Nothing
R/FunctionsForNoReplicationAnalysis.R
In r3Cseq: Analysis of Chromosome Conformation Capture and Next-generation Sequencing (3C-seq)
# TODO: These following functions were implemented to get interaction regions.
# Author: Supat Thongjuea
# Contact : supat.thongjuea@imm.ox.ac.uk or supat.thongjuea@gmail.com
#####
#####The functions below are completely migrated to BioC 3.9 on R 3.6
#####
setGeneric(
name="getCoverage",
def=function(object){
standardGeneric("getCoverage")
}
)
setMethod("getCoverage",
signature(object = "r3Cseq"),
function (object){
stop("This method has been removed!!.")
}
)
#####
setGeneric(
name="getRawReads",
def=function(object){
standardGeneric("getRawReads")
}
)
setMethod("getRawReads",
signature(object = "r3Cseq"),
function (object){
if(!is(object,"r3Cseq")){
stop("Need to initialize the r3Cseq object")
}
objName <- deparse(substitute(object))
if(isControlInvolved(object)==TRUE){
exp.bam.file <-alignedReadsBamExpFile(object)
contr.bam.file <-alignedReadsBamContrFile(object)
if(file.exists(exp.bam.file) ==FALSE){
stop(paste("Couldn't find file","-->",exp.bam.file))
}
if(file.exists(contr.bam.file) ==FALSE){
stop(paste("Couldn't find file","-->",contr.bam.file))
}
expLabeled<-expLabel(object)
controlLabeled<-contrLabel(object)
if(nchar(expLabeled)==0 & nchar(controlLabeled)==0){
stop("The package requires input of expLabel and contrLabel.")
}
print("start reading in ......")
what <- c("rname", "strand", "pos", "qwidth","seq")
param <- ScanBamParam(what=what,flag = scanBamFlag(isUnmappedQuery = FALSE))
exp.bam <- scanBam(exp.bam.file, param=param)
contr.bam <- scanBam(contr.bam.file, param=param)
exp.read.length <-width(exp.bam[[1]]$seq[1])
exp.GRanges<-GRanges(seqnames=as.vector(exp.bam[[1]]$rname),IRanges(start=exp.bam[[1]]$pos,width=exp.read.length),strand=exp.bam[[1]]$strand)
contr.read.length <-width(contr.bam[[1]]$seq[1])
contr.GRanges<-GRanges(seqnames=as.vector(contr.bam[[1]]$rname),IRanges(start=contr.bam[[1]]$pos,width=contr.read.length),strand=contr.bam[[1]]$strand)
expLibrarySize(object) <-length(exp.GRanges)
contrLibrarySize(object) <-length(contr.GRanges)
#######Remove chr.._random chromosomes############################################
#exp.GRanges<-exp.GRanges[seqnames(exp.GRanges) %in% paste('chr',c(seq(1,50),'X','Y'),sep='')]
#contr.GRanges<-contr.GRanges[seqnames(contr.GRanges) %in% paste('chr',c(seq(1,50),'X','Y'),sep='')]
#######assign raw read to the object########
expReadLength(object)<-exp.read.length
contrReadLength(object)<-contr.read.length
expRawData(object)<-exp.GRanges
contrRawData(object)<-contr.GRanges
assign(objName,object,envir=parent.frame())
invisible(1)
print("Raw reads processing is done.")
}
if(isControlInvolved(object)==FALSE){
exp.bam.file <-alignedReadsBamExpFile(object)
if(file.exists(exp.bam.file) ==FALSE){
stop(paste("Couldn't find file","-->",exp.bam.file))
}
expLabeled<-expLabel(object)
if(nchar(expLabeled)==0){
stop("The package requires input of expLabel.")
}
print("start reading in ......")
what <- c("rname", "strand", "pos", "qwidth","seq")
param <- ScanBamParam(what=what,flag = scanBamFlag(isUnmappedQuery = FALSE))
exp.bam <- scanBam(exp.bam.file, param=param)
exp.read.length <-width(exp.bam[[1]]$seq[1])
exp.GRanges<-GRanges(seqnames=as.vector(exp.bam[[1]]$rname),IRanges(start=exp.bam[[1]]$pos,width=exp.read.length),strand=exp.bam[[1]]$strand)
#######Remove chr.._random chromosomes############################################
#exp.GRanges<-exp.GRanges[seqnames(exp.GRanges) %in% paste('chr',c(seq(1,50),'X','Y'),sep='')]
##########assign raw read to the object######
expLibrarySize(object) <-length(exp.GRanges)
expReadLength(object)<-exp.read.length
expRawData(object)<-exp.GRanges
assign(objName,object,envir=parent.frame())
invisible(1)
print("Raw reads processing is done.")
}
}
)
########
setGeneric(
name="getReadCountPerRestrictionFragment",
def=function(object,getReadsMethod = c("wholeReads", "adjacentFragmentEndsReads"),
nFragmentExcludedReadsNearViewpoint=2){
standardGeneric("getReadCountPerRestrictionFragment")
}
)
setMethod("getReadCountPerRestrictionFragment",
signature(object = "r3Cseq"),
function (object,getReadsMethod,nFragmentExcludedReadsNearViewpoint){
objName <- deparse(substitute(object))
if(!is(object,"r3Cseq")){
stop("Need to initialize the r3Cseq object")
}
#####Select the read count methods#########
selected_methods <- match.arg(getReadsMethod)
if(selected_methods==""){
selected_methods<-"wholeReads"
}
######check the number of fragment to exclude reads around the viewpoint###
nExcludedFragments <- nFragmentExcludedReadsNearViewpoint
if(nExcludedFragments <0 | nExcludedFragments>5){
stop("The number of fragment that uses for removing reads around the viewpoint must be 0-5 restriction fragments.")
