Nothing
R/getBreakpoints.R
In AneuFinder: Analysis of Copy Number Variation in Single-Cell-Sequencing Data
Defines functions
annotateBreakpoints
refineBreakpoints
confidenceIntervals
getBreakpoints
Documented in
annotateBreakpoints
getBreakpoints
refineBreakpoints
#' Extract breakpoints
#'
#' Extract breakpoints with confidence intervals from an \code{\link{aneuHMM}} or \code{\link{aneuBiHMM}} object.
#'
#' Confidence intervals for breakpoints are estimated by going outwards from the breakpoint read by read, and performing a test of getting the observed or a more extreme outcome, given that the reads within the confidence interval belong to the other side of the breakpoint.
#'
#' @param model An \code{\link{aneuHMM}} or \code{\link{aneuBiHMM}} object or a file that contains such an object.
#' @param fragments A \code{\link{GRanges-class}} object with read fragments or a file that contains such an object.
#' @param confint Desired confidence interval for breakpoints. Set \code{confint=NULL} to disable confidence interval estimation.
#' @return A \code{\link{GRanges-class}} with breakpoint coordinates and confidence interals if \code{fragments} was specified.
#' @importFrom stats pnbinom pbinom pgeom dnbinom dbinom dgeom
#' @export
#'
#' @examples
#'## Get an example BED file with single-cell-sequencing reads
#'bedfile <- system.file("extdata", "KK150311_VI_07.bam.bed.gz", package="AneuFinderData")
#'## Bin the data into bin size 1Mp
#'readfragments <- binReads(bedfile, assembly='mm10', binsize=1e6,
#' chromosomes=c(1:19,'X','Y'), reads.return=TRUE)
#'binned <- binReads(bedfile, assembly='mm10', binsize=1e6,
#' chromosomes=c(1:19,'X','Y'))
#'## Fit the Hidden Markov Model
#'model <- findCNVs.strandseq(binned[[1]])
#'## Add confidence intervals
#'breakpoints <- getBreakpoints(model, readfragments)
#'
getBreakpoints <- function(model, fragments=NULL, confint=0.99) {
model <- loadFromFiles(model, check.class = c("aneuHMM", "aneuBiHMM"))[[1]]
if (is.null(model$segments)) {
gr <- GRanges(seqnames = character(), ranges = IRanges(), mstate.left=factor(), pstate.left=factor(), mstate.right=factor(), pstate.right=factor(), start.conf=integer(), end.conf=integer(), type=factor())
return(gr)
}
fragments <- loadFromFiles(fragments, check.class = 'GRanges')[[1]]
binsize <- mean(width(model$bincounts[[1]]))
## Get breakpoints
breaks <- model$segments
mcols(breaks) <- NULL
end(breaks) <- end(breaks) - 1
seqlengths(breaks) <- NA # to not have the chromosome end as gap
breaks <- gaps(breaks)
breaks <- breaks[strand(breaks)=='*']
seqlengths(breaks) <- seqlengths(model$segments)
# Add genotype transition
if (length(breaks) == 0) {
return(breaks)
}
ind <- findOverlaps(breaks, model$segments)
if (class(model) == "aneuBiHMM") {
breaks$mstate.left <- factor(NA, levels=levels(model$segments$mstate))
breaks$mstate.left[ind@from] <- model$segments$mstate[ind@to]
breaks$pstate.left <- factor(NA, levels=levels(model$segments$pstate))
breaks$pstate.left[ind@from] <- model$segments$pstate[ind@to]
breaks$mstate.right <- factor(NA, levels=levels(model$segments$mstate))
breaks$mstate.right[ind@from] <- model$segments$mstate[ind@to+1]
breaks$pstate.right <- factor(NA, levels=levels(model$segments$pstate))
breaks$pstate.right[ind@from] <- model$segments$pstate[ind@to+1]
} else if (class(model) == "aneuHMM") {
breaks$state.left <- factor(NA, levels=levels(model$segments$state))
breaks$state.left[ind@from] <- model$segments$state[ind@to]
breaks$state.right <- factor(NA, levels=levels(model$segments$state))
breaks$state.right[ind@from] <- model$segments$state[ind@to+1]
}
if (is.null(fragments) | is.null(confint)) {
breaks <- annotateBreakpoints(breaks)
return(breaks)
}
## Sort fragments
seqlevels(fragments) <- seqlevels(model$segments)
fragments <- sort(fragments, ignore.strand=TRUE)
fragments$p <- 0
## Distributions
distr <- model$distributions
if (class(distr) == 'list') {
distr <- distr[[1]]
}
breaks.conf <- confidenceIntervals(breaks = breaks, fragments = fragments, distr = distr, confint = confint, binsize = binsize)
breaks$start.conf <- start(breaks.conf)
breaks$end.conf <- end(breaks.conf)
breaks <- annotateBreakpoints(breaks)
return(breaks)
}
confidenceIntervals <- function(breaks, fragments, distr, confint, binsize) {
ptm <- startTimedMessage("Finding confidence intervals ...")
min.i1 <- 10
doublestranded <- 'mstate.left' %in% names(mcols(breaks))
## Do chromosomes one by one
breaks.conf <- GenomicRanges::GRangesList()
seqlevels(breaks.conf) <- seqlevels(breaks)
for (chrom in unique(seqnames(breaks))) {
# message("chrom = ", chrom)
cbreaks <- breaks[seqnames(breaks) == chrom]
cbreaks.mod <- cbreaks
cfrags <- fragments[seqnames(fragments) == chrom]
if (length(cbreaks) > 0) {
for (ibreak in 1:length(cbreaks)) {
# message(ibreak)
states <- as.data.frame(mcols(cbreaks[ibreak]))
if (doublestranded) {
mus <- array(NA, dim=c(2,2), dimnames=list(strand=c('-','+'), direction=c('left','right')))
mus['-','right'] <- distr[as.character(states[,'mstate.right']),'mu']
mus['+','right'] <- distr[as.character(states[,'pstate.right']),'mu']
mus['-','left'] <- distr[as.character(states[,'mstate.left']),'mu']
mus['+','left'] <- distr[as.character(states[,'pstate.left']),'mu']
} else {
mus <- array(NA, dim=c(1,2), dimnames=list(strand=c('*'), direction=c('left','right')))
mus['*','right'] <- distr[as.character(states[,'state.right']),'mu']
mus['*','left'] <- distr[as.character(states[,'state.left']),'mu']
}
# Directionality of test
left.is.bigger <- mus[,'left', drop=FALSE] >= mus[,'right', drop=FALSE]
right.is.bigger <- !left.is.bigger
## Left side
ind <- which(start(cfrags) < start(cbreaks)[ibreak])
if (ibreak > 1) {
max.bp <- start(cbreaks[ibreak]) - start(cbreaks[ibreak-1])
} else {
max.bp <- start(cbreaks[ibreak])
}
if (length(ind) > 0) {
ind <- ind[length(ind)]
p <- 1
ps <- numeric()
ps['0'] <- p
numReads <- c('*'=0, '-'=0, '+'=0)
if (!any(is.na(mus))) {
i1 <- -1
## Helpers for speed improvement
start.cfrags <- start(cfrags)
start.cbreaks <- start(cbreaks)
strand.cfrags <- as.character(strand(cfrags))
while (p > 1-confint | i1 <= min.i1) {
i1 <- i1+1
if (ind-i1 <= 0) {
i1 = i1 - 1
break
}
i1.bp <- - (start.cfrags[ind-i1] - start.cbreaks[ibreak])
if (i1.bp > max.bp) {
i1 = i1 - 1
break
}
strandofread <- strand.cfrags[ind-i1]
numReads[strandofread] <- numReads[strandofread] + 1 # assuming there are no * reads in the fragments
numReads['*'] <- numReads['*'] + 1
if (doublestranded) {
# Minus strand
dtype <- distr[as.character(states[,'mstate.right']), 'type']
if (dtype == 'dnbinom') {
