/
draw_fusions.R
executable file
·1389 lines (1295 loc) · 74.6 KB
/
draw_fusions.R
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#!/usr/bin/env Rscript
# print warnings as they happen instead of collecting them for after a loop ends
options(warn=1)
# define valid parameters
parameters <- list(
fusions=list("fusionsFile", "file", "fusions.tsv", T),
annotation=list("exonsFile", "file", "annotation.gtf", T),
output=list("outputFile", "string", "output.pdf", T),
alignments=list("alignmentsFile", "file", "Aligned.sortedByCoord.out.bam"),
cytobands=list("cytobandsFile", "file", "cytobands.tsv"),
minConfidenceForCircosPlot=list("minConfidenceForCircosPlot", "string", "medium"),
proteinDomains=list("proteinDomainsFile", "file", "protein_domains.gff3"),
sampleName=list("sampleName", "string", ""),
squishIntrons=list("squishIntrons", "bool", T),
printExonLabels=list("printExonLabels", "bool", T),
render3dEffect=list("render3dEffect", "bool", T),
pdfWidth=list("pdfWidth", "numeric", 11.692),
pdfHeight=list("pdfHeight", "numeric", 8.267),
color1=list("color1", "string", "#e5a5a5"),
color2=list("color2", "string", "#a7c4e5"),
mergeDomainsOverlappingBy=list("mergeDomainsOverlappingBy", "numeric", 0.9),
optimizeDomainColors=list("optimizeDomainColors", "bool", F),
fontSize=list("fontSize", "numeric", 1),
fontFamily=list("fontFamily", "string", "Helvetica"),
showIntergenicVicinity=list("showIntergenicVicinity", "string", "0"),
transcriptSelection=list("transcriptSelection", "string", "provided"),
fixedScale=list("fixedScale", "numeric", 0),
coverageRange=list("coverageRange", "string", "0")
)
# print help if necessary
args <- commandArgs(trailingOnly=T)
if (any(grepl("^--help", args)) || length(args) == 0) {
usage <- "Usage: draw_fusions.R"
for (parameter in names(parameters)) {
usage <- paste0(usage, " ")
if (length(parameters[[parameter]]) <= 3 || !parameters[[parameter]][[4]])
usage <- paste0(usage, "[")
usage <- paste0(usage, "--", parameter, "=", parameters[[parameter]][[3]])
if (length(parameters[[parameter]]) <= 3 || !parameters[[parameter]][[4]])
usage <- paste0(usage, "]")
}
message(usage)
quit("no", ifelse(length(args) == 0, 1, 0))
}
# make sure mandatory arguments are present
for (parameter in names(parameters))
if (length(parameters[[parameter]]) > 3 && parameters[[parameter]][[4]])
if (!any(grepl(paste0("^--", parameter, "="), args), perl=T))
stop(paste0("Missing mandatory argument: --", parameter))
# set default values
for (parameter in names(parameters))
assign(parameters[[parameter]][[1]], ifelse(parameters[[parameter]][[2]] == "file", "", parameters[[parameter]][[3]]))
# parse command-line parameters
for (arg in args) {
argName <- sub("=.*", "", sub("^--", "", arg, perl=T), perl=T)
argValue <- sub("^[^=]*=", "", arg, perl=T)
if (!(argName %in% names(parameters)) || !grepl("^--", arg, perl=T))
stop(paste("Unknown parameter:", arg))
if (parameters[[argName]][[2]] == "bool") {
if (argValue %in% c("TRUE", "T", "FALSE", "F")) {
assign(parameters[[argName]][[1]], as.logical(argValue))
} else {
stop(paste0("Invalid argument to --", argName))
}
} else if (parameters[[argName]][[2]] == "string") {
assign(parameters[[argName]][[1]], argValue)
} else if (parameters[[argName]][[2]] == "numeric") {
if (is.na(suppressWarnings(as.numeric(argValue))))
stop(paste0("Invalid argument to --", argName))
assign(parameters[[argName]][[1]], as.numeric(argValue))
} else if (parameters[[argName]][[2]] == "file") {
if (file.access(argValue) == -1)
stop(paste("Cannot read file:", argValue))
assign(parameters[[argName]][[1]], argValue)
}
}
# validate values of parameters
if (cytobandsFile == "")
warning("Missing parameter '--cytobands'. No ideograms and circos plots will be drawn.")
if (!(minConfidenceForCircosPlot %in% c("none", "low", "medium", "high")))
stop("Invalid argument to --minConfidenceForCircosPlot")
showIntergenicVicinity <- as.list(unlist(strsplit(showIntergenicVicinity, ",", fixed=T)))
if (!(length(showIntergenicVicinity) %in% c(1,4)))
stop(paste0("Invalid argument to --showIntergenicVicinity"))
showIntergenicVicinity <- lapply(showIntergenicVicinity, function(x) {
if (x == "closestGene") {
return("exon")
} else if (x == "closestProteinCodingGene") {
return("CDS")
} else if (is.na(suppressWarnings(as.numeric(x))) || as.numeric(x) < 0) {
stop(paste0("Invalid argument to --showIntergenicVicinity"))
} else {
return(as.numeric(x))
}
})
if (length(showIntergenicVicinity) == 1)
showIntergenicVicinity <- rep(showIntergenicVicinity, 4)
if (squishIntrons)
if (any(!is.numeric(unlist(showIntergenicVicinity))) || any(showIntergenicVicinity > 0))
stop("--squishIntrons must be disabled, when --showIntergenicVicinity is > 0")
if (!(transcriptSelection %in% c("coverage", "provided", "canonical")))
stop("Invalid argument to --transcriptSelection")
if (fixedScale < 0)
stop("Invalid argument to --fixedScale")
if (!(fontFamily %in% names(pdfFonts())))
stop(paste0("Unknown font: ", fontFamily, ". Available fonts: ", paste(names(pdfFonts()), collapse=", ")))
coverageRange <- suppressWarnings(as.numeric(unlist(strsplit(coverageRange, ",", fixed=T))))
if (!(length(coverageRange) %in% 1:2) || any(is.na(coverageRange)) || any(coverageRange < 0))
stop("Invalid argument to --coverageRange")
# check if required packages are installed
if (!suppressPackageStartupMessages(require(GenomicRanges)))
warning("Package 'GenomicRanges' is not installed. No protein domains and circos plots will be drawn.")
if (!suppressPackageStartupMessages(require(circlize)))
warning("Package 'circlize' is not installed. No circos plots will be drawn.")