}
if(length(expRawData(object))==0){
stop("No reads found, please run getRawReads function to process BAM file.")
}
if(isControlInvolved(object)==FALSE){
if(selected_methods=="wholeReads"){
####Get organism#######################
orgName <- organismName(object)
####Initialized Restriction Enzyme Object##
enzymeDb <-new("repbaseEnzyme")
resEnzyme <-restrictionEnzyme(object)
####Build GRanges for restriction site#####
print(paste("Fragmenting genome by....",resEnzyme,"....",sep=""))
wholeFragments<-getWholeGenomeRestrictionFragments(enzymeDb,resEnzyme,orgName)
wholeFragments.GRanges<-GRanges(seqnames=as.character(wholeFragments$chromosome),IRanges(start=wholeFragments$start,end=wholeFragments$end))
##########Count all reads located inside the fragments######
exp.GRanges<-expRawData(object)
exp.GRanges<-excludeReadsNearViewpoint(object,exp.GRanges,nExcludedFragments)
readsPerFragment <- countOverlaps(wholeFragments.GRanges,subject=exp.GRanges)
reads<-data.frame(nReads=readsPerFragment)
####migrate the countTable to GRanges because BioC3.9 will not support the RagnedData###
values(wholeFragments.GRanges)=reads
countTable<-wholeFragments.GRanges
countTable.filtered<-countTable[countTable$nReads >0,]
expReadCount(object)<-countTable.filtered
print("Count processing is done.")
}
if(selected_methods=="adjacentFragmentEndsReads"){
####Get organism#######################
orgName <- organismName(object)
exp.read.length<-expReadLength(object)
####Initialized Restriction Enzyme Object##
enzymeDb <-new("repbaseEnzyme")
resEnzyme <-restrictionEnzyme(object)
####Build GRanges for restriction site#####
print(paste("Fragmenting genome by....",resEnzyme,"....",sep=""))
wholeFragments<-getWholeGenomeRestrictionFragments(enzymeDb,resEnzyme,orgName)
wholeFragments.GRanges<-GRanges(seqnames=as.character(wholeFragments$chromosome),IRanges(start=wholeFragments$start,end=wholeFragments$end))
########Make GRanges for the 5' and 3' of RE#######
exp.GRanges<-expRawData(object)
exp.GRanges<-excludeReadsNearViewpoint(object,exp.GRanges,nExcludedFragments)
wholeFragments_5_prime.GRanges<-GRanges(seqnames =seqnames(wholeFragments.GRanges),
IRanges(start=start(wholeFragments.GRanges),
end=start(wholeFragments.GRanges)+exp.read.length))
wholeFragments_3_prime.GRanges<-GRanges(seqnames =seqnames(wholeFragments.GRanges),
IRanges(start=end(wholeFragments.GRanges)-exp.read.length,
end=end(wholeFragments.GRanges)))
readsPerFragment_5 <- countOverlaps(wholeFragments_5_prime.GRanges,subject=exp.GRanges)
readsPerFragment_3 <- countOverlaps(wholeFragments_3_prime.GRanges,subject=exp.GRanges)
combined_reads<-readsPerFragment_5+readsPerFragment_3
reads<-data.frame(nReads=combined_reads)
####migrate the countTable to GRanges because BioC3.9 will not support the RagnedData###
values(wholeFragments.GRanges)=reads
countTable<-wholeFragments.GRanges
countTable.filtered<-countTable[countTable$nReads >0,]
expReadCount(object)<-countTable.filtered
print("Count processing is done.")
}
assign(objName,object,envir=parent.frame())
invisible(1)
}
if(isControlInvolved(object)==TRUE){
if(selected_methods=="wholeReads"){
####Get organism#######################
orgName <- organismName(object)
####Initialized Restriction Enzyme Object##
enzymeDb <-new("repbaseEnzyme")
resEnzyme <-restrictionEnzyme(object)
####Build GRanges for restriction site#####
print(paste("Fragmenting genome by....",resEnzyme,"....",sep=""))
wholeFragments<-getWholeGenomeRestrictionFragments(enzymeDb,resEnzyme,orgName)
wholeFragments.GRanges<-GRanges(seqnames =as.character(wholeFragments$chromosome),IRanges(start=wholeFragments$start,end=wholeFragments$end))
##########Count all reads in the experiment, which are located inside the fragments######
exp.GRanges<-expRawData(object)
exp.GRanges<-excludeReadsNearViewpoint(object,exp.GRanges,nExcludedFragments)
readsPerFragment <- countOverlaps(wholeFragments.GRanges,subject=exp.GRanges)
exp.reads<-data.frame(nReads=readsPerFragment)
####migrate the countTable to GRanges because BioC3.9 will not support the RagnedData###
values(wholeFragments.GRanges)=exp.reads
exp.countTable<-wholeFragments.GRanges
exp.countTable.filtered<-exp.countTable[exp.countTable$nReads >0,]
expReadCount(object)<-exp.countTable.filtered
print("Count processing in the experiment is done.")
##########Count all reads in the control, which are located inside the fragments######
contr.GRanges<-contrRawData(object)
contr.GRanges<-excludeReadsNearViewpoint(object,contr.GRanges,nExcludedFragments)
readsPerFragment <- countOverlaps(wholeFragments.GRanges,subject=contr.GRanges)
contr.reads<-data.frame(nReads=readsPerFragment)
####migrate the countTable to GRanges because BioC3.9 will not support the RagnedData###
values(wholeFragments.GRanges)=contr.reads
contr.countTable<-wholeFragments.GRanges
contr.countTable.filtered<-contr.countTable[contr.countTable$nReads >0,]
contrReadCount(object)<-contr.countTable.filtered
print("Count processing in the control is done.")