p.minus <- stats::pnbinom(q = numReads['-'] - !right.is.bigger['-',], size = distr[as.character(states[,'mstate.right']), 'size'] * i1.bp/binsize, prob = distr[as.character(states[,'mstate.right']), 'prob'], lower.tail = right.is.bigger['-',])
} else if (dtype == 'dpois') {
p.minus <- stats::ppois(q = numReads['-'] - !right.is.bigger['-',], lambda = distr[as.character(states[,'mstate.right']), 'mu'] * i1.bp/binsize, lower.tail = right.is.bigger['-',])
} else if (dtype == 'dgeom') {
p.minus <- stats::pgeom(q = numReads['-'] - !right.is.bigger['-',], prob = dgeom.prob(distr[as.character(states[,'mstate.right']), 'mu'] * i1.bp/binsize), lower.tail = right.is.bigger['-',])
} else if (dtype == 'delta') {
p.minus <- c('-'=as.numeric(numReads['-'] == 0))
} else if (dtype == 'dbinom') {
p.minus <- stats::pbinom(q = numReads['-'] - !right.is.bigger['-',], size = round(distr[as.character(states[,'mstate.right']), 'size'] * i1.bp/binsize), prob = distr[as.character(states[,'mstate.right']), 'prob'], lower.tail = right.is.bigger['-',])
}
# Plus strand
dtype <- distr[as.character(states[,'pstate.right']), 'type']
if (dtype == 'dnbinom') {
p.plus <- stats::pnbinom(q = numReads['+'] - !right.is.bigger['+',], size = distr[as.character(states[,'pstate.right']), 'size'] * i1.bp/binsize, prob = distr[as.character(states[,'pstate.right']), 'prob'], lower.tail = right.is.bigger['+',])
} else if (dtype == 'dpois') {
p.plus <- stats::ppois(q = numReads['+'] - !right.is.bigger['+',], lambda = distr[as.character(states[,'pstate.right']), 'mu'] * i1.bp/binsize, lower.tail = right.is.bigger['+',])
} else if (dtype == 'dgeom') {
p.plus <- stats::pgeom(q = numReads['+'] - !right.is.bigger['+',], prob = dgeom.prob(distr[as.character(states[,'pstate.right']), 'mu'] * i1.bp/binsize), lower.tail = right.is.bigger['+',])
} else if (dtype == 'delta') {
p.plus <- c('+'=as.numeric(numReads['+'] == 0))
} else if (dtype == 'dbinom') {
p.plus <- stats::pbinom(q = numReads['+'] - !right.is.bigger['+',], size = round(distr[as.character(states[,'pstate.right']), 'size'] * i1.bp/binsize), prob = distr[as.character(states[,'pstate.right']), 'prob'], lower.tail = right.is.bigger['+',])
}
p <- c(p.minus, p.plus)
p[is.na(p)] <- 1
p <- p['-'] * p['+']
} else {
# Star strand
dtype <- distr[as.character(states[,'state.right']), 'type']
if (dtype == 'dnbinom') {
p <- stats::pnbinom(q = numReads['*'] - !right.is.bigger['*',], size = distr[as.character(states[,'state.right']), 'size'] * i1.bp/binsize, prob = distr[as.character(states[,'state.right']), 'prob'], lower.tail = right.is.bigger['*',])
} else if (dtype == 'dpois') {
p <- stats::ppois(q = numReads['*'] - !right.is.bigger['*',], lambda = distr[as.character(states[,'state.right']), 'mu'] * i1.bp/binsize, lower.tail = right.is.bigger['*',])
} else if (dtype == 'dgeom') {
p <- stats::pgeom(q = numReads['*'] - !right.is.bigger['*',], prob = dgeom.prob(distr[as.character(states[,'state.right']), 'mu'] * i1.bp/binsize), lower.tail = right.is.bigger['*',])
} else if (dtype == 'delta') {
p <- c('*'=as.numeric(numReads['*'] == 0))
} else if (dtype == 'dbinom') {
p <- stats::pbinom(q = numReads['*'] - !right.is.bigger['*',], size = round(distr[as.character(states[,'state.right']), 'size'] * i1.bp/binsize), prob = distr[as.character(states[,'state.right']), 'prob'], lower.tail = right.is.bigger['*',])
}
}
ps[as.character(i1)] <- p
}
if (i1 >= 0) {
revps <- rev(ps)
if (revps[1] < 1-confint) {
i1 <- as.numeric(names(revps)[which(revps >= 1-confint)[1] - 1]) # first one from the end that is below threshold
if (is.na(i1)) {
i1 <- as.numeric(names(ps)[1])
}
} else {
i1 <- as.numeric(names(revps)[1])
}
new.start <- end(cfrags)[ind-i1]
if (new.start < start(cbreaks.mod)[ibreak]) {
start(cbreaks.mod)[ibreak] <- new.start
}
}
}
}
## Right side
ind <- which(end(cfrags) > end(cbreaks)[ibreak])
if (ibreak < length(cbreaks)) {
max.bp <- end(cbreaks[ibreak+1]) - end(cbreaks[ibreak])
} else {
max.bp <- Inf
}
if (length(ind) > 0) {
ind <- ind[1]
p <- 1
ps <- numeric()
ps['0'] <- p
numReads <- c('*'=0, '-'=0, '+'=0)
if (!any(is.na(mus))) {
i1 <- -1
## Helpers for speed improvement
end.cfrags <- end(cfrags)
end.cbreaks <- end(cbreaks)
strand.cfrags <- as.character(strand(cfrags))
while (p > 1-confint | i1 <= min.i1) {
i1 <- i1+1
if (ind+i1 > length(cfrags)) {
i1 = i1 - 1
break
}
i1.bp <- end.cfrags[ind+i1] - end.cbreaks[ibreak]
if (i1.bp > max.bp) {
i1 = i1 - 1
break
}
strandofread <- strand.cfrags[ind+i1]
numReads[strandofread] <- numReads[strandofread] + 1 # assuming there are no * reads in the fragments
numReads['*'] <- numReads['*'] + 1
if (doublestranded) {
# Minus strand
dtype <- distr[as.character(states[,'mstate.left']), 'type']
if (dtype == 'dnbinom') {
p.minus <- stats::pnbinom(q = numReads['-'] - !left.is.bigger['-',], size = distr[as.character(states[,'mstate.left']), 'size'] * i1.bp/binsize, prob = distr[as.character(states[,'mstate.left']), 'prob'], lower.tail = left.is.bigger['-',])
} else if (dtype == 'dpois') {
p.minus <- stats::ppois(q = numReads['-'] - !left.is.bigger['-',], lambda = distr[as.character(states[,'mstate.left']), 'mu'] * i1.bp/binsize, lower.tail = left.is.bigger['-',])
} else if (dtype == 'dgeom') {
p.minus <- stats::pgeom(q = numReads['-'] - !left.is.bigger['-',], prob = dgeom.prob(distr[as.character(states[,'mstate.left']), 'mu'] * i1.bp/binsize), lower.tail = left.is.bigger['-',])
} else if (dtype == 'delta') {
p.minus <- c('-'=as.numeric(numReads['-'] == 0))
} else if (dtype == 'dbinom') {
p.minus <- stats::pbinom(q = numReads['-'] - !left.is.bigger['-',], size = round(distr[as.character(states[,'mstate.left']), 'size'] * i1.bp/binsize), prob = distr[as.character(states[,'mstate.left']), 'prob'], lower.tail = left.is.bigger['-',])
}
# Plus strand
dtype <- distr[as.character(states[,'pstate.left']), 'type']
if (dtype == 'dnbinom') {
p.plus <- stats::pnbinom(q = numReads['+'] - !left.is.bigger['+',], size = distr[as.character(states[,'pstate.left']), 'size'] * i1.bp/binsize, prob = distr[as.character(states[,'pstate.left']), 'prob'], lower.tail = left.is.bigger['+',])
} else if (dtype == 'dpois') {
p.plus <- stats::ppois(q = numReads['+'] - !left.is.bigger['+',], lambda = distr[as.character(states[,'pstate.left']), 'mu'] * i1.bp/binsize, lower.tail = left.is.bigger['+',])
} else if (dtype == 'dgeom') {
p.plus <- stats::pgeom(q = numReads['+'] - !left.is.bigger['+',], prob = dgeom.prob(distr[as.character(states[,'pstate.left']), 'mu'] * i1.bp/binsize), lower.tail = left.is.bigger['+',])
} else if (dtype == 'delta') {
p.plus <- c('+'=as.numeric(numReads['+'] == 0))
