list)
if(n > 1){
for( i in 1:(length(R.list)-1)) {
if((R.list[i] < 0 & R.list[i+1] > 0) | (R.list[i] > 0 & R.list[i+1] < 0)){
return( FALSE )
}
if(abs(R.list[i]) > (abs(R.list[i+1]) * 1.2)){
resultado <- FALSE
}
}
}
resultado
}
ids <- ls(envir = idHash)
replicates <- function( id ){
mainList <- get(id, envir = idHash)
if(compara(as.numeric(mainList$spotCy3), as.numeric(mainList$spotCy5)))
if(!is.null(spot$Symdesc))
list(Id = id, Symdesc = unique(mainList$spotSymdesc), Cy3 = mean(mainList$spotCy3), Cy5 = mean(mainList$spotCy5), BgCy3 = mainList$spotBgCy3[length(mainList$spotBgCy3)], BgCy5 = mainList$spotBgCy5[length(mainList$spotBgCy3)])
else
list(Id = id, Cy3 = mean(mainList$spotCy3), Cy5 = mean(mainList$spotCy5), BgCy3 = mainList$spotBgCy3[length(mainList$spotBgCy3)], BgCy5 = mainList$spotBgCy5[length(mainList$spotBgCy3)])
}
restCy3 <- restCy5 <- restBgCy3 <- restBgCy5 <- restId <- restSymdesc <- list()
for(i in 1:length(ids)){
rest <- replicates(ids[i])
restCy3 <- c(restCy3,rest$Cy3,recursive=TRUE)
restCy5 <- c(restCy5,rest$Cy5,recursive=TRUE)
restBgCy3 <- c(restBgCy3,rest$BgCy3,recursive=TRUE)
restBgCy5 <- c(restBgCy5,rest$BgCy5,recursive=TRUE)
restId <- c(restId,rest$Id,recursive=TRUE)
if(!is.null(spot$Symdesc))
restSymdesc <- c(restSymdesc,rest$Symdesc,recursive=TRUE)
}
if(!is.null(spot$Symdesc))
SD <- list(Cy3 = restCy3, Cy5 = restCy5, BgCy3 = restBgCy3, BgCy5 = restBgCy5, Id = restId, Symdesc = restSymdesc)
else
SD <- list(Cy3 = restCy3, Cy5 = restCy5, BgCy3 = restBgCy3, BgCy5 = restBgCy5, Id = restId)
spotName <- attr(mySpot, "name")
new("Spot", name = spotName, spotData = SD)
}
# Non-extreme values replicate filtering
alter.unique <- function(mySpot){
spot <- attr(mySpot, "spotData")
myHash <- new.env(hash=TRUE)
n <- length(spot$Cy3)
spot$Id <- toupper(spot$Id)
for(i in 1:n){
if(spot$Id[i] == ""){
spot$Id[i] <- "EMPTY"
}
if(exists( spot$Id[i], envir = myHash)){
idData <- get( spot$Id[i], envir = myHash)
if(!is.null(spot$Symdesc)){
assign(spot$Id[i], list(spotSymdesc = c(idData$spotSymdesc, spot$Symdesc[i], recursive = TRUE), spotCy3 = c( idData$spotCy3, spot$Cy3[i],recursive=TRUE) ,
spotCy5 = c( idData$spotCy5, spot$Cy5[i],recursive=TRUE),spotBgCy3 = c( idData$spotBgCy3, spot$BgCy3[i],recursive=TRUE),
spotBgCy5 = c( idData$spotBgCy5, spot$BgCy5[i],recursive=TRUE)), envir = myHash)
}
else{
assign(spot$Id[i], list(spotCy3 = c( idData$spotCy3, spot$Cy3[i]) ,
spotCy5 = c( idData$spotCy5, spot$Cy5[i]),spotBgCy3 = c( idData$spotBgCy3, spot$BgCy3[i]),
spotBgCy5 = c( idData$spotBgCy5, spot$BgCy5[i])), envir = myHash)
}
}
else{
if(!is.null(spot$Symdesc)){
assign(spot$Id[i], list(spotSymdesc = spot$Symdesc[i], spotCy3 = spot$Cy3[i] ,spotCy5 = spot$Cy5[i],spotBgCy3 = spot$BgCy3[i],