}
if(selected_methods=="adjacentFragmentEndsReads"){
####Get organism#######################
orgName <- organismName(object)
exp.read.length<-expReadLength(object)
####Initialized Restriction Enzyme Object##
enzymeDb <-new("repbaseEnzyme")
resEnzyme <-restrictionEnzyme(object)
####Build GRanges for restriction site#####
print(paste("Fragmenting genome by....",resEnzyme,"....",sep=""))
wholeFragments<-getWholeGenomeRestrictionFragments(enzymeDb,resEnzyme,orgName)
wholeFragments.GRanges<-GRanges(seqnames =as.character(wholeFragments$chromosome),IRanges(start=wholeFragments$start,end=wholeFragments$end))
########Make RangedData for the 5' and 3' of RE#######
exp.GRanges<-expRawData(object)
exp.GRanges<-excludeReadsNearViewpoint(object,exp.GRanges,nExcludedFragments)
wholeFragments_5_prime.GRanges<-GRanges(seqnames =seqnames(wholeFragments.GRanges),
IRanges(start=start(wholeFragments.GRanges),
end=start(wholeFragments.GRanges)+exp.read.length))
wholeFragments_3_prime.GRanges<-GRanges(seqnames =seqnames(wholeFragments.GRanges),
IRanges(start=end(wholeFragments.GRanges)-exp.read.length,
end=end(wholeFragments.GRanges)))
exp.readsPerFragment_5 <- countOverlaps(wholeFragments_5_prime.GRanges,subject=exp.GRanges)
exp.readsPerFragment_3 <- countOverlaps(wholeFragments_3_prime.GRanges,subject=exp.GRanges)
exp.combined_reads<-exp.readsPerFragment_5+exp.readsPerFragment_3
exp.reads<-data.frame(nReads=exp.combined_reads)
####migrate the countTable to GRanges because BioC3.9 will not support the RagnedData###
values(wholeFragments.GRanges)=exp.reads
exp.countTable<-wholeFragments.GRanges
exp.countTable.filtered<-exp.countTable[exp.countTable$nReads >0,]
expReadCount(object)<-exp.countTable.filtered
print("Count processing in the experiment is done.")
contr.GRanges<-contrRawData(object)
contr.GRanges<-excludeReadsNearViewpoint(object,contr.GRanges,nExcludedFragments)
contr.readsPerFragment_5 <- countOverlaps(wholeFragments_5_prime.GRanges,subject=contr.GRanges)
contr.readsPerFragment_3 <- countOverlaps(wholeFragments_3_prime.GRanges,subject=contr.GRanges)
contr.combined_reads<-contr.readsPerFragment_5+contr.readsPerFragment_3
contr.reads<-data.frame(nReads=contr.combined_reads)
####migrate the countTable to GRanges because BioC3.9 will not support the RagnedData###
values(wholeFragments.GRanges)=contr.reads
contr.countTable<-wholeFragments.GRanges
contr.countTable.filtered<-contr.countTable[contr.countTable$nReads >0,]
contrReadCount(object)<-contr.countTable.filtered
print("Count processing in the control is done.")
}
assign(objName,object,envir=parent.frame())
invisible(1)
}
}
)
##########
setGeneric(
name="getReadCountPerWindow",
def=function(object,windowSize=5e3,nFragmentExcludedReadsNearViewpoint=2,mode=c("non-overlapping")){
standardGeneric("getReadCountPerWindow")
}
)
setMethod("getReadCountPerWindow",
signature(object = "r3Cseq"),
function (object,windowSize,nFragmentExcludedReadsNearViewpoint,mode){
objName <- deparse(substitute(object))
if(!is(object,"r3Cseq")){
stop("Need to initialize the r3Cseq object")
}
######check the number of fragment to exclude reads around the viewpoint###
nExcludedFragments <- nFragmentExcludedReadsNearViewpoint
if(nExcludedFragments <0 | nExcludedFragments>5){
stop("The number of fragment that uses for removing reads around the viewpoint must be 0-5 restriction fragments.")
}
if(length(expRawData(object))==0){
stop("No reads found, please run getRawReads function to process BAM file.")
}
#####Select the read count methods#########
mode <- match.arg(mode)
if(mode==""){
mode<-"non-overlapping"
}
#####Select the read count methods#########
if(isControlInvolved(object)==FALSE){
wholeFragments.GRanges<-getFragmentsPerWindow(object,windowSize,mode)
##########Count all reads located inside the fragments######
exp.GRanges<-expRawData(object)
exp.GRanges<-excludeReadsNearViewpoint(object,exp.GRanges,nExcludedFragments)
readsPerFragment <- countOverlaps(wholeFragments.GRanges,subject=exp.GRanges)
reads<-data.frame(nReads=readsPerFragment)
####migrate the countTable to GRanges because BioC3.9 will not support the RagnedData###
values(wholeFragments.GRanges)<-reads
countTable<-wholeFragments.GRanges
countTable.filtered<-countTable[countTable$nReads >0,]
expReadCount(object)<-countTable.filtered
expRPM(object)<-GRanges()
print("Count processing is done.")