} else if (dtype == 'dbinom') {
p.plus <- stats::pbinom(q = numReads['+'] - !left.is.bigger['+',], size = round(distr[as.character(states[,'pstate.left']), 'size'] * i1.bp/binsize), prob = distr[as.character(states[,'pstate.left']), 'prob'], lower.tail = left.is.bigger['+',])
}
p <- c(p.minus, p.plus)
p[is.na(p)] <- 1
p <- p['-'] * p['+']
} else {
# Star strand
dtype <- distr[as.character(states[,'state.left']), 'type']
if (dtype == 'dnbinom') {
p <- stats::pnbinom(q = numReads['*'] - !left.is.bigger['*',], size = distr[as.character(states[,'state.left']), 'size'] * i1.bp/binsize, prob = distr[as.character(states[,'state.left']), 'prob'], lower.tail = left.is.bigger['*',])
} else if (dtype == 'dpois') {
p <- stats::ppois(q = numReads['*'] - !left.is.bigger['*',], lambda = distr[as.character(states[,'state.left']), 'mu'] * i1.bp/binsize, lower.tail = left.is.bigger['*',])
} else if (dtype == 'dgeom') {
p <- stats::pgeom(q = numReads['*'] - !left.is.bigger['*',], prob = dgeom.prob(distr[as.character(states[,'state.left']), 'mu'] * i1.bp/binsize), lower.tail = left.is.bigger['*',])
} else if (dtype == 'delta') {
p <- c('*'=as.numeric(numReads['*'] == 0))
} else if (dtype == 'dbinom') {
p <- stats::pbinom(q = numReads['*'] - !left.is.bigger['*',], size = round(distr[as.character(states[,'state.left']), 'size'] * i1.bp/binsize), prob = distr[as.character(states[,'state.left']), 'prob'], lower.tail = left.is.bigger['*',])
}
}
ps[as.character(i1)] <- p
}
if (i1 >= 0) {
revps <- rev(ps)
if (revps[1] < 1-confint) {
i1 <- as.numeric(names(revps)[which(revps >= 1-confint)[1] - 1]) # first one from the end that is below threshold
if (is.na(i1)) {
i1 <- as.numeric(names(ps)[1])
}
} else {
i1 <- as.numeric(names(revps)[1])
}
new.end <- start(cfrags)[ind+i1]
if (new.end > end(cbreaks.mod)[ibreak]) {
end(cbreaks.mod)[ibreak] <- new.end
}
}
}
}
}
}
breaks.conf[[chrom]] <- cbreaks.mod
}
breaks.conf <- unlist(breaks.conf, use.names = FALSE)
stopTimedMessage(ptm)
return(breaks.conf)
}
#' Refine breakpoints
#'
#' Refine breakpoints with confidence intervals from an initial estimate (from \code{\link{getBreakpoints}}).
#'
#' Breakpoints are refined by shifting the breakpoint within its initial confidence interval read by read and maximizing the probability of observing the left-right read distribution.
#'
#' @param model An \code{\link{aneuBiHMM}} object or a file that contains such an object.
#' @param fragments A \code{\link{GRanges-class}} object with read fragments or a file that contains such an object.
#' @param breakpoints A \code{\link{GRanges-class}} object with breakpoints and confidence intervals, as returned by function \code{\link{getBreakpoints}}.
#' @param confint Desired confidence interval for breakpoints.
#' @return An \code{\link{aneuBiHMM}} with adjusted breakpoint coordinates and confidence interals, bins and segments.
#' @export
#'
#' @examples
#'## Get an example BED file with single-cell-sequencing reads
#'bedfile <- system.file("extdata", "KK150311_VI_07.bam.bed.gz", package="AneuFinderData")
#'## Bin the data into bin size 1Mp
#'readfragments <- binReads(bedfile, assembly='mm10', binsize=1e6,
#' chromosomes=c(1:19,'X','Y'), reads.return=TRUE)
#'binned <- binReads(bedfile, assembly='mm10', binsize=1e6,
#' chromosomes=c(1:19,'X','Y'))
#'## Fit the Hidden Markov Model
#'model <- findCNVs.strandseq(binned[[1]])
#'## Add confidence intervals
#'breakpoints <- getBreakpoints(model, readfragments)
#'## Refine breakpoints
#'refined.model <- refineBreakpoints(model, readfragments, breakpoints)
#'
refineBreakpoints <- function(model, fragments, breakpoints = model$breakpoints, confint=0.99) {
ptm <- startTimedMessage("Refining breakpoints ...")
model <- loadFromFiles(model, check.class = c("aneuHMM", "aneuBiHMM"))[[1]]
doublestranded <- 'mstate.left' %in% names(mcols(breakpoints))
fragments <- loadFromFiles(fragments, check.class = 'GRanges')[[1]]
binsize <- mean(width(model$bincounts[[1]]))
breaks <- breakpoints
if (is.null(breaks)) {
stopTimedMessage(ptm)
warning(paste0(model$ID, ": No breakpoints found. Cannot refine breakpoints."))
return(model)
}
if (is.null(breaks$start.conf)) {
stopTimedMessage(ptm)
warning(paste0(model$ID, ": No confidence intervals found. Cannot refine breakpoints."))
return(model)
}
if (length(breaks) == 0) {
stopTimedMessage(ptm)
return(model)
}
## Sort fragments
seqlevels(fragments) <- seqlevels(model$segments)
fragments <- sort(fragments, ignore.strand=TRUE)
fragments$p <- 0
## Distributions
distr <- model$distributions
if (class(distr) == 'list') {
distr <- distr[[1]]
}
## Do chromosomes one by one
breaks.refined <- GenomicRanges::GRangesList()
seqlevels(breaks.refined) <- seqlevels(breaks)
for (chrom in unique(seqnames(breaks))) {
# message("chrom = ", chrom)
cbreaks <- breaks[seqnames(breaks) == chrom]
cbreaks.mod <- cbreaks
cfrags <- fragments[seqnames(fragments) == chrom]
if (length(cbreaks) > 0) {
for (ibreak in 1:length(cbreaks)) {
# message(ibreak, "/", length(cbreaks))
## Go through confidence interval left to right
states <- as.data.frame(mcols(cbreaks[ibreak]))
ind <- which( (start(cfrags) >= cbreaks[ibreak]$start.conf) & (end(cfrags) <= cbreaks[ibreak]$end.conf) )
frags.ind <- cfrags[ind]
if (length(ind) > 0) {
numReads <- array(NA, dim=c(3,2,length(ind)+1), dimnames=list(strand=c('+','-','*'), direction=c('left','right'), ind=0:length(ind)))
ps <- array(NA, dim=c(length(ind)+1), dimnames=list(ind=0:length(ind)))
break.coords <- array(NA, dim=c(length(ind)+1), dimnames=list(ind=0:length(ind)))
## Helpers for speed improvement
end.frags.ind <- end(frags.ind)
start.frags.ind <- start(frags.ind)
strand.frags.ind <- strand(frags.ind)
length.frags.ind <- length(frags.ind)
length.ind <- length(ind)
start.conf.cbreaks.ibreak <- cbreaks[ibreak]$start.conf
end.conf.cbreaks.ibreak <- cbreaks[ibreak]$end.conf
for (i1 in 0:length.ind) {
i1c <- as.character(i1)
if (i1 > 0 & i1 < length.ind) {
break.coord <- end.frags.ind[i1] + (start.frags.ind[i1+1] - end.frags.ind[i1]) / 2
numReads[, 'left', i1c] <- table(strand.frags.ind[1:i1])[c('+','-','*')]
numReads[, 'right', i1c] <- table(strand.frags.ind[(i1+1):length.frags.ind])[c('+','-','*')]
} else if (i1 == length.ind) {
break.coord <- end.conf.cbreaks.ibreak
numReads[, 'left', i1c] <- table(strand.frags.ind[1:i1])[c('+','-','*')]
numReads[, 'right', i1c] <- 0
} else {
break.coord <- start.conf.cbreaks.ibreak
numReads[, 'left', i1c] <- 0
numReads[, 'right', i1c] <- table(strand.frags.ind)[c('+','-','*')]
}
numReads['*', , i1c] <- colSums(numReads[c('+','-'), , i1c]) # assuming there are no * reads in the fragments