spotBgCy5 = spot$BgCy5[i]), envir = myHash)
}
else{
assign(spot$Id[i], list(spotCy3 = spot$Cy3[i] ,spotCy5 = spot$Cy5[i],spotBgCy3 = spot$BgCy3[i],
spotBgCy5 = spot$BgCy5[i]), envir = myHash)
}
}
}
ids <- ls(envir = myHash)
Cy3.list <- Cy5.list <- BgCy3.list <- BgCy5.list <- Id.list <- Symdesc.list <- list()
Cy3s <- Cy5s <- BgCy3s <- BgCy5s <- Ids <- Symdescs <- list()
replicates2 <- function(id){
mainList <- get(id, envir = myHash)
Cy3.list <- mainList$spotCy3
Cy5.list <- mainList$spotCy5
BgCy3.list <- mainList$spotBgCy3
BgCy5.list <- mainList$spotBgCy5
if(!is.null(spot$Symdesc))
Symdesc.list <- mainList$spotSymdesc
R.list <- log2(Cy5.list)-log2(Cy3.list)
x.prueba <- R.list[R.list < 0]
Cy3s <- Cy5s <- BgCy3s <- BgCy5s <- ids <- Symdescs <- list()
if(length(x.prueba) == 0 || length(x.prueba) == length(Cy3.list)){
if(length(x.prueba) == 0){ # all positive
bueno.index <- as.numeric(which(R.list == max(R.list)))
}
else{# all negative
bueno.index <- as.numeric(which(R.list == min(R.list)))
}
Cy3s <- Cy3.list[bueno.index]
Cy5s <- Cy5.list[bueno.index]
BgCy3s <- BgCy3.list[bueno.index]
BgCy5s <- BgCy5.list[bueno.index]
if(!is.null(spot$Symdesc))
Symdescs <- Symdesc.list[bueno.index]
ids <- id
if(!is.null(spot$Symdesc))
list(Cy3 = Cy3s[1], Cy5 = Cy5s[1], BgCy3 = BgCy3s[1], BgCy5 = BgCy5s[1], Id = ids, Symdesc = Symdescs[1])
else
list(Cy3 = Cy3s[1], Cy5 = Cy5s[1], BgCy3 = BgCy3s[1], BgCy5 = BgCy5s[1], Id = ids)
}
}
restCy3 <- restCy5 <- restBgCy3 <- restBgCy5 <- restId <- restSymdesc <- list()
for(i in 1:length(ids)){
rest <- replicates2(ids[i])
restCy3 <- c(restCy3,rest$Cy3,recursive=TRUE)
restCy5 <- c(restCy5,rest$Cy5,recursive=TRUE)
restBgCy3 <- c(restBgCy3,rest$BgCy3,recursive=TRUE)
restBgCy5 <- c(restBgCy5,rest$BgCy5,recursive=TRUE)
restId <- c(restId,rest$Id,recursive=TRUE)
if(!is.null(spot$Symdesc)){
restSymdesc <- c(restSymdesc,rest$Symdesc,recursive=TRUE)
}
}
if(!is.null(spot$Symdesc))
SD <- list(Cy3 = restCy3, Cy5 = restCy5, BgCy3 = restBgCy3, BgCy5 = restBgCy5, Id = restId, Symdesc = restSymdesc)
else
SD <- list(Cy3 = restCy3, Cy5 = restCy5, BgCy3 = restBgCy3, BgCy5 = restBgCy5, Id = restId)
spotName <- attr(mySpot, "name")
new("Spot", name = spotName, spotData = SD)
}
# Remove replicated observations, geometric mean
spotUnique <- function(mySpot){
spot <- attr(mySpot, "spotData")
idHash <- new.env(hash=TRUE)
n <- length(spot$Cy3)
spot$Id <- toupper(spot$Id)
for(i in 1:n){
if(spot$Id[i] == ""){
spot$Id[i] <- "EMPTY"
}
if(exists( spot$Id[i], envir = idHash)){
idData <- get( spot$Id[i], envir = idHash)
if(!is.null(spot$Symdesc)){