assign(objName,object,envir=parent.frame())
invisible(1)
}
if(isControlInvolved(object)==TRUE){
wholeFragments.GRanges<-getFragmentsPerWindow(object,windowSize,mode)
##########Count all reads located inside the fragments######
exp.GRanges<-expRawData(object)
exp.GRanges<-excludeReadsNearViewpoint(object,exp.GRanges,nExcludedFragments)
exp.readsPerFragment <- countOverlaps(wholeFragments.GRanges,subject=exp.GRanges)
exp.reads<-data.frame(nReads=exp.readsPerFragment)
####migrate the countTable to GRanges because BioC3.9 will not support the RagnedData###
values(wholeFragments.GRanges)<-exp.reads
exp.countTable<-wholeFragments.GRanges
exp.countTable.filtered<-exp.countTable[exp.countTable$nReads >0,]
expReadCount(object)<-exp.countTable.filtered
expRPM(object)<-GRanges()
contr.GRanges<-contrRawData(object)
contr.GRanges<-excludeReadsNearViewpoint(object,contr.GRanges,nExcludedFragments)
contr.readsPerFragment <- countOverlaps(wholeFragments.GRanges,subject=contr.GRanges)
contr.reads<-data.frame(nReads=contr.readsPerFragment)
####migrate the countTable to GRanges because BioC3.9 will not support the RagnedData###
values(wholeFragments.GRanges)<-contr.reads
contr.countTable<-wholeFragments.GRanges
contr.countTable.filtered<-contr.countTable[contr.countTable$nReads >0,]
contrReadCount(object)<-contr.countTable.filtered
contrRPM(object)<-GRanges()
print("Count processing is done.")
assign(objName,object,envir=parent.frame())
invisible(1)
}
}
)
##############
setGeneric(
name="calculateRPM",
def=function(object,normalized_method=c("powerlawFittedRPM","normalRPM")){
standardGeneric("calculateRPM")
}
)
setMethod("calculateRPM",
signature(object = "r3Cseq"),
function (object,normalized_method){
if(!is(object,"r3Cseq")){
stop("Need the r3Cseq object")
}
#####Select the method#########
selected_methods <- match.arg(normalized_method)
if(selected_methods==""){
selected_methods<-"powerlawFittedRPM"
}
if(selected_methods=="powerlawFittedRPM"){
if(isControlInvolved(object)==FALSE){
objName <- deparse(substitute(object))
expReadCounts <-expReadCount(object)
if(length(expReadCounts)>0){
coef<-getPowerLawFittedCoeficient(expReadCounts$nReads)
expReadCounts$RPMs<-powerLawFittedRPM(expReadCounts$nReads,coef)
expRPM(object) <-expReadCounts
assign(objName,object,envir=parent.frame())
invisible(1)
print(paste("Normal RPM calculation is done."))
}else{
stop("No reads count per regions found in the r3Cseq object.")
}
}
if(isControlInvolved(object)==TRUE){
expReadCounts <-expReadCount(object)
contrReadCounts <-contrReadCount(object)
if(length(expReadCounts)>0){
objName <- deparse(substitute(object))
exp.coef <- getPowerLawFittedCoeficient(expReadCounts$nReads)
contr.coef <- getPowerLawFittedCoeficient(contrReadCounts$nReads)
expReadCounts$RPMs <- powerLawFittedRPM(expReadCounts$nReads,exp.coef)
contrReadCounts$RPMs <-powerLawFittedRPM(contrReadCounts$nReads,contr.coef)
expRPM(object) <-expReadCounts
contrRPM(object) <-contrReadCounts
assign(objName,object,envir=parent.frame())
invisible(1)
print(paste("Normal RPM calculation is done."))
}else{
stop("No reads count per regions found in the r3Cseq object.")
}
}
}
if(selected_methods=="normalRPM"){
if(isControlInvolved(object)==FALSE){
objName <- deparse(substitute(object))
lib.size<-expLibrarySize(object)
expReadCounts <-expReadCount(object)
if(length(expReadCounts)>0){
expReadCounts$RPMs <- normalcalRPM(expReadCounts$nReads,lib.size)
expRPM(object) <-expReadCounts
assign(objName,object,envir=parent.frame())
invisible(1)
print(paste("Normal RPM calculation is done."))
}else{
stop("No reads count per regions found in the r3Cseq object.")
}
}
if(isControlInvolved(object)==TRUE){
expReadCounts <-expReadCount(object)
contrReadCounts <-contrReadCount(object)
if(length(expReadCounts)>0){
objName <- deparse(substitute(object))
expLibSize <- expLibrarySize(object)
contrLibSize <- contrLibrarySize(object)
expReadCounts<-expReadCount(object)
contrReadCounts<-contrReadCount(object)
expRPMs <- normalcalRPM(expReadCounts$nReads,expLibSize)
contrRPMs <- normalcalRPM(contrReadCounts$nReads,contrLibSize)
expReadCounts$RPMs <- expRPMs
contrReadCounts$RPMs <-contrRPMs
expRPM(object) <-expReadCounts
contrRPM(object) <-contrReadCounts
assign(objName,object,envir=parent.frame())
invisible(1)
print(paste("Normal RPM calculation is done."))
}else{
stop("No reads count per regions found in the r3Cseq object.")
}
}
}
}
)
##################
setGeneric(
name="getInteractions",
def=function(object,smoothing.parameter=0.1,fdr=0.05){
standardGeneric("getInteractions")
}
)
setMethod("getInteractions",
signature(object = "r3Cseq"),
function (object,smoothing.parameter,fdr){
if(!is(object,"r3Cseq")){
stop("Need the r3Cseq object")
}
if(smoothing.parameter <0.06 | smoothing.parameter>0.4){
stop("The smoothing parameter must be 0.06-0.4 (default=0.1).")