if (alignmentsFile != "")
if (!suppressPackageStartupMessages(require(GenomicAlignments)))
stop("Package 'GenomicAlignments' must be installed when '--alignments' is used")
# define colors
changeColorBrightness <- function(color, delta) {
rgb(
min(255,max(0,col2rgb(color)["red",]+delta)),
min(255,max(0,col2rgb(color)["green",]+delta)),
min(255,max(0,col2rgb(color)["blue",]+delta)),
maxColorValue=255
)
}
getDarkColor <- function(color) { changeColorBrightness(color, -100) }
getBrightColor <- function(color) { changeColorBrightness(color, +190) }
darkColor1 <- getDarkColor(color1)
darkColor2 <- getDarkColor(color2)
circosColors <- c(translocation="#000000", duplication="#00bb00", deletion="#ff0000", inversion="#0000ff")
# convenience functions to add/remove "chr" prefix
addChr <- function(contig) {
ifelse(contig == "MT", "chrM", paste0("chr", contig))
}
removeChr <- function(contig) {
sub("^chr", "", sub("^chrM", "MT", contig, perl=T), perl=T)
}
# convenience function to check if a value is between two others
between <- function(value, start, end) {
value >= start & value <= end
}
# read fusions
fusions <- read.table(fusionsFile, stringsAsFactors=F, sep="\t", header=T, comment.char="", quote="")
if (colnames(fusions)[1] == "X.gene1") { # Arriba output
colnames(fusions)[colnames(fusions) %in% c("X.gene1", "strand1.gene.fusion.", "strand2.gene.fusion.")] <- c("gene1", "strand1", "strand2")
fusions$display_contig1 <- sub(":[^:]*$", "", fusions$breakpoint1, perl=T)
fusions$display_contig2 <- sub(":[^:]*$", "", fusions$breakpoint2, perl=T)
fusions$contig1 <- removeChr(fusions$display_contig1)
fusions$contig2 <- removeChr(fusions$display_contig2)
fusions$breakpoint1 <- as.numeric(sub(".*:", "", fusions$breakpoint1, perl=T))
fusions$breakpoint2 <- as.numeric(sub(".*:", "", fusions$breakpoint2, perl=T))
fusions$split_reads1 <- fusions$split_reads1
fusions$split_reads2 <- fusions$split_reads2
fusions$type <- sub(".*(translocation|duplication|deletion|inversion).*", "\\1", fusions$type, perl=T)
fusions$fusion_transcript <- gsub("[()^$]", "", fusions$fusion_transcript)
} else if (colnames(fusions)[1] == "X.FusionName") { # STAR-Fusion
fusions$gene1 <- sub("\\^.*", "", fusions$LeftGene, perl=T)
fusions$gene2 <- sub("\\^.*", "", fusions$RightGene, perl=T)
fusions$strand1 <- sub(".*:(.)$", "\\1/\\1", fusions$LeftBreakpoint, perl=T)
fusions$strand2 <- sub(".*:(.)$", "\\1/\\1", fusions$RightBreakpoint, perl=T)
fusions$display_contig1 <- sub(":[^:]*:[^:]*$", "", fusions$LeftBreakpoint, perl=T)
fusions$display_contig2 <- sub(":[^:]*:[^:]*$", "", fusions$RightBreakpoint, perl=T)
fusions$contig1 <- removeChr(fusions$display_contig1)
fusions$contig2 <- removeChr(fusions$display_contig2)
fusions$breakpoint1 <- as.numeric(sub(".*:([^:]*):[^:]*$", "\\1", fusions$LeftBreakpoint, perl=T))
fusions$breakpoint2 <- as.numeric(sub(".*:([^:]*):[^:]*$", "\\1", fusions$RightBreakpoint, perl=T))
fusions$direction1 <- ifelse(grepl(":\\+$", fusions$LeftBreakpoint, perl=T), "downstream", "upstream")
fusions$direction2 <- ifelse(grepl(":\\+$", fusions$RightBreakpoint, perl=T), "upstream", "downstream")
fusions$gene_id1 <- sub(".*\\^", "", fusions$LeftGene, perl=T)
fusions$gene_id2 <- sub(".*\\^", "", fusions$RightGene, perl=T)
fusions$transcript_id1 <- ifelse(rep(!("CDS_LEFT_ID" %in% colnames(fusions)), nrow(fusions)), ".", fusions$CDS_LEFT_ID)
fusions$transcript_id2 <- ifelse(rep(!("CDS_RIGHT_ID" %in% colnames(fusions)), nrow(fusions)), ".", fusions$CDS_RIGHT_ID)
fusions$fusion_transcript <- ifelse(rep(!("FUSION_CDS" %in% colnames(fusions)), nrow(fusions)), ".", toupper(sub("([a-z]*)", "\\1|", fusions$FUSION_CDS, perl=T)))
fusions$reading_frame <- ifelse(rep(!("PROT_FUSION_TYPE" %in% colnames(fusions)), nrow(fusions)), ".", ifelse(fusions$PROT_FUSION_TYPE == "INFRAME", "in-frame", ifelse(fusions$PROT_FUSION_TYPE == "FRAMESHIFT", "out-of-frame", ".")))
fusions$split_reads <- fusions$JunctionReadCount
fusions$discordant_mates <- fusions$SpanningFragCount
fusions$site1 <- rep("exon", nrow(fusions))
fusions$site2 <- rep("exon", nrow(fusions))
fusions$confidence <- rep("high", nrow(fusions))
fusions$type <- ifelse(fusions$contig1 != fusions$contig2, "translocation", ifelse(fusions$direction1 == fusions$direction2, "inversion", ifelse((fusions$direction1 == "downstream") == (fusions$breakpoint1 < fusions$breakpoint2), "deletion", "duplication")))
} else {
stop("Unrecognized fusion file format")
}
pdf(outputFile, onefile=T, width=pdfWidth, height=pdfHeight, title=ifelse(sampleName != "", sampleName, fusionsFile))
par(family=fontFamily)
if (nrow(fusions) == 0) {
plot(0, 0, type="l", xaxt="n", yaxt="n", xlab="", ylab="")
text(0, 0, "empty input file")
warning("empty input file")
dev.off()
quit("no")
}
# read cytoband annotation
cytobands <- NULL
if (cytobandsFile != "") {
cytobands <- read.table(cytobandsFile, header=T, colClasses=c("character", "numeric", "numeric", "character", "character"))
cytobands <- cytobands[order(cytobands$contig, cytobands$start, cytobands$end),]
}
# read exon annotation
message("Loading annotation")
exons <- scan(exonsFile, what=list(contig="",src="",type="",start=0,end=0,score="",strand="",frame="",attributes=""), sep="\t", comment.char="#", quote='"', multi.line=F)
attr(exons, "row.names") <- .set_row_names(length(exons[[1]]))
class(exons) <- "data.frame"
exons <- exons[exons$type %in% c("exon","CDS"),c("contig","type","start","end","strand","attributes")]
exons$contig <- removeChr(exons$contig)
parseGtfAttribute <- function(attribute, gtf) {
parsed <- sub(paste0(".*", attribute, "[ =]([^;]+).*"), "\\1", gtf$attributes, perl=T)
failedToParse <- parsed == gtf$attributes
if (any(failedToParse)) {
warning(paste0("Failed to parse '", attribute, "' attribute of ", sum(failedToParse), " record(s)."))