assign(spot$Id[i], list(spotSymdesc = c(idData$spotSymdesc, spot$Symdesc[i], recursive = TRUE), spotCy3 = c( idData$spotCy3, spot$Cy3[i],recursive=TRUE) ,
spotCy5 = c( idData$spotCy5, spot$Cy5[i],recursive=TRUE),spotBgCy3 = c( idData$spotBgCy3, spot$BgCy3[i],recursive=TRUE),
spotBgCy5 = c( idData$spotBgCy5, spot$BgCy5[i],recursive=TRUE)), envir = idHash)
}
else{
assign(spot$Id[i], list(spotCy3 = c( idData$spotCy3, spot$Cy3[i],recursive=TRUE) ,
spotCy5 = c( idData$spotCy5, spot$Cy5[i],recursive=TRUE),spotBgCy3 = c( idData$spotBgCy3, spot$BgCy3[i],recursive=TRUE),
spotBgCy5 = c( idData$spotBgCy5, spot$BgCy5[i],recursive=TRUE)), envir = idHash)
}
}
else{
if(!is.null(spot$Symdesc)){
assign(spot$Id[i], list(spotSymdesc = spot$Symdesc[i], spotCy3 = spot$Cy3[i] ,spotCy5 = spot$Cy5[i],spotBgCy3 = spot$BgCy3[i],
spotBgCy5 = spot$BgCy5[i]), envir = idHash)
}
else{
assign(spot$Id[i], list(spotCy3 = spot$Cy3[i] ,spotCy5 = spot$Cy5[i],spotBgCy3 = spot$BgCy3[i],
spotBgCy5 = spot$BgCy5[i]), envir = idHash)
}
}
}
geometric.mean <- function(x){
prod(x^(1/length(x)))
}
ids <- ls(envir = idHash)
replicates3 <- function(id){
mainList <- get(id, envir = idHash)
Cy3.list <- mainList$spotCy3
Cy5.list <- mainList$spotCy5
BgCy3.list <- mainList$spotBgCy3
BgCy5.list <- mainList$spotBgCy5
Symdesc.list <- mainList$spotSymdesc
Cy3 <- Cy5 <- BgCy3 <- BgCy5 <- Id <- Symdesc <- list()
tmp1 <- geometric.mean(Cy3.list)
tmp2 <- geometric.mean(Cy5.list)
if(tmp1 != 0 && tmp2 != 0){
Cy3 <- c(Cy3, round(tmp1,1), recursive=TRUE)
Cy5 <- c(Cy5, round(tmp2,1), recursive=TRUE)
BgCy3 <- c(BgCy3, BgCy3.list[length(mainList$spotBgCy3)],recursive=TRUE)
BgCy5 <- c(BgCy5, BgCy5.list[length(mainList$spotBgCy5)],recursive=TRUE)
Id <- c(Id, id, recursive=TRUE)
Symdesc <- c(Symdesc, Symdesc.list[1], recursive=TRUE)
}
list(Cy3 = Cy3, Cy5 = Cy5, BgCy3 = BgCy3, BgCy5 = BgCy5, Id = Id, Symdesc = Symdesc)
}
restCy3 <- restCy5 <- restBgCy3 <- restBgCy5 <- restId <- restSymdesc <- list()
for(i in 1:length(ids)){
rest <- replicates3(ids[i])
restCy3 <- c(restCy3,rest$Cy3,recursive=TRUE)
restCy5 <- c(restCy5,rest$Cy5,recursive=TRUE)
restBgCy3 <- c(restBgCy3,rest$BgCy3,recursive=TRUE)
restBgCy5 <- c(restBgCy5,rest$BgCy5,recursive=TRUE)
restId <- c(restId,rest$Id,recursive=TRUE)
if(!is.null(spot$Symdesc))
restSymdesc <- c(restSymdesc, rest$Symdesc, recursive=TRUE)
}
if(!is.null(spot$Symdesc))
SD <- list(Cy3 = restCy3, Cy5 = restCy5, BgCy3 = restBgCy3, BgCy5 = restBgCy5, Id = restId, Symdesc = restSymdesc)
else
SD <- list(Cy3 = restCy3, Cy5 = restCy5, BgCy3 = restBgCy3, BgCy5 = restBgCy5, Id = restId)
spotName <- attr(mySpot, "name")
new("Spot", name = spotName, spotData = SD)
}
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