}
if(isControlInvolved(object)==FALSE){
expRPM.RangedData <-expRPM(object)
if(length(expRPM.RangedData)>0){
objName <- deparse(substitute(object))
expInteraction<-assign3CseqSigContact(object,expRPM.RangedData,smoothing.parameter,fdr)
expInteractionRegions(object) <-expInteraction
assign(objName,object,envir=parent.frame())
invisible(1)
print("Calculation is done. Use function 'expInteractionRegions' or 'contrInteractionRegions' to get the result.")
}else{
stop("No RPM found in the r3Cseq object, you have to run the function 'getReadCountPerRestrictionFragment' following by the function 'calculateRPM'.")
}
}
if(isControlInvolved(object)==TRUE){
expRPM.RangedData <-expRPM(object)
contrRPM.RangedData <-contrRPM(object)
if(length(expRPM.RangedData)>0){
objName <- deparse(substitute(object))
expInteraction<-assign3CseqSigContact(object,expRPM.RangedData,smoothing.parameter,fdr)
expInteractionRegions(object) <-expInteraction
##calculate in the control##
contrInteraction<-assign3CseqSigContact(object,contrRPM.RangedData,smoothing.parameter,fdr)
contrInteractionRegions(object) <-contrInteraction
assign(objName,object,envir=parent.frame())
invisible(1)
print("Calculation is done. Use function 'expInteractionRegions' or 'contrInteractionRegions' to get the result.")
}else{
stop("No RPM found in the r3Cseq object, you have to run the function 'getReadCountPerRestrictionFragment' following by the function 'calculateRPM'.")
}
}
}
)
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# TODO: These following functions were implemented to get interaction regions.
# Author: Supat Thongjuea
# Contact : supat.thongjuea@imm.ox.ac.uk or supat.thongjuea@gmail.com
#####
#####The functions below are completely migrated to BioC 3.9 on R 3.6
#####
setGeneric(
name="getCoverage",
def=function(object){
standardGeneric("getCoverage")
}
)
setMethod("getCoverage",
signature(object = "r3Cseq"),
function (object){
stop("This method has been removed!!.")
}
)
#####
setGeneric(
name="getRawReads",
def=function(object){
standardGeneric("getRawReads")
}
)
setMethod("getRawReads",
signature(object = "r3Cseq"),
function (object){
if(!is(object,"r3Cseq")){
stop("Need to initialize the r3Cseq object")
}
objName <- deparse(substitute(object))
if(isControlInvolved(object)==TRUE){
exp.bam.file <-alignedReadsBamExpFile(object)
contr.bam.file <-alignedReadsBamContrFile(object)
if(file.exists(exp.bam.file) ==FALSE){
stop(paste("Couldn't find file","-->",exp.bam.file))
}
if(file.exists(contr.bam.file) ==FALSE){
stop(paste("Couldn't find file","-->",contr.bam.file))
}
expLabeled<-expLabel(object)
controlLabeled<-contrLabel(object)
if(nchar(expLabeled)==0 & nchar(controlLabeled)==0){
stop("The package requires input of expLabel and contrLabel.")
}
print("start reading in ......")
what <- c("rname", "strand", "pos", "qwidth","seq")
param <- ScanBamParam(what=what,flag = scanBamFlag(isUnmappedQuery = FALSE))
exp.bam <- scanBam(exp.bam.file, param=param)
contr.bam <- scanBam(contr.bam.file, param=param)
exp.read.length <-width(exp.bam[[1]]$seq[1])
exp.GRanges<-GRanges(seqnames=as.vector(exp.bam[[1]]$rname),IRanges(start=exp.bam[[1]]$pos,width=exp.read.length),strand=exp.bam[[1]]$strand)
contr.read.length <-width(contr.bam[[1]]$seq[1])
contr.GRanges<-GRanges(seqnames=as.vector(contr.bam[[1]]$rname),IRanges(start=contr.bam[[1]]$pos,width=contr.read.length),strand=contr.bam[[1]]$strand)
expLibrarySize(object) <-length(exp.GRanges)
contrLibrarySize(object) <-length(contr.GRanges)
#######Remove chr.._random chromosomes############################################
#exp.GRanges<-exp.GRanges[seqnames(exp.GRanges) %in% paste('chr',c(seq(1,50),'X','Y'),sep='')]
#contr.GRanges<-contr.GRanges[seqnames(contr.GRanges) %in% paste('chr',c(seq(1,50),'X','Y'),sep='')]
#######assign raw read to the object########
expReadLength(object)<-exp.read.length
contrReadLength(object)<-contr.read.length
expRawData(object)<-exp.GRanges
contrRawData(object)<-contr.GRanges
assign(objName,object,envir=parent.frame())
invisible(1)
print("Raw reads processing is done.")
}
if(isControlInvolved(object)==FALSE){
exp.bam.file <-alignedReadsBamExpFile(object)
if(file.exists(exp.bam.file) ==FALSE){
stop(paste("Couldn't find file","-->",exp.bam.file))
}
expLabeled<-expLabel(object)
if(nchar(expLabeled)==0){
stop("The package requires input of expLabel.")