parsed <- ifelse(failedToParse, "", parsed)
}
return(parsed)
}
exons$geneID <- parseGtfAttribute("gene_id", exons)
exons$geneName <- parseGtfAttribute("gene_name", exons)
exons$geneName <- ifelse(exons$geneName == "", exons$geneID, exons$geneName)
exons$transcript <- parseGtfAttribute("transcript_id", exons)
exons$exonNumber <- ifelse(rep(printExonLabels, nrow(exons)), parseGtfAttribute("exon_number", exons), "")
# read protein domain annotation
proteinDomains <- NULL
if (proteinDomainsFile != "") {
message("Loading protein domains")
proteinDomains <- scan(proteinDomainsFile, what=list(contig="",src="",type="",start=0,end=0,score="",strand="",frame="",attributes=""), sep="\t", comment.char="", quote="", multi.line=F)
attr(proteinDomains, "row.names") <- .set_row_names(length(proteinDomains[[1]]))
class(proteinDomains) <- "data.frame"
proteinDomains$color <- parseGtfAttribute("color", proteinDomains)
proteinDomains$proteinDomainName <- sapply(parseGtfAttribute("Name", proteinDomains), URLdecode)
proteinDomains$proteinDomainID <- parseGtfAttribute("protein_domain_id", proteinDomains)
}
# insert dummy annotations for intergenic breakpoints
if (any(fusions$site1 == "intergenic" | fusions$site2 == "intergenic")) {
intergenicBreakpoints <- rbind(
setNames(fusions[fusions$site1 == "intergenic",c("gene1", "strand1", "contig1", "breakpoint1")], c("gene", "strand", "contig", "breakpoint")),
setNames(fusions[fusions$site2 == "intergenic",c("gene2", "strand2", "contig2", "breakpoint2")], c("gene", "strand", "contig", "breakpoint"))
)
exons <- rbind(exons, data.frame(
contig=intergenicBreakpoints$contig,
type="intergenic",
start=sapply(intergenicBreakpoints$breakpoint-1000, max, 1),
end=intergenicBreakpoints$breakpoint+1000,
strand=".",
attributes="",
geneName=intergenicBreakpoints$gene,
geneID=paste0(intergenicBreakpoints$contig, ":", intergenicBreakpoints$breakpoint),
transcript=paste0(intergenicBreakpoints$contig, ":", intergenicBreakpoints$breakpoint),
exonNumber="intergenic"
))
fusions[fusions$site1 == "intergenic","gene_id1"] <- paste0(fusions[fusions$site1 == "intergenic","contig1"], ":", fusions[fusions$site1 == "intergenic","breakpoint1"])
fusions[fusions$site2 == "intergenic","gene_id2"] <- paste0(fusions[fusions$site2 == "intergenic","contig2"], ":", fusions[fusions$site2 == "intergenic","breakpoint2"])
}
drawVerticalGradient <- function(left, right, y, color, selection=NULL) {
# check if gradient should only be drawn in part of the region
if (!is.null(selection)) {
y <- y[selection]
left <- left[selection]
right <- right[selection]
}
# draw gradient
for (i in 1:length(y)) {
polygon(
c(left[1:i], right[1:i]),
c(y[1:i], y[i:1]),
border=NA,
col=rgb(col2rgb(color)["red",], col2rgb(color)["green",], col2rgb(color)["blue",], col2rgb(color, alpha=T)["alpha",]*(1/length(y)), max=255)
)
}
}
if (!render3dEffect) # nullify function, if no 3D effect should be drawn
drawVerticalGradient <- function(left, right, y, color, selection=NULL) { }
drawCurlyBrace <- function(left, right, top, bottom, tip) {
smoothness <- 20
x <- cumsum(exp(-seq(-2.5, 2.5, len=smoothness)^2))
x <- x/max(x)
y <- seq(top, bottom, len=smoothness)
lines(left+(tip-left)+x*(left-tip), y)
lines(tip+x*(right-tip), y)
}
drawIdeogram <- function(adjust, left, right, y, cytobands, contig, breakpoint) {
# define design of ideogram
bandColors <- setNames(rgb(100:0, 100:0, 100:0, maxColorValue=100), paste0("gpos", 0:100))
bandColors <- c(bandColors, gneg="#ffffff", acen="#ec4f4f", stalk="#0000ff")
cytobands$color <- bandColors[cytobands$giemsa]
arcSteps <- 30 # defines roundness of arc
curlyBraceHeight <- 0.03
ideogramHeight <- 0.04
ideogramWidth <- 0.4
# extract bands of given contig
bands <- cytobands[cytobands$contig==contig,]
if (nrow(bands) == 0) {
warning(paste("Ideogram of contig", contig, "cannot be drawn, because no Giemsa staining information is available."))
return(NULL)
}
# scale width of ideogram to fit inside given region
bands$left <- bands$start / max(cytobands$end) * ideogramWidth
bands$right <- bands$end / max(cytobands$end) * ideogramWidth
# left/right-align cytobands
offset <- ifelse(adjust=="left", left, right - max(bands$right))
bands$left <- bands$left + offset
bands$right <- bands$right + offset
# draw curly braces
tip <- min(bands$left) + (max(bands$right)-min(bands$left)) / (max(bands$end)-min(bands$start)) * breakpoint
drawCurlyBrace(left, right, y-0.05+curlyBraceHeight, y-0.05, tip)
# draw title of chromosome
text((max(bands$right)+min(bands$left))/2, y+0.07, paste("chromosome", contig), font=2, cex=fontSize, adj=c(0.5,0))
# draw name of band
bandName <- bands[which(between(breakpoint, bands$start, bands$end)), "name"]
text(tip, y+0.03, bandName, cex=fontSize, adj=c(0.5,0))
# draw start of chromosome
leftArcX <- bands[1,"left"] + (1+cos(seq(pi/2,1.5*pi,len=arcSteps))) * (bands[1,"right"]-bands[1,"left"])
leftArcY <- y + sin(seq(pi/2,1.5*pi,len=arcSteps)) * (ideogramHeight/2)
polygon(leftArcX, leftArcY, col=bands[1,"color"])
# draw bands
centromereStart <- NULL
centromereEnd <- NULL
for (band in 2:(nrow(bands)-1)) {
if (bands[band,"giemsa"] != "acen") {
rect(bands[band,"left"], y-ideogramHeight/2, bands[band,"right"], y+ideogramHeight/2, col=bands[band,"color"])
} else { # draw centromere
if (is.null(centromereStart)) {
polygon(c(bands[band,"left"], bands[band,"right"], bands[band,"left"]), c(y-ideogramHeight/2, y, y+ideogramHeight/2), col=bands[band,"color"])
centromereStart <- bands[band,"left"]
} else {
polygon(c(bands[band,"right"], bands[band,"left"], bands[band,"right"]), c(y-ideogramHeight/2, y, y+ideogramHeight/2), col=bands[band,"color"])
centromereEnd <- bands[band,"right"]
}
}
}
# draw end of chromosome
band <- nrow(bands)
rightArcX <- bands[band,"right"] - (1+cos(seq(1.5*pi,pi/2,len=arcSteps))) * (bands[band,"right"]-bands[band,"left"])
rightArcY <- y + sin(seq(pi/2,1.5*pi,len=arcSteps)) * ideogramHeight/2
polygon(rightArcX, rightArcY, col=bands[band,"color"])
# if there is no centromere, make an artificial one with length zero
if (is.null(centromereStart) || is.null(centromereEnd)) {
centromereStart <- bands[1,"right"]
centromereEnd <- bands[1,"right"]
}
# draw gradients for 3D effect
drawVerticalGradient(leftArcX, rep(centromereStart, arcSteps), leftArcY, rgb(0,0,0,0.8), 1:round(arcSteps*0.4)) # black from top on p-arm
drawVerticalGradient(leftArcX, rep(centromereStart, arcSteps), leftArcY, rgb(1,1,1,0.7), round(arcSteps*0.4):round(arcSteps*0.1)) # white to top on p-arm
drawVerticalGradient(leftArcX, rep(centromereStart, arcSteps), leftArcY, rgb(1,1,1,0.7), round(arcSteps*0.4):round(arcSteps*0.6)) # white to bottom on p-arm