i1.bp <- break.coord - start.conf.cbreaks.ibreak
if (doublestranded) {
ptable <- numReads[c('+','-'),,1, drop=FALSE]
} else {
ptable <- numReads[c('*'),,1, drop=FALSE]
}
for (strand in dimnames(ptable)[[1]]) {
for (direction in dimnames(numReads)[[2]]) {
if (doublestranded) {
select <- paste0(c('-'='mstate.', '+'='pstate.')[strand], direction)
} else {
select <- paste0('state.', direction)
}
dtype <- distr[as.character(states[,select]), 'type']
if (dtype == 'dnbinom') {
p <- stats::dnbinom(x = numReads[strand, direction, i1c], size = distr[as.character(states[,select]), 'size'] * i1.bp/binsize, prob = distr[as.character(states[,select]), 'prob'])
} else if (dtype == 'dpois') {
p <- stats::dpois(x = numReads[strand, direction, i1c], lambda = distr[as.character(states[,select]), 'mu'] * i1.bp/binsize)
} else if (dtype == 'dgeom') {
p <- stats::dgeom(x = numReads[strand, direction, i1c], prob = dgeom.prob(distr[as.character(states[,select]), 'mu'] * i1.bp/binsize))
} else if (dtype == 'delta') {
p <- as.numeric(numReads[strand, direction, i1c] == 0)
} else if (dtype == 'dbinom') {
p <- stats::dbinom(x = numReads[strand, direction, i1c], size = round(distr[as.character(states[,select]), 'size'] * i1.bp/binsize), prob = distr[as.character(states[,select]), 'prob'])
}
ptable[strand, direction, ] <- p
}
}
ps[i1c] <- prod(ptable)
break.coords[i1c] <- break.coord
}
new.break <- break.coords[names(ps)[which.max(ps)]]
ranges(cbreaks.mod)[ibreak] <- IRanges(start = new.break, end = new.break)
}
} # end loop ibreak
}
breaks.refined[[chrom]] <- cbreaks.mod
} # end loop chrom
breaks.refined <- unlist(breaks.refined, use.names = FALSE)
stopTimedMessage(ptm)
## Adjust bins and segments of model
ptm <- startTimedMessage("Adjusting bins and segments ...")
# Resize breaks to segments
new.segments <- GenomicRanges::GRangesList()
seqlevels(new.segments) <- seqlevels(model$segments)
new.breaks <- GenomicRanges::GRangesList()
seqlevels(new.breaks) <- seqlevels(breaks.refined)
for (chrom in seqlevels(breaks.refined)) {
cbreaks <- breaks.refined[breaks.refined@seqnames == chrom]
csegs <- model$segments[model$segments@seqnames == chrom]
new.starts <- c(1, end(cbreaks) + 1)
new.ends <- c(end(cbreaks), seqlengths(cbreaks)[chrom])
new.widths <- new.ends - new.starts
# Remove segments and breaks with negative width
ind.remove <- which(new.widths < 0)
if (length(ind.remove) > 0) {
csegs <- csegs[-ind.remove]
cbreaks <- cbreaks[-ind.remove]
# Adjust segment coordinates with breakpoints
ranges(csegs) <- IRanges(start = new.starts[-ind.remove], end = new.ends[-ind.remove])
} else {
# Adjust segment coordinates with breakpoints
ranges(csegs) <- IRanges(start = new.starts, end = new.ends)
}
new.segments[[chrom]] <- csegs
new.breaks[[chrom]] <- cbreaks
}
new.segments <- unlist(new.segments, use.names = FALSE)
new.breaks <- unlist(new.breaks, use.names = FALSE)
# Segmentation step for segments to get rid of consecutive same states
if (doublestranded) {
new.segments$state.temp <- paste(new.segments$mcopy.number, new.segments$pcopy.number)
} else {
new.segments$state.temp <- new.segments$copy.number
}
suppressMessages(
new.segments <- as(collapseBins(as.data.frame(new.segments), column2collapseBy='state.temp', columns2drop='width'), 'GRanges')
)
seqlevels(new.segments) <- seqlevels(model$segments) # correct order from as()
seqlengths(new.segments) <- seqlengths(model$segments)[names(seqlengths(new.segments))]
new.segments$state.temp <- NULL
model$segments <- new.segments
# Reassign bin states
ind <- findOverlaps(model$bins, new.segments, select='first')
if (doublestranded) {
mcols(model$bins)[c('state','mstate','pstate','copy.number','mcopy.number','pcopy.number')] <- mcols(new.segments)[ind,c('state','mstate','pstate','copy.number','mcopy.number','pcopy.number')]
} else {
mcols(model$bins)[c('state','copy.number')] <- mcols(new.segments)[ind,c('state','copy.number')]
}
stopTimedMessage(ptm)
## New breakpoints and confidence intervals
model$breakpoints <- getBreakpoints(model = model, fragments = fragments, confint = confint)
return(model)
}
#' Annotate breakpoints
#'
#' Annotate breakpoints as sister-chromatid-exchange (SCE), copy-number-breakpoint (CNB).
#'
#' @param breakpoints A \code{\link{GRanges-class}} as returned by \code{\link{getBreakpoints}}.
#' @return The input \code{\link{GRanges-class}} with additinal column 'type'.
#'
#' @examples
#'## Get an example BED file with single-cell-sequencing reads
#'bedfile <- system.file("extdata", "KK150311_VI_07.bam.bed.gz", package="AneuFinderData")
#'## Bin the data into bin size 1Mp
#'readfragments <- binReads(bedfile, assembly='mm10', binsize=1e6,
#' chromosomes=c(1:19,'X','Y'), reads.return=TRUE)
#'binned <- binReads(bedfile, assembly='mm10', binsize=1e6,
#' chromosomes=c(1:19,'X','Y'))
#'## Fit the Hidden Markov Model
#'model <- findCNVs.strandseq(binned[[1]])
#'## Add confidence intervals
#'breakpoints <- getBreakpoints(model, readfragments)
#'
annotateBreakpoints <- function(breakpoints) {
ptm <- startTimedMessage("Annotating breakpoints ...")
doublestranded <- 'mstate.left' %in% names(mcols(breakpoints))
## Get copy-numbers from states
if (doublestranded) {
statedf <- mcols(breakpoints)[,c('mstate.left','pstate.left','mstate.right','pstate.right')]
} else {
statedf <- mcols(breakpoints)[,c('state.left','state.right')]
}
statelevels <- unique(unlist(lapply(statedf, levels)))
multiplicity <- suppressWarnings( initializeStates(statelevels)$multiplicity )
copydf <- lapply(statedf, function(x) { multiplicity[x] })
copydf <- as(copydf, 'DataFrame')
if (doublestranded) {
copydf$state.left <- copydf$mstate.left + copydf$pstate.left
copydf$state.right <- copydf$mstate.right + copydf$pstate.right
}
## Categorize breaks
breakpoints$type <- factor('other', levels=c('CNB','SCE','CNB+SCE','other'))
is.cnb <- copydf$state.left != copydf$state.right
breakpoints$type[is.cnb] <- 'CNB'
if (doublestranded) {
is.sce <- ( copydf$mstate.left != copydf$mstate.right ) & ( copydf$pstate.left != copydf$pstate.right )
is.cnb.sce <- is.cnb & is.sce
breakpoints$type[is.sce] <- 'SCE'
breakpoints$type[is.cnb.sce] <- 'CNB+SCE'
}
stopTimedMessage(ptm)
return(breakpoints)
}
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Defines functions annotateBreakpoints refineBreakpoints confidenceIntervals getBreakpoints
Documented in annotateBreakpoints getBreakpoints refineBreakpoints
#' Extract breakpoints
#'
#' Extract breakpoints with confidence intervals from an \code{\link{aneuHMM}} or \code{\link{aneuBiHMM}} object.