}
print("start reading in ......")
what <- c("rname", "strand", "pos", "qwidth","seq")
param <- ScanBamParam(what=what,flag = scanBamFlag(isUnmappedQuery = FALSE))
exp.bam <- scanBam(exp.bam.file, param=param)
exp.read.length <-width(exp.bam[[1]]$seq[1])
exp.GRanges<-GRanges(seqnames=as.vector(exp.bam[[1]]$rname),IRanges(start=exp.bam[[1]]$pos,width=exp.read.length),strand=exp.bam[[1]]$strand)
#######Remove chr.._random chromosomes############################################
#exp.GRanges<-exp.GRanges[seqnames(exp.GRanges) %in% paste('chr',c(seq(1,50),'X','Y'),sep='')]
##########assign raw read to the object######
expLibrarySize(object) <-length(exp.GRanges)
expReadLength(object)<-exp.read.length
expRawData(object)<-exp.GRanges
assign(objName,object,envir=parent.frame())
invisible(1)
print("Raw reads processing is done.")
}
}
)
########
setGeneric(
name="getReadCountPerRestrictionFragment",
def=function(object,getReadsMethod = c("wholeReads", "adjacentFragmentEndsReads"),
nFragmentExcludedReadsNearViewpoint=2){
standardGeneric("getReadCountPerRestrictionFragment")
}
)
setMethod("getReadCountPerRestrictionFragment",
signature(object = "r3Cseq"),
function (object,getReadsMethod,nFragmentExcludedReadsNearViewpoint){
objName <- deparse(substitute(object))
if(!is(object,"r3Cseq")){
stop("Need to initialize the r3Cseq object")
}
#####Select the read count methods#########
selected_methods <- match.arg(getReadsMethod)
if(selected_methods==""){
selected_methods<-"wholeReads"
}
######check the number of fragment to exclude reads around the viewpoint###
nExcludedFragments <- nFragmentExcludedReadsNearViewpoint
if(nExcludedFragments <0 | nExcludedFragments>5){
stop("The number of fragment that uses for removing reads around the viewpoint must be 0-5 restriction fragments.")
}
if(length(expRawData(object))==0){
stop("No reads found, please run getRawReads function to process BAM file.")
}
if(isControlInvolved(object)==FALSE){
if(selected_methods=="wholeReads"){
####Get organism#######################
orgName <- organismName(object)
####Initialized Restriction Enzyme Object##
enzymeDb <-new("repbaseEnzyme")
resEnzyme <-restrictionEnzyme(object)
####Build GRanges for restriction site#####
print(paste("Fragmenting genome by....",resEnzyme,"....",sep=""))
wholeFragments<-getWholeGenomeRestrictionFragments(enzymeDb,resEnzyme,orgName)
wholeFragments.GRanges<-GRanges(seqnames=as.character(wholeFragments$chromosome),IRanges(start=wholeFragments$start,end=wholeFragments$end))
##########Count all reads located inside the fragments######
exp.GRanges<-expRawData(object)
exp.GRanges<-excludeReadsNearViewpoint(object,exp.GRanges,nExcludedFragments)
readsPerFragment <- countOverlaps(wholeFragments.GRanges,subject=exp.GRanges)
reads<-data.frame(nReads=readsPerFragment)
####migrate the countTable to GRanges because BioC3.9 will not support the RagnedData###
values(wholeFragments.GRanges)=reads
countTable<-wholeFragments.GRanges
countTable.filtered<-countTable[countTable$nReads >0,]
expReadCount(object)<-countTable.filtered
print("Count processing is done.")
}
if(selected_methods=="adjacentFragmentEndsReads"){
####Get organism#######################
orgName <- organismName(object)
exp.read.length<-expReadLength(object)
####Initialized Restriction Enzyme Object##
enzymeDb <-new("repbaseEnzyme")
resEnzyme <-restrictionEnzyme(object)
####Build GRanges for restriction site#####
print(paste("Fragmenting genome by....",resEnzyme,"....",sep=""))
wholeFragments<-getWholeGenomeRestrictionFragments(enzymeDb,resEnzyme,orgName)
wholeFragments.GRanges<-GRanges(seqnames=as.character(wholeFragments$chromosome),IRanges(start=wholeFragments$start,end=wholeFragments$end))
########Make GRanges for the 5' and 3' of RE#######
exp.GRanges<-expRawData(object)
exp.GRanges<-excludeReadsNearViewpoint(object,exp.GRanges,nExcludedFragments)
wholeFragments_5_prime.GRanges<-GRanges(seqnames =seqnames(wholeFragments.GRanges),
IRanges(start=start(wholeFragments.GRanges),
end=start(wholeFragments.GRanges)+exp.read.length))
wholeFragments_3_prime.GRanges<-GRanges(seqnames =seqnames(wholeFragments.GRanges),
IRanges(start=end(wholeFragments.GRanges)-exp.read.length,
end=end(wholeFragments.GRanges)))
readsPerFragment_5 <- countOverlaps(wholeFragments_5_prime.GRanges,subject=exp.GRanges)
readsPerFragment_3 <- countOverlaps(wholeFragments_3_prime.GRanges,subject=exp.GRanges)
combined_reads<-readsPerFragment_5+readsPerFragment_3
reads<-data.frame(nReads=combined_reads)
####migrate the countTable to GRanges because BioC3.9 will not support the RagnedData###
values(wholeFragments.GRanges)=reads
countTable<-wholeFragments.GRanges
countTable.filtered<-countTable[countTable$nReads >0,]
expReadCount(object)<-countTable.filtered
print("Count processing is done.")