drawVerticalGradient(leftArcX, rep(centromereStart, arcSteps), leftArcY, rgb(0,0,0,0.9), arcSteps:round(arcSteps*0.5)) # black from bottom on p-arm
drawVerticalGradient(rightArcX, rep(centromereEnd, arcSteps), rightArcY, rgb(0,0,0,0.8), 1:round(arcSteps*0.4)) # black from top on q-arm
drawVerticalGradient(rightArcX, rep(centromereEnd, arcSteps), rightArcY, rgb(1,1,1,0.7), round(arcSteps*0.4):round(arcSteps*0.1)) # white to top on q-arm
drawVerticalGradient(rightArcX, rep(centromereEnd, arcSteps), rightArcY, rgb(1,1,1,0.7), round(arcSteps*0.4):round(arcSteps*0.6)) # white to bottom on q-arm
drawVerticalGradient(rightArcX, rep(centromereEnd, arcSteps), rightArcY, rgb(0,0,0,0.9), arcSteps:round(arcSteps*0.5)) # black from bottom on q-arm
}
drawCoverage <- function(left, right, y, coverage, start, end, color) {
maxResolution <- 5000 # max number of data points to draw coverage
# draw coverage as bars
if (!is.null(coverage)) {
coverageData <- as.numeric(coverage[IRanges(sapply(start, max, min(start(coverage))), sapply(end, min, max(end(coverage))))])
# downsample to maxResolution, if there are too many data points
coverageData <- aggregate(coverageData, by=list(round(1:length(coverageData) * (right-left) * maxResolution/length(coverageData))), mean)$x
polygon(c(left, seq(left, right, length.out=length(coverageData)), right), c(y, y+coverageData*0.1, y), col=color, border=NA)
}
}
drawStrand <- function(left, right, y, color, strand) {
if (strand %in% c("+", "-")) {
# draw strand
lines(c(left+0.001, right-0.001), c(y, y), col=color, lwd=2)
lines(c(left+0.001, right-0.001), c(y, y), col=rgb(1,1,1,0.1), lwd=1)
# indicate orientation
if (right - left > 0.01)
for (i in seq(left+0.005, right-0.005, by=sign(right-left-2*0.005)*0.01)) {
arrows(i, y, i+0.001*ifelse(strand=="+", 1, -1), y, col=color, length=0.05, lwd=2, angle=60)
arrows(i, y, i+0.001*ifelse(strand=="+", 1, -1), y, col=rgb(1,1,1,0.1), length=0.05, lwd=1, angle=60)
}
}
}
drawExon <- function(left, right, y, color, title, type) {
gradientSteps <- 10 # defines smoothness of gradient
exonHeight <- 0.03
if (type == "CDS") {
# draw coding regions as thicker bars
rect(left, y+exonHeight, right, y+exonHeight/2-0.001, col=color, border=NA)
rect(left, y-exonHeight, right, y-exonHeight/2+0.001, col=color, border=NA)
# draw border
lines(c(left, left, right, right), c(y+exonHeight/2, y+exonHeight, y+exonHeight, y+exonHeight/2), col=getDarkColor(color), lend=2)
lines(c(left, left, right, right), c(y-exonHeight/2, y-exonHeight, y-exonHeight, y-exonHeight/2), col=getDarkColor(color), lend=2)
# draw gradients for 3D effect
drawVerticalGradient(rep(left, gradientSteps), rep(right, gradientSteps), seq(y+0.03, y+0.015, len=gradientSteps), rgb(0,0,0,0.2))
drawVerticalGradient(rep(left, gradientSteps), rep(right, gradientSteps), seq(y-0.03, y-0.015, len=gradientSteps), rgb(0,0,0,0.3))
} else if (type == "exon") {
rect(left, y+exonHeight/2, right, y-exonHeight/2, col=color, border=getDarkColor(color))
# draw gradients for 3D effect
drawVerticalGradient(rep(left, gradientSteps), rep(right, gradientSteps), seq(y, y+exonHeight/2, len=gradientSteps), rgb(1,1,1,0.6))
drawVerticalGradient(rep(left, gradientSteps), rep(right, gradientSteps), seq(y, y-exonHeight/2, len=gradientSteps), rgb(1,1,1,0.6))
# add exon label
text((left+right)/2, y, title, cex=0.9*fontSize)
}
}
drawCircos <- function(fusion, fusions, cytobands, minConfidenceForCircosPlot, circosColors) {
# check if Giemsa staining information is available
for (contig in unlist(fusions[fusion,c("contig1", "contig2")])) {
if (!any(cytobands$contig==contig)) {
warning(paste0("Circos plot cannot be drawn, because no Giemsa staining information is available for contig ", contig, "."))
# draw empty plots as placeholder
plot(0, 0, type="l", xlim=c(0, 1), ylim=c(0, 1), bty="n", xaxt="n", yaxt="n", xlab="", ylab="")
plot(0, 0, type="l", xlim=c(0, 1), ylim=c(0, 1), bty="n", xaxt="n", yaxt="n", xlab="", ylab="")
return(NULL)
}
}
# initialize with empty circos plot
circos.clear()
circos.initializeWithIdeogram(cytoband=cytobands, plotType=NULL)
# use gene names as labels or <contig>:<position> for intergenic breakpoints
geneLabels <- data.frame(
contig=c(fusions[fusion,"contig1"], fusions[fusion,"contig2"]),
start=c(fusions[fusion,"breakpoint1"], fusions[fusion,"breakpoint2"])
)
geneLabels$end <- geneLabels$start + 1
geneLabels$gene <- c(fusions[fusion,"gene1"], fusions[fusion,"gene2"])
geneLabels$gene <- ifelse(c(fusions[fusion,"site1"], fusions[fusion,"site2"]) == "intergenic", paste0(c(fusions[fusion,"display_contig1"], fusions[fusion,"display_contig2"]), ":", geneLabels$start), geneLabels$gene)
# draw gene labels
circos.genomicLabels(geneLabels, labels.column=4, side="outside", cex=fontSize, labels_height=0.27)
# draw chromosome labels in connector plot
for (contig in unique(cytobands$contig)) {
set.current.cell(track.index=2, sector.index=contig) # draw in gene label connector track (track.index=2)
circos.text(CELL_META$xcenter, CELL_META$ycenter, contig, cex=0.85)
}
# draw ideograms
circos.genomicIdeogram(cytoband=cytobands)
# draw arcs
confidenceRank <- c(low=0, medium=1, high=2)
for (i in c(setdiff(1:nrow(fusions), fusion), fusion)) { # draw fusion of interest last, such that its arc is on top
f <- fusions[i,]
if (any(cytobands$contig==f$contig1) && any(cytobands$contig==f$contig2)) # ignore viral contigs, because we have no cytoband information for them
if (minConfidenceForCircosPlot != "none" && confidenceRank[f$confidence] >= confidenceRank[minConfidenceForCircosPlot] || i==fusion)
circos.link(
f$contig1, f$breakpoint1,
f$contig2, f$breakpoint2,
lwd=2, col=ifelse(i==fusion, circosColors[f$type], getBrightColor(circosColors[f$type]))
)
}
# draw legend
plot(0, 0, type="l", xlim=c(0, 1), ylim=c(0, 1), bty="n", xaxt="n", yaxt="n", ylab="", xlab="")
legend(x="top", legend=names(circosColors), col=sapply(circosColors, getBrightColor), lwd=3, ncol=2, box.lty=0)
}
drawProteinDomains <- function(fusion, exons1, exons2, proteinDomains, color1, color2, mergeDomainsOverlappingBy, optimizeDomainColors) {
exonHeight <- 0.2
exonsY <- 0.5
geneNamesY <- exonsY - exonHeight/2 - 0.05
# find coding exons
codingExons1 <- exons1[exons1$type == "CDS" & fusion$site1 != "intergenic",]
codingExons2 <- exons2[exons2$type == "CDS" & fusion$site2 != "intergenic",]
# cut off coding regions beyond breakpoint
if (fusion$direction1 == "upstream") {
codingExons1 <- codingExons1[codingExons1$end >= fusion$breakpoint1,]
codingExons1$start <- ifelse(codingExons1$start < fusion$breakpoint1, fusion$breakpoint1, codingExons1$start)
} else {
codingExons1 <- codingExons1[codingExons1$start <= fusion$breakpoint1,]
codingExons1$end <- ifelse(codingExons1$end > fusion$breakpoint1, fusion$breakpoint1, codingExons1$end)
}