#'
#' Confidence intervals for breakpoints are estimated by going outwards from the breakpoint read by read, and performing a test of getting the observed or a more extreme outcome, given that the reads within the confidence interval belong to the other side of the breakpoint.
#'
#' @param model An \code{\link{aneuHMM}} or \code{\link{aneuBiHMM}} object or a file that contains such an object.
#' @param fragments A \code{\link{GRanges-class}} object with read fragments or a file that contains such an object.
#' @param confint Desired confidence interval for breakpoints. Set \code{confint=NULL} to disable confidence interval estimation.
#' @return A \code{\link{GRanges-class}} with breakpoint coordinates and confidence interals if \code{fragments} was specified.
#' @importFrom stats pnbinom pbinom pgeom dnbinom dbinom dgeom
#' @export
#'
#' @examples
#'## Get an example BED file with single-cell-sequencing reads
#'bedfile <- system.file("extdata", "KK150311_VI_07.bam.bed.gz", package="AneuFinderData")
#'## Bin the data into bin size 1Mp
#'readfragments <- binReads(bedfile, assembly='mm10', binsize=1e6,
#' chromosomes=c(1:19,'X','Y'), reads.return=TRUE)
#'binned <- binReads(bedfile, assembly='mm10', binsize=1e6,
#' chromosomes=c(1:19,'X','Y'))
#'## Fit the Hidden Markov Model
#'model <- findCNVs.strandseq(binned[[1]])
#'## Add confidence intervals
#'breakpoints <- getBreakpoints(model, readfragments)
#'
getBreakpoints <- function(model, fragments=NULL, confint=0.99) {
model <- loadFromFiles(model, check.class = c("aneuHMM", "aneuBiHMM"))[[1]]
if (is.null(model$segments)) {
gr <- GRanges(seqnames = character(), ranges = IRanges(), mstate.left=factor(), pstate.left=factor(), mstate.right=factor(), pstate.right=factor(), start.conf=integer(), end.conf=integer(), type=factor())
return(gr)
}
fragments <- loadFromFiles(fragments, check.class = 'GRanges')[[1]]
binsize <- mean(width(model$bincounts[[1]]))
## Get breakpoints
breaks <- model$segments
mcols(breaks) <- NULL
end(breaks) <- end(breaks) - 1
seqlengths(breaks) <- NA # to not have the chromosome end as gap
breaks <- gaps(breaks)
breaks <- breaks[strand(breaks)=='*']
seqlengths(breaks) <- seqlengths(model$segments)
# Add genotype transition
if (length(breaks) == 0) {
return(breaks)
}
ind <- findOverlaps(breaks, model$segments)
if (class(model) == "aneuBiHMM") {
breaks$mstate.left <- factor(NA, levels=levels(model$segments$mstate))
breaks$mstate.left[ind@from] <- model$segments$mstate[ind@to]
breaks$pstate.left <- factor(NA, levels=levels(model$segments$pstate))
breaks$pstate.left[ind@from] <- model$segments$pstate[ind@to]
breaks$mstate.right <- factor(NA, levels=levels(model$segments$mstate))
breaks$mstate.right[ind@from] <- model$segments$mstate[ind@to+1]
breaks$pstate.right <- factor(NA, levels=levels(model$segments$pstate))
breaks$pstate.right[ind@from] <- model$segments$pstate[ind@to+1]
} else if (class(model) == "aneuHMM") {
breaks$state.left <- factor(NA, levels=levels(model$segments$state))
breaks$state.left[ind@from] <- model$segments$state[ind@to]
breaks$state.right <- factor(NA, levels=levels(model$segments$state))
breaks$state.right[ind@from] <- model$segments$state[ind@to+1]
}
if (is.null(fragments) | is.null(confint)) {
breaks <- annotateBreakpoints(breaks)
return(breaks)
}
## Sort fragments
seqlevels(fragments) <- seqlevels(model$segments)
fragments <- sort(fragments, ignore.strand=TRUE)
fragments$p <- 0
## Distributions
distr <- model$distributions
if (class(distr) == 'list') {
distr <- distr[[1]]
}
breaks.conf <- confidenceIntervals(breaks = breaks, fragments = fragments, distr = distr, confint = confint, binsize = binsize)
breaks$start.conf <- start(breaks.conf)
breaks$end.conf <- end(breaks.conf)
breaks <- annotateBreakpoints(breaks)
return(breaks)
}
confidenceIntervals <- function(breaks, fragments, distr, confint, binsize) {
ptm <- startTimedMessage("Finding confidence intervals ...")
min.i1 <- 10
doublestranded <- 'mstate.left' %in% names(mcols(breaks))
## Do chromosomes one by one
breaks.conf <- GenomicRanges::GRangesList()
seqlevels(breaks.conf) <- seqlevels(breaks)
for (chrom in unique(seqnames(breaks))) {
# message("chrom = ", chrom)
cbreaks <- breaks[seqnames(breaks) == chrom]
cbreaks.mod <- cbreaks
cfrags <- fragments[seqnames(fragments) == chrom]
if (length(cbreaks) > 0) {
for (ibreak in 1:length(cbreaks)) {
# message(ibreak)
states <- as.data.frame(mcols(cbreaks[ibreak]))
if (doublestranded) {
mus <- array(NA, dim=c(2,2), dimnames=list(strand=c('-','+'), direction=c('left','right')))
mus['-','right'] <- distr[as.character(states[,'mstate.right']),'mu']
mus['+','right'] <- distr[as.character(states[,'pstate.right']),'mu']
mus['-','left'] <- distr[as.character(states[,'mstate.left']),'mu']
mus['+','left'] <- distr[as.character(states[,'pstate.left']),'mu']
} else {
mus <- array(NA, dim=c(1,2), dimnames=list(strand=c('*'), direction=c('left','right')))
mus['*','right'] <- distr[as.character(states[,'state.right']),'mu']
mus['*','left'] <- distr[as.character(states[,'state.left']),'mu']
}
# Directionality of test
left.is.bigger <- mus[,'left', drop=FALSE] >= mus[,'right', drop=FALSE]
right.is.bigger <- !left.is.bigger
## Left side
ind <- which(start(cfrags) < start(cbreaks)[ibreak])
if (ibreak > 1) {
max.bp <- start(cbreaks[ibreak]) - start(cbreaks[ibreak-1])
} else {
max.bp <- start(cbreaks[ibreak])
}
if (length(ind) > 0) {
ind <- ind[length(ind)]
p <- 1
ps <- numeric()
ps['0'] <- p
numReads <- c('*'=0, '-'=0, '+'=0)
if (!any(is.na(mus))) {
i1 <- -1
## Helpers for speed improvement
start.cfrags <- start(cfrags)
start.cbreaks <- start(cbreaks)
strand.cfrags <- as.character(strand(cfrags))
while (p > 1-confint | i1 <= min.i1) {
i1 <- i1+1
if (ind-i1 <= 0) {
i1 = i1 - 1
break
}
i1.bp <- - (start.cfrags[ind-i1] - start.cbreaks[ibreak])
if (i1.bp > max.bp) {
i1 = i1 - 1
break
}
strandofread <- strand.cfrags[ind-i1]
numReads[strandofread] <- numReads[strandofread] + 1 # assuming there are no * reads in the fragments
numReads['*'] <- numReads['*'] + 1
if (doublestranded) {
# Minus strand
dtype <- distr[as.character(states[,'mstate.right']), 'type']
if (dtype == 'dnbinom') {
p.minus <- stats::pnbinom(q = numReads['-'] - !right.is.bigger['-',], size = distr[as.character(states[,'mstate.right']), 'size'] * i1.bp/binsize, prob = distr[as.character(states[,'mstate.right']), 'prob'], lower.tail = right.is.bigger['-',])