}
assign(objName,object,envir=parent.frame())
invisible(1)
}
if(isControlInvolved(object)==TRUE){
if(selected_methods=="wholeReads"){
####Get organism#######################
orgName <- organismName(object)
####Initialized Restriction Enzyme Object##
enzymeDb <-new("repbaseEnzyme")
resEnzyme <-restrictionEnzyme(object)
####Build GRanges for restriction site#####
print(paste("Fragmenting genome by....",resEnzyme,"....",sep=""))
wholeFragments<-getWholeGenomeRestrictionFragments(enzymeDb,resEnzyme,orgName)
wholeFragments.GRanges<-GRanges(seqnames =as.character(wholeFragments$chromosome),IRanges(start=wholeFragments$start,end=wholeFragments$end))
##########Count all reads in the experiment, which are located inside the fragments######
exp.GRanges<-expRawData(object)
exp.GRanges<-excludeReadsNearViewpoint(object,exp.GRanges,nExcludedFragments)
readsPerFragment <- countOverlaps(wholeFragments.GRanges,subject=exp.GRanges)
exp.reads<-data.frame(nReads=readsPerFragment)
####migrate the countTable to GRanges because BioC3.9 will not support the RagnedData###
values(wholeFragments.GRanges)=exp.reads
exp.countTable<-wholeFragments.GRanges
exp.countTable.filtered<-exp.countTable[exp.countTable$nReads >0,]
expReadCount(object)<-exp.countTable.filtered
print("Count processing in the experiment is done.")
##########Count all reads in the control, which are located inside the fragments######
contr.GRanges<-contrRawData(object)
contr.GRanges<-excludeReadsNearViewpoint(object,contr.GRanges,nExcludedFragments)
readsPerFragment <- countOverlaps(wholeFragments.GRanges,subject=contr.GRanges)
contr.reads<-data.frame(nReads=readsPerFragment)
####migrate the countTable to GRanges because BioC3.9 will not support the RagnedData###
values(wholeFragments.GRanges)=contr.reads
contr.countTable<-wholeFragments.GRanges
contr.countTable.filtered<-contr.countTable[contr.countTable$nReads >0,]
contrReadCount(object)<-contr.countTable.filtered
print("Count processing in the control is done.")
}
if(selected_methods=="adjacentFragmentEndsReads"){
####Get organism#######################
orgName <- organismName(object)
exp.read.length<-expReadLength(object)
####Initialized Restriction Enzyme Object##
enzymeDb <-new("repbaseEnzyme")
resEnzyme <-restrictionEnzyme(object)
####Build GRanges for restriction site#####
print(paste("Fragmenting genome by....",resEnzyme,"....",sep=""))
wholeFragments<-getWholeGenomeRestrictionFragments(enzymeDb,resEnzyme,orgName)
wholeFragments.GRanges<-GRanges(seqnames =as.character(wholeFragments$chromosome),IRanges(start=wholeFragments$start,end=wholeFragments$end))
########Make RangedData for the 5' and 3' of RE#######
exp.GRanges<-expRawData(object)
exp.GRanges<-excludeReadsNearViewpoint(object,exp.GRanges,nExcludedFragments)
wholeFragments_5_prime.GRanges<-GRanges(seqnames =seqnames(wholeFragments.GRanges),
IRanges(start=start(wholeFragments.GRanges),
end=start(wholeFragments.GRanges)+exp.read.length))
wholeFragments_3_prime.GRanges<-GRanges(seqnames =seqnames(wholeFragments.GRanges),
IRanges(start=end(wholeFragments.GRanges)-exp.read.length,
end=end(wholeFragments.GRanges)))
exp.readsPerFragment_5 <- countOverlaps(wholeFragments_5_prime.GRanges,subject=exp.GRanges)
exp.readsPerFragment_3 <- countOverlaps(wholeFragments_3_prime.GRanges,subject=exp.GRanges)
exp.combined_reads<-exp.readsPerFragment_5+exp.readsPerFragment_3
exp.reads<-data.frame(nReads=exp.combined_reads)
####migrate the countTable to GRanges because BioC3.9 will not support the RagnedData###
values(wholeFragments.GRanges)=exp.reads
exp.countTable<-wholeFragments.GRanges
exp.countTable.filtered<-exp.countTable[exp.countTable$nReads >0,]
expReadCount(object)<-exp.countTable.filtered
print("Count processing in the experiment is done.")
contr.GRanges<-contrRawData(object)
contr.GRanges<-excludeReadsNearViewpoint(object,contr.GRanges,nExcludedFragments)
contr.readsPerFragment_5 <- countOverlaps(wholeFragments_5_prime.GRanges,subject=contr.GRanges)
contr.readsPerFragment_3 <- countOverlaps(wholeFragments_3_prime.GRanges,subject=contr.GRanges)
contr.combined_reads<-contr.readsPerFragment_5+contr.readsPerFragment_3
contr.reads<-data.frame(nReads=contr.combined_reads)
####migrate the countTable to GRanges because BioC3.9 will not support the RagnedData###
values(wholeFragments.GRanges)=contr.reads
contr.countTable<-wholeFragments.GRanges
contr.countTable.filtered<-contr.countTable[contr.countTable$nReads >0,]
contrReadCount(object)<-contr.countTable.filtered
print("Count processing in the control is done.")
}
assign(objName,object,envir=parent.frame())
invisible(1)
}
}
)
##########
setGeneric(
name="getReadCountPerWindow",
def=function(object,windowSize=5e3,nFragmentExcludedReadsNearViewpoint=2,mode=c("non-overlapping")){
standardGeneric("getReadCountPerWindow")
}
)
setMethod("getReadCountPerWindow",
signature(object = "r3Cseq"),
function (object,windowSize,nFragmentExcludedReadsNearViewpoint,mode){
objName <- deparse(substitute(object))
if(!is(object,"r3Cseq")){
stop("Need to initialize the r3Cseq object")
}
######check the number of fragment to exclude reads around the viewpoint###
nExcludedFragments <- nFragmentExcludedReadsNearViewpoint
if(nExcludedFragments <0 | nExcludedFragments>5){
stop("The number of fragment that uses for removing reads around the viewpoint must be 0-5 restriction fragments.")