if (fusion$direction2 == "upstream") {
codingExons2 <- codingExons2[codingExons2$end >= fusion$breakpoint2,]
codingExons2$start <- ifelse(codingExons2$start < fusion$breakpoint2, fusion$breakpoint2, codingExons2$start)
} else {
codingExons2 <- codingExons2[codingExons2$start <= fusion$breakpoint2,]
codingExons2$end <- ifelse(codingExons2$end > fusion$breakpoint2, fusion$breakpoint2, codingExons2$end)
}
# find overlapping domains
exonsGRanges1 <- GRanges(codingExons1$contig, IRanges(codingExons1$start, codingExons1$end), strand=codingExons1$strand)
exonsGRanges2 <- GRanges(codingExons2$contig, IRanges(codingExons2$start, codingExons2$end), strand=codingExons2$strand)
domainsGRanges <- GRanges(proteinDomains$contig, IRanges(proteinDomains$start, proteinDomains$end), strand=proteinDomains$strand)
domainsGRanges$proteinDomainName <- proteinDomains$proteinDomainName
domainsGRanges$proteinDomainID <- proteinDomains$proteinDomainID
domainsGRanges$color <- proteinDomains$color
domainsGRanges <- domainsGRanges[suppressWarnings(unique(queryHits(findOverlaps(domainsGRanges, union(exonsGRanges1, exonsGRanges2)))))]
# group overlapping domains by domain ID
domainsGRangesList <- GRangesList(lapply(unique(domainsGRanges$proteinDomainID), function(x) { domainsGRanges[domainsGRanges$proteinDomainID == x] }))
# trim protein domains to exon boundaries
trimDomains <- function(domainsGRangesList, exonsGRanges) {
do.call(
"rbind",
lapply(
domainsGRangesList,
function(x) {
intersected <- as.data.frame(reduce(suppressWarnings(intersect(x, exonsGRanges))))
if (nrow(intersected) > 0) {
intersected$proteinDomainName <- head(x$proteinDomainName, 1)
intersected$proteinDomainID <- head(x$proteinDomainID, 1)
intersected$color <- head(x$color, 1)
} else {
intersected$proteinDomainName <- character()
intersected$proteinDomainID <- character()
intersected$color <- character()
}
return(intersected)
}
)
)
}
retainedDomains1 <- trimDomains(domainsGRangesList, exonsGRanges1)
retainedDomains2 <- trimDomains(domainsGRangesList, exonsGRanges2)
# calculate length of coding exons
codingExons1$length <- codingExons1$end - codingExons1$start + 1
codingExons2$length <- codingExons2$end - codingExons2$start + 1
# abort, if there are no coding regions
if (sum(exons1$type == "CDS") + sum(exons2$type == "CDS") == 0) {
text(0.5, 0.5, "Genes are not protein-coding.")
return(NULL)
}
codingLength1 <- sum(codingExons1$length)
codingLength2 <- sum(codingExons2$length)
if (codingLength1 + codingLength2 == 0) {
text(0.5, 0.5, "No coding regions retained in fusion transcript.")
return(NULL)
}
if ((codingLength1 == 0 || grepl("\\.$", fusion$strand1)) && (codingLength2 == 0 || grepl("\\.$", fusion$strand2))) {
text(0.5, 0.5, "Failed to determine retained protein domains due to lack of strand information.")
return(NULL)
}
antisenseTranscription1 <- sub("/.*", "", fusion$strand1) != sub(".*/", "", fusion$strand1)
antisenseTranscription2 <- sub("/.*", "", fusion$strand2) != sub(".*/", "", fusion$strand2)
if ((codingLength1 == 0 || antisenseTranscription1) && (codingLength2 == 0 || antisenseTranscription2)) {
text(0.5, 0.5, "No coding regions due to antisense transcription.")
return(NULL)
}
# remove introns from protein domains
removeIntronsFromProteinDomains <- function(codingExons, retainedDomains) {
if (nrow(codingExons) == 0) return(NULL)
cumulativeIntronLength <- 0
previousExonEnd <- 0
for (exon in 1:nrow(codingExons)) {
if (codingExons[exon,"start"] > previousExonEnd)
cumulativeIntronLength <- cumulativeIntronLength + codingExons[exon,"start"] - previousExonEnd
domainsInExon <- which(between(retainedDomains$start, codingExons[exon,"start"], codingExons[exon,"end"]))
retainedDomains[domainsInExon,"start"] <- retainedDomains[domainsInExon,"start"] - cumulativeIntronLength
domainsInExon <- which(between(retainedDomains$end, codingExons[exon,"start"], codingExons[exon,"end"]))
retainedDomains[domainsInExon,"end"] <- retainedDomains[domainsInExon,"end"] - cumulativeIntronLength
previousExonEnd <- codingExons[exon,"end"]
}
# merge adjacent domains
retainedDomains <- do.call(
"rbind",
lapply(
unique(retainedDomains$proteinDomainID),
function(x) {
domain <- retainedDomains[retainedDomains$proteinDomainID == x,]
merged <- reduce(GRanges(domain$seqnames, IRanges(domain$start, domain$end), strand=domain$strand))
merged$proteinDomainName <- head(domain$proteinDomainName, 1)
merged$proteinDomainID <- head(domain$proteinDomainID, 1)
merged$color <- head(domain$color, 1)
return(as.data.frame(merged))
}
)
)
return(retainedDomains)
}
retainedDomains1 <- removeIntronsFromProteinDomains(codingExons1, retainedDomains1)
retainedDomains2 <- removeIntronsFromProteinDomains(codingExons2, retainedDomains2)
# abort, if no domains are retained
if (is.null(retainedDomains1) && is.null(retainedDomains2)) {
text(0.5, 0.5, "No protein domains retained in fusion.")
return(NULL)
}
# merge domains with similar coordinates
mergeSimilarDomains <- function(domains, mergeDomainsOverlappingBy) {
if (is.null(domains)) return(domains)
merged <- domains[F,] # create empty data frame
domains <- domains[order(domains$end - domains$start, decreasing=T),] # start with bigger domains => bigger domains are retained
for (domain in rownames(domains)) {
if (!any((abs(merged$start - domains[domain,"start"]) + abs(merged$end - domains[domain,"end"])) / (domains[domain,"end"] - domains[domain,"start"] + 1) <= 1-mergeDomainsOverlappingBy))
merged <- rbind(merged, domains[domain,])
}
return(merged)
}
retainedDomains1 <- mergeSimilarDomains(retainedDomains1, mergeDomainsOverlappingBy)
retainedDomains2 <- mergeSimilarDomains(retainedDomains2, mergeDomainsOverlappingBy)
# if desired, reassign colors to protein domains to maximize contrast
if (optimizeDomainColors) {
uniqueDomains <- unique(c(retainedDomains1$proteinDomainID, retainedDomains2$proteinDomainID))
# make rainbow of pretty pastell colors
colors <- rainbow(length(uniqueDomains))
colors <- apply(col2rgb(colors), 2, function(x) { 0.3 + y/255 * 0.7 }) # make pastell colors
colors <- apply(colors, 2, function(x) {rgb(x["red"], x["green"], x["blue"])}) # convert back to rgb
# reassign colors
names(colors) <- uniqueDomains
retainedDomains1$color <- colors[retainedDomains1$proteinDomainID]
retainedDomains2$color <- colors[retainedDomains2$proteinDomainID]
}
# reverse exons and protein domains, if on the reverse strand
if (any(codingExons1$strand == "-")) {
codingExons1$length <- rev(codingExons1$length)
temp <- retainedDomains1$end
retainedDomains1$end <- codingLength1 - retainedDomains1$start
retainedDomains1$start <- codingLength1 - temp
}
if (any(codingExons2$strand == "-")) {
codingExons2$length <- rev(codingExons2$length)
temp <- retainedDomains2$end