} else if (dtype == 'dpois') {
p.minus <- stats::ppois(q = numReads['-'] - !right.is.bigger['-',], lambda = distr[as.character(states[,'mstate.right']), 'mu'] * i1.bp/binsize, lower.tail = right.is.bigger['-',])
} else if (dtype == 'dgeom') {
p.minus <- stats::pgeom(q = numReads['-'] - !right.is.bigger['-',], prob = dgeom.prob(distr[as.character(states[,'mstate.right']), 'mu'] * i1.bp/binsize), lower.tail = right.is.bigger['-',])
} else if (dtype == 'delta') {
p.minus <- c('-'=as.numeric(numReads['-'] == 0))
} else if (dtype == 'dbinom') {
p.minus <- stats::pbinom(q = numReads['-'] - !right.is.bigger['-',], size = round(distr[as.character(states[,'mstate.right']), 'size'] * i1.bp/binsize), prob = distr[as.character(states[,'mstate.right']), 'prob'], lower.tail = right.is.bigger['-',])
}
# Plus strand
dtype <- distr[as.character(states[,'pstate.right']), 'type']
if (dtype == 'dnbinom') {
p.plus <- stats::pnbinom(q = numReads['+'] - !right.is.bigger['+',], size = distr[as.character(states[,'pstate.right']), 'size'] * i1.bp/binsize, prob = distr[as.character(states[,'pstate.right']), 'prob'], lower.tail = right.is.bigger['+',])
} else if (dtype == 'dpois') {
p.plus <- stats::ppois(q = numReads['+'] - !right.is.bigger['+',], lambda = distr[as.character(states[,'pstate.right']), 'mu'] * i1.bp/binsize, lower.tail = right.is.bigger['+',])
} else if (dtype == 'dgeom') {
p.plus <- stats::pgeom(q = numReads['+'] - !right.is.bigger['+',], prob = dgeom.prob(distr[as.character(states[,'pstate.right']), 'mu'] * i1.bp/binsize), lower.tail = right.is.bigger['+',])
} else if (dtype == 'delta') {
p.plus <- c('+'=as.numeric(numReads['+'] == 0))
} else if (dtype == 'dbinom') {
p.plus <- stats::pbinom(q = numReads['+'] - !right.is.bigger['+',], size = round(distr[as.character(states[,'pstate.right']), 'size'] * i1.bp/binsize), prob = distr[as.character(states[,'pstate.right']), 'prob'], lower.tail = right.is.bigger['+',])
}
p <- c(p.minus, p.plus)
p[is.na(p)] <- 1
p <- p['-'] * p['+']
} else {
# Star strand
dtype <- distr[as.character(states[,'state.right']), 'type']
if (dtype == 'dnbinom') {
p <- stats::pnbinom(q = numReads['*'] - !right.is.bigger['*',], size = distr[as.character(states[,'state.right']), 'size'] * i1.bp/binsize, prob = distr[as.character(states[,'state.right']), 'prob'], lower.tail = right.is.bigger['*',])
} else if (dtype == 'dpois') {
p <- stats::ppois(q = numReads['*'] - !right.is.bigger['*',], lambda = distr[as.character(states[,'state.right']), 'mu'] * i1.bp/binsize, lower.tail = right.is.bigger['*',])
} else if (dtype == 'dgeom') {
p <- stats::pgeom(q = numReads['*'] - !right.is.bigger['*',], prob = dgeom.prob(distr[as.character(states[,'state.right']), 'mu'] * i1.bp/binsize), lower.tail = right.is.bigger['*',])
} else if (dtype == 'delta') {
p <- c('*'=as.numeric(numReads['*'] == 0))
} else if (dtype == 'dbinom') {
p <- stats::pbinom(q = numReads['*'] - !right.is.bigger['*',], size = round(distr[as.character(states[,'state.right']), 'size'] * i1.bp/binsize), prob = distr[as.character(states[,'state.right']), 'prob'], lower.tail = right.is.bigger['*',])
}
}
ps[as.character(i1)] <- p
}
if (i1 >= 0) {
revps <- rev(ps)
if (revps[1] < 1-confint) {
i1 <- as.numeric(names(revps)[which(revps >= 1-confint)[1] - 1]) # first one from the end that is below threshold
if (is.na(i1)) {
i1 <- as.numeric(names(ps)[1])
}
} else {
i1 <- as.numeric(names(revps)[1])
}
new.start <- end(cfrags)[ind-i1]
if (new.start < start(cbreaks.mod)[ibreak]) {
start(cbreaks.mod)[ibreak] <- new.start
}
}
}
}
## Right side
ind <- which(end(cfrags) > end(cbreaks)[ibreak])
if (ibreak < length(cbreaks)) {
max.bp <- end(cbreaks[ibreak+1]) - end(cbreaks[ibreak])
} else {
max.bp <- Inf
}
if (length(ind) > 0) {
ind <- ind[1]
p <- 1
ps <- numeric()
ps['0'] <- p
numReads <- c('*'=0, '-'=0, '+'=0)
if (!any(is.na(mus))) {
i1 <- -1
## Helpers for speed improvement
end.cfrags <- end(cfrags)
end.cbreaks <- end(cbreaks)
strand.cfrags <- as.character(strand(cfrags))
while (p > 1-confint | i1 <= min.i1) {
i1 <- i1+1
if (ind+i1 > length(cfrags)) {
i1 = i1 - 1
break
}
i1.bp <- end.cfrags[ind+i1] - end.cbreaks[ibreak]
if (i1.bp > max.bp) {
i1 = i1 - 1
break
}
strandofread <- strand.cfrags[ind+i1]
numReads[strandofread] <- numReads[strandofread] + 1 # assuming there are no * reads in the fragments
numReads['*'] <- numReads['*'] + 1
if (doublestranded) {
# Minus strand
dtype <- distr[as.character(states[,'mstate.left']), 'type']
if (dtype == 'dnbinom') {
p.minus <- stats::pnbinom(q = numReads['-'] - !left.is.bigger['-',], size = distr[as.character(states[,'mstate.left']), 'size'] * i1.bp/binsize, prob = distr[as.character(states[,'mstate.left']), 'prob'], lower.tail = left.is.bigger['-',])
} else if (dtype == 'dpois') {
p.minus <- stats::ppois(q = numReads['-'] - !left.is.bigger['-',], lambda = distr[as.character(states[,'mstate.left']), 'mu'] * i1.bp/binsize, lower.tail = left.is.bigger['-',])
} else if (dtype == 'dgeom') {
p.minus <- stats::pgeom(q = numReads['-'] - !left.is.bigger['-',], prob = dgeom.prob(distr[as.character(states[,'mstate.left']), 'mu'] * i1.bp/binsize), lower.tail = left.is.bigger['-',])
} else if (dtype == 'delta') {
p.minus <- c('-'=as.numeric(numReads['-'] == 0))
} else if (dtype == 'dbinom') {
p.minus <- stats::pbinom(q = numReads['-'] - !left.is.bigger['-',], size = round(distr[as.character(states[,'mstate.left']), 'size'] * i1.bp/binsize), prob = distr[as.character(states[,'mstate.left']), 'prob'], lower.tail = left.is.bigger['-',])
}
# Plus strand
dtype <- distr[as.character(states[,'pstate.left']), 'type']
if (dtype == 'dnbinom') {
p.plus <- stats::pnbinom(q = numReads['+'] - !left.is.bigger['+',], size = distr[as.character(states[,'pstate.left']), 'size'] * i1.bp/binsize, prob = distr[as.character(states[,'pstate.left']), 'prob'], lower.tail = left.is.bigger['+',])
} else if (dtype == 'dpois') {
p.plus <- stats::ppois(q = numReads['+'] - !left.is.bigger['+',], lambda = distr[as.character(states[,'pstate.left']), 'mu'] * i1.bp/binsize, lower.tail = left.is.bigger['+',])
} else if (dtype == 'dgeom') {
p.plus <- stats::pgeom(q = numReads['+'] - !left.is.bigger['+',], prob = dgeom.prob(distr[as.character(states[,'pstate.left']), 'mu'] * i1.bp/binsize), lower.tail = left.is.bigger['+',])
} else if (dtype == 'delta') {
p.plus <- c('+'=as.numeric(numReads['+'] == 0))
} else if (dtype == 'dbinom') {