retainedDomains2$end <- codingLength2 - retainedDomains2$start
retainedDomains2$start <- codingLength2 - temp
}
# normalize length to 1
codingExons1$length <- codingExons1$length / (codingLength1 + codingLength2)
codingExons2$length <- codingExons2$length / (codingLength1 + codingLength2)
retainedDomains1$start <- retainedDomains1$start / (codingLength1 + codingLength2)
retainedDomains1$end <- retainedDomains1$end / (codingLength1 + codingLength2)
retainedDomains2$start <- retainedDomains2$start / (codingLength1 + codingLength2)
retainedDomains2$end <- retainedDomains2$end / (codingLength1 + codingLength2)
# draw coding regions
rect(0, exonsY-exonHeight/2, sum(codingExons1$length), exonsY+exonHeight/2, col=color1, border=NA)
rect(sum(codingExons1$length), exonsY-exonHeight/2, sum(codingExons1$length) + sum(codingExons2$length), exonsY+exonHeight/2, col=color2, border=NA)
# indicate exon boundaries as dotted lines
exonBoundaries <- cumsum(c(codingExons1$length, codingExons2$length))
if (length(exonBoundaries) > 1) {
exonBoundaries <- exonBoundaries[1:(length(exonBoundaries)-1)]
for (exonBoundary in exonBoundaries)
lines(c(exonBoundary, exonBoundary), c(exonsY-exonHeight, exonsY+exonHeight), col="white", lty=3)
}
# find overlapping domains
# nest if one is contained in another
# stack if they overlap partially
nestDomains <- function(domains) {
if (length(unlist(domains)) == 0) return(domains)
domains <- domains[order(domains$end - domains$start, decreasing=T),]
rownames(domains) <- 1:nrow(domains)
# find nested domains and make tree structure
domains$parent <- 0
for (domain in rownames(domains))
domains[domains$start >= domains[domain,"start"] & domains$end <= domains[domain,"end"] & rownames(domains) != domain,"parent"] <- domain
# find partially overlapping domains
maxOverlappingDomains <- max(1, as.integer(coverage(IRanges(domains$start*10e6, domains$end*10e6))))
padding <- 1 / maxOverlappingDomains * 0.4
domains$y <- 0
domains$height <- 0
adjustPositionAndHeight <- function(parentDomain, y, height, padding, e) {
for (domain in which(e$domains$parent == parentDomain)) {
overlappingDomains <- which((between(e$domains$start, e$domains[domain,"start"], e$domains[domain,"end"]) |
between(e$domains$end , e$domains[domain,"start"], e$domains[domain,"end"])) &
e$domains$parent == parentDomain)
e$domains[domain,"height"] <- height/length(overlappingDomains) - padding * (length(overlappingDomains)-1) / length(overlappingDomains)
e$domains[domain,"y"] <- y + (which(domain==overlappingDomains)-1) * (e$domains[domain,"height"] + padding)
adjustPositionAndHeight(domain, e$domains[domain,"y"]+padding, e$domains[domain,"height"]-2*padding, padding, e)
}
}
adjustPositionAndHeight(0, 0, 1, padding, environment())
domains <- domains[order(domains$height, decreasing=T),] # draw nested domains last
return(domains)
}
retainedDomains1 <- nestDomains(retainedDomains1)
retainedDomains2 <- nestDomains(retainedDomains2)
retainedDomains1$y <- exonsY - exonHeight/2 + 0.025 + (exonHeight-2*0.025) * retainedDomains1$y
retainedDomains2$y <- exonsY - exonHeight/2 + 0.025 + (exonHeight-2*0.025) * retainedDomains2$y
retainedDomains1$height <- retainedDomains1$height * (exonHeight-2*0.025)
retainedDomains2$height <- retainedDomains2$height * (exonHeight-2*0.025)
# draw domains
drawProteinDomainRect <- function(left, bottom, right, top, color) {
rect(left, bottom, right, top, col=color, border=getDarkColor(color))
# draw gradients for 3D effect
gradientSteps <- 20
drawVerticalGradient(rep(left, gradientSteps), rep(right, gradientSteps), seq(top, bottom, len=gradientSteps), rgb(1,1,1,0.7))
drawVerticalGradient(rep(left, gradientSteps), rep(right, gradientSteps), seq(bottom, bottom+(top-bottom)*0.4, len=gradientSteps), rgb(0,0,0,0.1))
}
if (length(unlist(retainedDomains1)) > 0)
for (domain in 1:nrow(retainedDomains1))
drawProteinDomainRect(retainedDomains1[domain,"start"], retainedDomains1[domain,"y"], retainedDomains1[domain,"end"], retainedDomains1[domain,"y"]+retainedDomains1[domain,"height"], retainedDomains1[domain,"color"])
if (length(unlist(retainedDomains2)) > 0)
for (domain in 1:nrow(retainedDomains2))
drawProteinDomainRect(sum(codingExons1$length)+retainedDomains2[domain,"start"], retainedDomains2[domain,"y"], sum(codingExons1$length)+retainedDomains2[domain,"end"], retainedDomains2[domain,"y"]+retainedDomains2[domain,"height"], retainedDomains2[domain,"color"])
# draw gene names, if there are coding exons
if (codingLength1 > 0)
text(sum(codingExons1$length)/2, geneNamesY, fusion$gene1, font=2, cex=fontSize)
if (codingLength2 > 0)
text(sum(codingExons1$length)+sum(codingExons2$length)/2, geneNamesY, fusion$gene2, font=2, cex=fontSize)
# calculate how many non-adjacent unique domains there are
# we need this info to know where to place labels vertically
countUniqueDomains <- function(domains) {
uniqueDomains <- 0
if (length(unlist(domains)) > 0) {
uniqueDomains <- 1
if (nrow(domains) > 1) {
previousDomain <- domains[1,"proteinDomainID"]
for (domain in 2:nrow(domains)) {
if (previousDomain != domains[domain,"proteinDomainID"])
uniqueDomains <- uniqueDomains + 1
previousDomain <- domains[domain,"proteinDomainID"]
}
}
}
return(uniqueDomains)
}
if (length(unlist(retainedDomains1)) > 0)
retainedDomains1 <- retainedDomains1[order(retainedDomains1$start),]
uniqueDomains1 <- countUniqueDomains(retainedDomains1)
if (length(unlist(retainedDomains2)) > 0)
retainedDomains2 <- retainedDomains2[order(retainedDomains2$end, decreasing=T),]
uniqueDomains2 <- countUniqueDomains(retainedDomains2)
# draw title of plot
titleY <- exonsY + exonHeight/2 + (uniqueDomains1 + 2) * 0.05
text(0.5, titleY+0.01, "RETAINED PROTEIN DOMAINS", adj=c(0.5, 0), font=2, cex=fontSize)
text(0.5, titleY, ifelse(fusion$reading_frame %in% c("in-frame", "out-of-frame"), paste(fusion$reading_frame, "fusion"), ifelse(fusion$reading_frame == "stop-codon", "stop codon before fusion junction", "reading frame unclear")), adj=c(0.5, 1), cex=fontSize)
# draw domain labels for gene1
if (length(unlist(retainedDomains1)) > 0) {
previousConnectorX <- -1
previousLabelX <- -1
labelY <- exonsY + exonHeight/2 + uniqueDomains1 * 0.05
for (domain in 1:nrow(retainedDomains1)) {
# if possible avoid overlapping lines of labels
connectorX <- min(retainedDomains1[domain,"start"] + 0.01, (retainedDomains1[domain,"start"] + retainedDomains1[domain,"end"])/2)
if (connectorX - previousConnectorX < 0.01 && retainedDomains1[domain,"end"] > previousConnectorX + 0.01)
connectorX <- previousConnectorX + 0.01
labelX <- max(connectorX, previousLabelX) + 0.02
# use a signle label for adjacent domains of same type
adjacentDomainsOfSameType <- domain + 1 <= nrow(retainedDomains1) && retainedDomains1[domain+1,"proteinDomainID"] == retainedDomains1[domain,"proteinDomainID"]