p.plus <- stats::pbinom(q = numReads['+'] - !left.is.bigger['+',], size = round(distr[as.character(states[,'pstate.left']), 'size'] * i1.bp/binsize), prob = distr[as.character(states[,'pstate.left']), 'prob'], lower.tail = left.is.bigger['+',])
}
p <- c(p.minus, p.plus)
p[is.na(p)] <- 1
p <- p['-'] * p['+']
} else {
# Star strand
dtype <- distr[as.character(states[,'state.left']), 'type']
if (dtype == 'dnbinom') {
p <- stats::pnbinom(q = numReads['*'] - !left.is.bigger['*',], size = distr[as.character(states[,'state.left']), 'size'] * i1.bp/binsize, prob = distr[as.character(states[,'state.left']), 'prob'], lower.tail = left.is.bigger['*',])
} else if (dtype == 'dpois') {
p <- stats::ppois(q = numReads['*'] - !left.is.bigger['*',], lambda = distr[as.character(states[,'state.left']), 'mu'] * i1.bp/binsize, lower.tail = left.is.bigger['*',])
} else if (dtype == 'dgeom') {
p <- stats::pgeom(q = numReads['*'] - !left.is.bigger['*',], prob = dgeom.prob(distr[as.character(states[,'state.left']), 'mu'] * i1.bp/binsize), lower.tail = left.is.bigger['*',])
} else if (dtype == 'delta') {
p <- c('*'=as.numeric(numReads['*'] == 0))
} else if (dtype == 'dbinom') {
p <- stats::pbinom(q = numReads['*'] - !left.is.bigger['*',], size = round(distr[as.character(states[,'state.left']), 'size'] * i1.bp/binsize), prob = distr[as.character(states[,'state.left']), 'prob'], lower.tail = left.is.bigger['*',])
}
}
ps[as.character(i1)] <- p
}
if (i1 >= 0) {
revps <- rev(ps)
if (revps[1] < 1-confint) {
i1 <- as.numeric(names(revps)[which(revps >= 1-confint)[1] - 1]) # first one from the end that is below threshold
if (is.na(i1)) {
i1 <- as.numeric(names(ps)[1])
}
} else {
i1 <- as.numeric(names(revps)[1])
}
new.end <- start(cfrags)[ind+i1]
if (new.end > end(cbreaks.mod)[ibreak]) {
end(cbreaks.mod)[ibreak] <- new.end
}
}
}
}
}
}
breaks.conf[[chrom]] <- cbreaks.mod
}
breaks.conf <- unlist(breaks.conf, use.names = FALSE)
stopTimedMessage(ptm)
return(breaks.conf)
}
#' Refine breakpoints
#'
#' Refine breakpoints with confidence intervals from an initial estimate (from \code{\link{getBreakpoints}}).
#'
#' Breakpoints are refined by shifting the breakpoint within its initial confidence interval read by read and maximizing the probability of observing the left-right read distribution.
#'
#' @param model An \code{\link{aneuBiHMM}} object or a file that contains such an object.
#' @param fragments A \code{\link{GRanges-class}} object with read fragments or a file that contains such an object.
#' @param breakpoints A \code{\link{GRanges-class}} object with breakpoints and confidence intervals, as returned by function \code{\link{getBreakpoints}}.
#' @param confint Desired confidence interval for breakpoints.
#' @return An \code{\link{aneuBiHMM}} with adjusted breakpoint coordinates and confidence interals, bins and segments.
#' @export
#'
#' @examples
#'## Get an example BED file with single-cell-sequencing reads
#'bedfile <- system.file("extdata", "KK150311_VI_07.bam.bed.gz", package="AneuFinderData")
#'## Bin the data into bin size 1Mp
#'readfragments <- binReads(bedfile, assembly='mm10', binsize=1e6,
#' chromosomes=c(1:19,'X','Y'), reads.return=TRUE)
#'binned <- binReads(bedfile, assembly='mm10', binsize=1e6,
#' chromosomes=c(1:19,'X','Y'))
#'## Fit the Hidden Markov Model
#'model <- findCNVs.strandseq(binned[[1]])
#'## Add confidence intervals
#'breakpoints <- getBreakpoints(model, readfragments)
#'## Refine breakpoints
#'refined.model <- refineBreakpoints(model, readfragments, breakpoints)
#'
refineBreakpoints <- function(model, fragments, breakpoints = model$breakpoints, confint=0.99) {
ptm <- startTimedMessage("Refining breakpoints ...")
model <- loadFromFiles(model, check.class = c("aneuHMM", "aneuBiHMM"))[[1]]
doublestranded <- 'mstate.left' %in% names(mcols(breakpoints))
fragments <- loadFromFiles(fragments, check.class = 'GRanges')[[1]]
binsize <- mean(width(model$bincounts[[1]]))
breaks <- breakpoints
if (is.null(breaks)) {
stopTimedMessage(ptm)
warning(paste0(model$ID, ": No breakpoints found. Cannot refine breakpoints."))
return(model)
}
if (is.null(breaks$start.conf)) {
stopTimedMessage(ptm)
warning(paste0(model$ID, ": No confidence intervals found. Cannot refine breakpoints."))
return(model)
}
if (length(breaks) == 0) {
stopTimedMessage(ptm)
return(model)
}
## Sort fragments
seqlevels(fragments) <- seqlevels(model$segments)
fragments <- sort(fragments, ignore.strand=TRUE)
fragments$p <- 0
## Distributions
distr <- model$distributions
if (class(distr) == 'list') {
distr <- distr[[1]]
}
## Do chromosomes one by one
breaks.refined <- GenomicRanges::GRangesList()
seqlevels(breaks.refined) <- seqlevels(breaks)
for (chrom in unique(seqnames(breaks))) {
# message("chrom = ", chrom)
cbreaks <- breaks[seqnames(breaks) == chrom]
cbreaks.mod <- cbreaks
cfrags <- fragments[seqnames(fragments) == chrom]
if (length(cbreaks) > 0) {
for (ibreak in 1:length(cbreaks)) {
# message(ibreak, "/", length(cbreaks))
## Go through confidence interval left to right
states <- as.data.frame(mcols(cbreaks[ibreak]))
ind <- which( (start(cfrags) >= cbreaks[ibreak]$start.conf) & (end(cfrags) <= cbreaks[ibreak]$end.conf) )
frags.ind <- cfrags[ind]
if (length(ind) > 0) {
numReads <- array(NA, dim=c(3,2,length(ind)+1), dimnames=list(strand=c('+','-','*'), direction=c('left','right'), ind=0:length(ind)))
ps <- array(NA, dim=c(length(ind)+1), dimnames=list(ind=0:length(ind)))
break.coords <- array(NA, dim=c(length(ind)+1), dimnames=list(ind=0:length(ind)))
## Helpers for speed improvement
end.frags.ind <- end(frags.ind)
start.frags.ind <- start(frags.ind)
strand.frags.ind <- strand(frags.ind)
length.frags.ind <- length(frags.ind)
length.ind <- length(ind)
start.conf.cbreaks.ibreak <- cbreaks[ibreak]$start.conf
end.conf.cbreaks.ibreak <- cbreaks[ibreak]$end.conf
for (i1 in 0:length.ind) {
i1c <- as.character(i1)
if (i1 > 0 & i1 < length.ind) {
break.coord <- end.frags.ind[i1] + (start.frags.ind[i1+1] - end.frags.ind[i1]) / 2
numReads[, 'left', i1c] <- table(strand.frags.ind[1:i1])[c('+','-','*')]
numReads[, 'right', i1c] <- table(strand.frags.ind[(i1+1):length.frags.ind])[c('+','-','*')]
} else if (i1 == length.ind) {
break.coord <- end.conf.cbreaks.ibreak
numReads[, 'left', i1c] <- table(strand.frags.ind[1:i1])[c('+','-','*')]
numReads[, 'right', i1c] <- 0
} else {
break.coord <- start.conf.cbreaks.ibreak
numReads[, 'left', i1c] <- 0