if (adjacentDomainsOfSameType) {
labelX <- retainedDomains1[domain+1,"start"] + 0.015
} else {
text(labelX, labelY, retainedDomains1[domain,"proteinDomainName"], adj=c(0,0.5), col=getDarkColor(retainedDomains1[domain,"color"]), cex=fontSize)
}
lines(c(labelX-0.005, connectorX, connectorX), c(labelY, labelY, retainedDomains1[domain,"y"]+retainedDomains1[domain,"height"]), col=getDarkColor(retainedDomains1[domain,"color"]))
if (!adjacentDomainsOfSameType)
labelY <- labelY - 0.05
previousConnectorX <- connectorX
previousLabelX <- labelX
}
}
# draw domain labels for gene2
if (length(unlist(retainedDomains2)) > 0) {
previousConnectorX <- 100
previousLabelX <- 100
labelY <- exonsY - exonHeight/2 - (uniqueDomains2+1) * 0.05
for (domain in 1:nrow(retainedDomains2)) {
# if possible avoid overlapping connector lines of labels
connectorX <- sum(codingExons1$length) + max(retainedDomains2[domain,"end"] - 0.01, (retainedDomains2[domain,"start"] + retainedDomains2[domain,"end"])/2)
if (previousConnectorX - connectorX < 0.01 && sum(codingExons1$length) + retainedDomains2[domain,"start"] < previousConnectorX - 0.01)
connectorX <- previousConnectorX - 0.01
labelX <- min(connectorX, previousLabelX) - 0.02
# use a signle label for adjacent domains of same type
adjacentDomainsOfSameType <- domain + 1 <= nrow(retainedDomains2) && retainedDomains2[domain+1,"proteinDomainID"] == retainedDomains2[domain,"proteinDomainID"]
if (adjacentDomainsOfSameType) {
labelX <- sum(codingExons1$length) + retainedDomains2[domain+1,"end"] - 0.015
} else {
text(labelX, labelY, retainedDomains2[domain,"proteinDomainName"], adj=c(1,0.5), col=getDarkColor(retainedDomains2[domain,"color"]), cex=fontSize)
}
lines(c(labelX+0.005, connectorX, connectorX), c(labelY, labelY, retainedDomains2[domain,"y"]), col=getDarkColor(retainedDomains2[domain,"color"]))
if (!adjacentDomainsOfSameType)
labelY <- labelY + 0.05
previousConnectorX <- connectorX
previousLabelX <- labelX
}
}
}
findExons <- function(exons, contig, geneID, direction, breakpoint, coverage, transcriptId, transcriptSelection) {
# use the provided transcript if desired
if (transcriptSelection == "provided" && transcriptId != "." && transcriptId != "") {
candidateExons <- exons[exons$transcript == transcriptId,]
if (nrow(candidateExons) == 0) {
warning(paste0("Unknown transcript given in fusions file (", transcriptId, "), selecting a different one"))
} else {
return(candidateExons)
}
}
if (transcriptSelection == "canonical") {
candidateExons <- exons[exons$geneID == geneID & exons$contig == contig,]
} else {
# look for exon with breakpoint as splice site
transcripts <- exons[exons$geneID == geneID & exons$contig == contig & exons$type == "exon" & (direction == "downstream" & abs(exons$end - breakpoint) <= 2 | direction == "upstream" & abs(exons$start - breakpoint) <= 2),"transcript"]
candidateExons <- exons[exons$transcript %in% transcripts,]
# if none was found, use all exons of the gene closest to the breakpoint
if (nrow(candidateExons) == 0)
candidateExons <- exons[exons$geneID == geneID & exons$contig == contig,]
# if we have coverage information, use the transcript with the highest coverage if there are multiple hits
if (!is.null(coverage)) {
highestCoverage <- -1
transcriptWithHighestCoverage <- NULL
lengthOfTranscriptWithHighestCoverage <- 0
for (transcript in unique(candidateExons$transcript)) {
exonsOfTranscript <- candidateExons[candidateExons$transcript==transcript,]
exonsOfTranscript$start <- sapply(exonsOfTranscript$start, max, min(start(coverage)))
exonsOfTranscript$end <- sapply(exonsOfTranscript$end, min, max(end(coverage)))
lengthOfTranscript <- sum(exonsOfTranscript$end - exonsOfTranscript$start + 1)
coverageSum <- sum(as.numeric(coverage[IRanges(exonsOfTranscript$start, exonsOfTranscript$end)]))
# we prefer shorter transcripts over longer ones, because otherwise there is a bias towards transcripts with long UTRs
# => a longer transcript must have substantially higher coverage to replace a shorter one
substantialDifference <- (1 - min(lengthOfTranscript, lengthOfTranscriptWithHighestCoverage) / max(lengthOfTranscript, lengthOfTranscriptWithHighestCoverage)) / 10
if (lengthOfTranscript > lengthOfTranscriptWithHighestCoverage && coverageSum * (1-substantialDifference) > highestCoverage ||
lengthOfTranscript < lengthOfTranscriptWithHighestCoverage && coverageSum > highestCoverage * (1-substantialDifference)) {
highestCoverage <- coverageSum
transcriptWithHighestCoverage <- transcript
lengthOfTranscriptWithHighestCoverage <- lengthOfTranscript
}
}
if (highestCoverage > 0)
candidateExons <- candidateExons[candidateExons$transcript==transcriptWithHighestCoverage,]
}
# if the gene has multiple transcripts, search for transcripts which encompass the breakpoint
if (length(unique(candidateExons$transcript)) > 1) {
transcriptStart <- aggregate(candidateExons$start, by=list(candidateExons$transcript), min)
rownames(transcriptStart) <- transcriptStart[,1]
transcriptEnd <- aggregate(candidateExons$end, by=list(candidateExons$transcript), max)
rownames(transcriptEnd) <- transcriptEnd[,1]
encompassingExons <- between(breakpoint, transcriptStart[candidateExons$transcript,2], transcriptEnd[candidateExons$transcript,2])
if (any(encompassingExons))
candidateExons <- candidateExons[encompassingExons,]
}
}
# find the consensus transcript, if there are multiple hits
if (length(unique(candidateExons$transcript)) > 1) {
consensusTranscript <-
ifelse(grepl("appris_principal_1", candidateExons$attributes), 12,
ifelse(grepl("appris_principal_2", candidateExons$attributes), 11,
ifelse(grepl("appris_principal_3", candidateExons$attributes), 10,
ifelse(grepl("appris_principal_4", candidateExons$attributes), 9,
ifelse(grepl("appris_principal_5", candidateExons$attributes), 8,
ifelse(grepl("appris_principal", candidateExons$attributes), 7,
ifelse(grepl("appris_candidate_longest", candidateExons$attributes), 6,
ifelse(grepl("appris_candidate", candidateExons$attributes), 5,
ifelse(grepl("appris_alternative_1", candidateExons$attributes), 4,
ifelse(grepl("appris_alternative_2", candidateExons$attributes), 3,
ifelse(grepl("appris_alternative", candidateExons$attributes), 2,
ifelse(grepl("CCDS", candidateExons$attributes), 1,
0
))))))))))))
candidateExons <- candidateExons[consensusTranscript == max(consensusTranscript),]
}
# use the transcript with the longest coding sequence, if there are still multiple hits
if (length(unique(candidateExons$transcript)) > 1) {
codingSequenceLength <- ifelse(candidateExons$type == "CDS", candidateExons$end - candidateExons$start, 0)