numReads[, 'right', i1c] <- table(strand.frags.ind)[c('+','-','*')]
}
numReads['*', , i1c] <- colSums(numReads[c('+','-'), , i1c]) # assuming there are no * reads in the fragments
i1.bp <- break.coord - start.conf.cbreaks.ibreak
if (doublestranded) {
ptable <- numReads[c('+','-'),,1, drop=FALSE]
} else {
ptable <- numReads[c('*'),,1, drop=FALSE]
}
for (strand in dimnames(ptable)[[1]]) {
for (direction in dimnames(numReads)[[2]]) {
if (doublestranded) {
select <- paste0(c('-'='mstate.', '+'='pstate.')[strand], direction)
} else {
select <- paste0('state.', direction)
}
dtype <- distr[as.character(states[,select]), 'type']
if (dtype == 'dnbinom') {
p <- stats::dnbinom(x = numReads[strand, direction, i1c], size = distr[as.character(states[,select]), 'size'] * i1.bp/binsize, prob = distr[as.character(states[,select]), 'prob'])
} else if (dtype == 'dpois') {
p <- stats::dpois(x = numReads[strand, direction, i1c], lambda = distr[as.character(states[,select]), 'mu'] * i1.bp/binsize)
} else if (dtype == 'dgeom') {
p <- stats::dgeom(x = numReads[strand, direction, i1c], prob = dgeom.prob(distr[as.character(states[,select]), 'mu'] * i1.bp/binsize))
} else if (dtype == 'delta') {
p <- as.numeric(numReads[strand, direction, i1c] == 0)
} else if (dtype == 'dbinom') {
p <- stats::dbinom(x = numReads[strand, direction, i1c], size = round(distr[as.character(states[,select]), 'size'] * i1.bp/binsize), prob = distr[as.character(states[,select]), 'prob'])
}
ptable[strand, direction, ] <- p
}
}
ps[i1c] <- prod(ptable)
break.coords[i1c] <- break.coord
}
new.break <- break.coords[names(ps)[which.max(ps)]]
ranges(cbreaks.mod)[ibreak] <- IRanges(start = new.break, end = new.break)
}
} # end loop ibreak
}
breaks.refined[[chrom]] <- cbreaks.mod
} # end loop chrom
breaks.refined <- unlist(breaks.refined, use.names = FALSE)
stopTimedMessage(ptm)
## Adjust bins and segments of model
ptm <- startTimedMessage("Adjusting bins and segments ...")
# Resize breaks to segments
new.segments <- GenomicRanges::GRangesList()
seqlevels(new.segments) <- seqlevels(model$segments)
new.breaks <- GenomicRanges::GRangesList()
seqlevels(new.breaks) <- seqlevels(breaks.refined)
for (chrom in seqlevels(breaks.refined)) {
cbreaks <- breaks.refined[breaks.refined@seqnames == chrom]
csegs <- model$segments[model$segments@seqnames == chrom]
new.starts <- c(1, end(cbreaks) + 1)
new.ends <- c(end(cbreaks), seqlengths(cbreaks)[chrom])
new.widths <- new.ends - new.starts
# Remove segments and breaks with negative width
ind.remove <- which(new.widths < 0)
if (length(ind.remove) > 0) {
csegs <- csegs[-ind.remove]
cbreaks <- cbreaks[-ind.remove]
# Adjust segment coordinates with breakpoints
ranges(csegs) <- IRanges(start = new.starts[-ind.remove], end = new.ends[-ind.remove])
} else {
# Adjust segment coordinates with breakpoints
ranges(csegs) <- IRanges(start = new.starts, end = new.ends)
}
new.segments[[chrom]] <- csegs
new.breaks[[chrom]] <- cbreaks
}
new.segments <- unlist(new.segments, use.names = FALSE)
new.breaks <- unlist(new.breaks, use.names = FALSE)
# Segmentation step for segments to get rid of consecutive same states
if (doublestranded) {
new.segments$state.temp <- paste(new.segments$mcopy.number, new.segments$pcopy.number)
} else {
new.segments$state.temp <- new.segments$copy.number
}
suppressMessages(
new.segments <- as(collapseBins(as.data.frame(new.segments), column2collapseBy='state.temp', columns2drop='width'), 'GRanges')
)
seqlevels(new.segments) <- seqlevels(model$segments) # correct order from as()
seqlengths(new.segments) <- seqlengths(model$segments)[names(seqlengths(new.segments))]
new.segments$state.temp <- NULL
model$segments <- new.segments
# Reassign bin states
ind <- findOverlaps(model$bins, new.segments, select='first')
if (doublestranded) {
mcols(model$bins)[c('state','mstate','pstate','copy.number','mcopy.number','pcopy.number')] <- mcols(new.segments)[ind,c('state','mstate','pstate','copy.number','mcopy.number','pcopy.number')]
} else {
mcols(model$bins)[c('state','copy.number')] <- mcols(new.segments)[ind,c('state','copy.number')]
}
stopTimedMessage(ptm)
## New breakpoints and confidence intervals
model$breakpoints <- getBreakpoints(model = model, fragments = fragments, confint = confint)
return(model)
}
#' Annotate breakpoints
#'
#' Annotate breakpoints as sister-chromatid-exchange (SCE), copy-number-breakpoint (CNB).
#'
#' @param breakpoints A \code{\link{GRanges-class}} as returned by \code{\link{getBreakpoints}}.
#' @return The input \code{\link{GRanges-class}} with additinal column 'type'.
#'
#' @examples
#'## Get an example BED file with single-cell-sequencing reads
#'bedfile <- system.file("extdata", "KK150311_VI_07.bam.bed.gz", package="AneuFinderData")
#'## Bin the data into bin size 1Mp
#'readfragments <- binReads(bedfile, assembly='mm10', binsize=1e6,
#' chromosomes=c(1:19,'X','Y'), reads.return=TRUE)
#'binned <- binReads(bedfile, assembly='mm10', binsize=1e6,
#' chromosomes=c(1:19,'X','Y'))
#'## Fit the Hidden Markov Model
#'model <- findCNVs.strandseq(binned[[1]])
#'## Add confidence intervals
#'breakpoints <- getBreakpoints(model, readfragments)
#'
annotateBreakpoints <- function(breakpoints) {
ptm <- startTimedMessage("Annotating breakpoints ...")
doublestranded <- 'mstate.left' %in% names(mcols(breakpoints))
## Get copy-numbers from states
if (doublestranded) {
statedf <- mcols(breakpoints)[,c('mstate.left','pstate.left','mstate.right','pstate.right')]
} else {
statedf <- mcols(breakpoints)[,c('state.left','state.right')]
}
statelevels <- unique(unlist(lapply(statedf, levels)))
multiplicity <- suppressWarnings( initializeStates(statelevels)$multiplicity )
copydf <- lapply(statedf, function(x) { multiplicity[x] })
copydf <- as(copydf, 'DataFrame')
if (doublestranded) {
copydf$state.left <- copydf$mstate.left + copydf$pstate.left
copydf$state.right <- copydf$mstate.right + copydf$pstate.right
}
## Categorize breaks
breakpoints$type <- factor('other', levels=c('CNB','SCE','CNB+SCE','other'))
is.cnb <- copydf$state.left != copydf$state.right
breakpoints$type[is.cnb] <- 'CNB'
if (doublestranded) {
is.sce <- ( copydf$mstate.left != copydf$mstate.right ) & ( copydf$pstate.left != copydf$pstate.right )
is.cnb.sce <- is.cnb & is.sce
breakpoints$type[is.sce] <- 'SCE'
breakpoints$type[is.cnb.sce] <- 'CNB+SCE'
}
stopTimedMessage(ptm)
return(breakpoints)
}
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