totalCodingSequenceLength <- aggregate(codingSequenceLength, by=list(candidateExons$transcript), sum)
rownames(totalCodingSequenceLength) <- totalCodingSequenceLength[,1]
candidateExons <- candidateExons[totalCodingSequenceLength[candidateExons$transcript,2] == max(totalCodingSequenceLength[,2]),]
}
# use the transcript with the longest overall sequence, if there are still multiple hits
if (length(unique(candidateExons$transcript)) > 1) {
exonLength <- candidateExons$end - candidateExons$start
totalExonLength <- aggregate(exonLength, by=list(candidateExons$transcript), sum)
rownames(totalExonLength) <- totalExonLength[,1]
candidateExons <- candidateExons[totalExonLength[candidateExons$transcript,2] == max(totalExonLength[,2]),]
}
# if there are still multiple hits, select the first one
candidateExons <- unique(candidateExons[candidateExons$transcript == head(unique(candidateExons$transcript), 1),])
return(candidateExons)
}
findClosestGene <- function(exons, contig, breakpoint, extraConditions) {
# find exons near breakpoint (extraConditions must define what is considered "near")
closestExons <- exons[exons$contig == contig & extraConditions,] # find closest exon
closestExons <- exons[exons$contig == contig & exons$geneID %in% closestExons$geneID,] # select all exons of closest gene
# when more than one gene found with the given name, use the closest one
if (length(unique(closestExons$geneID)) > 1) { # more than one gene found with the given name => use the closest one
distanceToBreakpoint <- aggregate(1:nrow(closestExons), by=list(closestExons$geneID), function(x) { min(abs(closestExons[x,"start"]-breakpoint), abs(closestExons[x,"end"]-breakpoint)) })
closestGene <- head(distanceToBreakpoint[distanceToBreakpoint[,2] == min(distanceToBreakpoint[,2]),1], 1)
closestExons <- closestExons[closestExons$geneID == closestGene,]
}
# when no gene was found, return default values
if (nrow(closestExons) == 0) {
return(IRanges(max(1, breakpoint-1000), breakpoint+1000))
} else {
return(IRanges(min(closestExons$start), max(closestExons$end)))
}
}
# main loop starts here
for (fusion in 1:nrow(fusions)) {
message(paste0("Drawing fusion #", fusion, ": ", fusions[fusion,"gene1"], ":", fusions[fusion,"gene2"]))
# if showIntergenicVicinity is a number, take it as is
# if it is a keyword (closestGene/closestProteinCodingGene), determine the range dynamically
showVicinity <- rep(0, 4)
if (fusions[fusion,"site1"] == "intergenic") {
showVicinity[1] <- ifelse(
is.numeric(showIntergenicVicinity[[1]]),
showIntergenicVicinity[[1]],
fusions[fusion,"breakpoint1"] - start(findClosestGene(exons, fusions[fusion,"contig1"], fusions[fusion,"breakpoint1"], exons$end < fusions[fusion,"breakpoint1"] & exons$type == showIntergenicVicinity[[1]]))
)
showVicinity[2] <- ifelse(
is.numeric(showIntergenicVicinity[[2]]),
showIntergenicVicinity[[2]],
end(findClosestGene(exons, fusions[fusion,"contig1"], fusions[fusion,"breakpoint1"], exons$start > fusions[fusion,"breakpoint1"] & exons$type == showIntergenicVicinity[[2]])) - fusions[fusion,"breakpoint1"]
)
}
if (fusions[fusion,"site2"] == "intergenic") {
showVicinity[3] <- ifelse(
is.numeric(showIntergenicVicinity[[3]]),
showIntergenicVicinity[[3]],
fusions[fusion,"breakpoint2"] - start(findClosestGene(exons, fusions[fusion,"contig2"], fusions[fusion,"breakpoint2"], exons$end < fusions[fusion,"breakpoint2"] & exons$type == showIntergenicVicinity[[3]]))
)
showVicinity[4] <- ifelse(
is.numeric(showIntergenicVicinity[[4]]),
showIntergenicVicinity[[4]],
end(findClosestGene(exons, fusions[fusion,"contig2"], fusions[fusion,"breakpoint2"], exons$start > fusions[fusion,"breakpoint2"] & exons$type == showIntergenicVicinity[[4]])) - fusions[fusion,"breakpoint2"]
)
}
# compute coverage from alignments file
coverage1 <- NULL
coverage2 <- NULL
if (alignmentsFile != "") {
# determine range in which we need to compute the coverage
determineCoverageRegion <- function(exons, geneID, contig, breakpoint, showVicinityLeft, showVicinityRight) {
closestGene <- findClosestGene(exons, contig, breakpoint, exons$geneID == geneID)
return(IRanges(min(start(closestGene), breakpoint-showVicinityLeft), max(end(closestGene), breakpoint+showVicinityRight)))
}
coverageRegion1 <- determineCoverageRegion(exons, fusions[fusion,"gene_id1"], fusions[fusion,"contig1"], fusions[fusion,"breakpoint1"], showVicinity[1], showVicinity[2])
coverageRegion2 <- determineCoverageRegion(exons, fusions[fusion,"gene_id2"], fusions[fusion,"contig2"], fusions[fusion,"breakpoint2"], showVicinity[3], showVicinity[4])
# function which reads alignments from BAM file with & without "chr" prefix
readCoverage <- function(alignmentsFile, contig, coverageRegion) {
coverageData <- tryCatch(
{
alignments <- readGAlignments(alignmentsFile, param=ScanBamParam(which=GRanges(contig, coverageRegion)))
coverage(alignments)[[contig]]
},
error=function(e) {
alignments <- readGAlignments(alignmentsFile, param=ScanBamParam(which=GRanges(addChr(contig), coverageRegion)))
coverage(alignments)[[addChr(contig)]]
}
)
if (exists("alignments")) rm(alignments)
return(coverageData)
}
# get coverage track
coverage1 <- readCoverage(alignmentsFile, fusions[fusion,"contig1"], coverageRegion1)
coverage2 <- readCoverage(alignmentsFile, fusions[fusion,"contig2"], coverageRegion2)
# shrink coverage range to chromosome boundaries to avoid subscript out of bounds errors
coverageRegion1 <- IRanges(max(start(coverageRegion1), min(start(coverage1))), min(end(coverageRegion1), max(end(coverage1))))
coverageRegion2 <- IRanges(max(start(coverageRegion2), min(start(coverage2))), min(end(coverageRegion2), max(end(coverage2))))
}
# find all exons belonging to the fused genes
exons1 <- findExons(exons, fusions[fusion,"contig1"], fusions[fusion,"gene_id1"], fusions[fusion,"direction1"], fusions[fusion,"breakpoint1"], coverage1, fusions[fusion,"transcript_id1"], transcriptSelection)
if (nrow(exons1) == 0) {
par(mfrow=c(1,1))
plot(0, 0, type="l", xaxt="n", yaxt="n", xlab="", ylab="")
text(0, 0, paste0("exon coordinates of ", fusions[fusion,"gene1"], " not found in\n", exonsFile))
warning(paste("exon coordinates of", fusions[fusion,"gene1"], "not found"))
next
}
exons2 <- findExons(exons, fusions[fusion,"contig2"], fusions[fusion,"gene_id2"], fusions[fusion,"direction2"], fusions[fusion,"breakpoint2"], coverage2, fusions[fusion,"transcript_id2"], transcriptSelection)
if (nrow(exons2) == 0) {
par(mfrow=c(1,1))
plot(0, 0, type="l", xaxt="n", yaxt="n", xlab="", ylab="")
text(0, 0, paste0("exon coordinates of ", fusions[fusion,"gene2"], " not found in\n", exonsFile))
warning(paste("exon coordinates of", fusions[fusion,"gene2"], "not found"))
next
}
# in case of intergenic breakpoints, show the vicinity
if (sum(showVicinity) > 0) {