}
if(length(expRawData(object))==0){
stop("No reads found, please run getRawReads function to process BAM file.")
}
#####Select the read count methods#########
mode <- match.arg(mode)
if(mode==""){
mode<-"non-overlapping"
}
#####Select the read count methods#########
if(isControlInvolved(object)==FALSE){
wholeFragments.GRanges<-getFragmentsPerWindow(object,windowSize,mode)
##########Count all reads located inside the fragments######
exp.GRanges<-expRawData(object)
exp.GRanges<-excludeReadsNearViewpoint(object,exp.GRanges,nExcludedFragments)
readsPerFragment <- countOverlaps(wholeFragments.GRanges,subject=exp.GRanges)
reads<-data.frame(nReads=readsPerFragment)
####migrate the countTable to GRanges because BioC3.9 will not support the RagnedData###
values(wholeFragments.GRanges)<-reads
countTable<-wholeFragments.GRanges
countTable.filtered<-countTable[countTable$nReads >0,]
expReadCount(object)<-countTable.filtered
expRPM(object)<-GRanges()
print("Count processing is done.")
assign(objName,object,envir=parent.frame())
invisible(1)
}
if(isControlInvolved(object)==TRUE){
wholeFragments.GRanges<-getFragmentsPerWindow(object,windowSize,mode)
##########Count all reads located inside the fragments######
exp.GRanges<-expRawData(object)
exp.GRanges<-excludeReadsNearViewpoint(object,exp.GRanges,nExcludedFragments)
exp.readsPerFragment <- countOverlaps(wholeFragments.GRanges,subject=exp.GRanges)
exp.reads<-data.frame(nReads=exp.readsPerFragment)
####migrate the countTable to GRanges because BioC3.9 will not support the RagnedData###
values(wholeFragments.GRanges)<-exp.reads
exp.countTable<-wholeFragments.GRanges
exp.countTable.filtered<-exp.countTable[exp.countTable$nReads >0,]
expReadCount(object)<-exp.countTable.filtered
expRPM(object)<-GRanges()
contr.GRanges<-contrRawData(object)
contr.GRanges<-excludeReadsNearViewpoint(object,contr.GRanges,nExcludedFragments)
contr.readsPerFragment <- countOverlaps(wholeFragments.GRanges,subject=contr.GRanges)
contr.reads<-data.frame(nReads=contr.readsPerFragment)
####migrate the countTable to GRanges because BioC3.9 will not support the RagnedData###
values(wholeFragments.GRanges)<-contr.reads
contr.countTable<-wholeFragments.GRanges
contr.countTable.filtered<-contr.countTable[contr.countTable$nReads >0,]
contrReadCount(object)<-contr.countTable.filtered
contrRPM(object)<-GRanges()
print("Count processing is done.")
assign(objName,object,envir=parent.frame())
invisible(1)
}
}
)
##############
setGeneric(
name="calculateRPM",
def=function(object,normalized_method=c("powerlawFittedRPM","normalRPM")){
standardGeneric("calculateRPM")
}
)
setMethod("calculateRPM",
signature(object = "r3Cseq"),
function (object,normalized_method){
if(!is(object,"r3Cseq")){
stop("Need the r3Cseq object")
}
#####Select the method#########
selected_methods <- match.arg(normalized_method)
if(selected_methods==""){
selected_methods<-"powerlawFittedRPM"
}
if(selected_methods=="powerlawFittedRPM"){
if(isControlInvolved(object)==FALSE){
objName <- deparse(substitute(object))
expReadCounts <-expReadCount(object)
if(length(expReadCounts)>0){
coef<-getPowerLawFittedCoeficient(expReadCounts$nReads)
expReadCounts$RPMs<-powerLawFittedRPM(expReadCounts$nReads,coef)
expRPM(object) <-expReadCounts
assign(objName,object,envir=parent.frame())
invisible(1)
print(paste("Normal RPM calculation is done."))
}else{
stop("No reads count per regions found in the r3Cseq object.")
}
}
if(isControlInvolved(object)==TRUE){
expReadCounts <-expReadCount(object)
contrReadCounts <-contrReadCount(object)
if(length(expReadCounts)>0){
objName <- deparse(substitute(object))
exp.coef <- getPowerLawFittedCoeficient(expReadCounts$nReads)
contr.coef <- getPowerLawFittedCoeficient(contrReadCounts$nReads)
expReadCounts$RPMs <- powerLawFittedRPM(expReadCounts$nReads,exp.coef)
contrReadCounts$RPMs <-powerLawFittedRPM(contrReadCounts$nReads,contr.coef)
expRPM(object) <-expReadCounts
contrRPM(object) <-contrReadCounts
assign(objName,object,envir=parent.frame())
invisible(1)
print(paste("Normal RPM calculation is done."))
}else{
stop("No reads count per regions found in the r3Cseq object.")
}
}
}
if(selected_methods=="normalRPM"){
if(isControlInvolved(object)==FALSE){
objName <- deparse(substitute(object))
lib.size<-expLibrarySize(object)
expReadCounts <-expReadCount(object)
if(length(expReadCounts)>0){
expReadCounts$RPMs <- normalcalRPM(expReadCounts$nReads,lib.size)
expRPM(object) <-expReadCounts
assign(objName,object,envir=parent.frame())
invisible(1)
print(paste("Normal RPM calculation is done."))
}else{
stop("No reads count per regions found in the r3Cseq object.")
}
}
if(isControlInvolved(object)==TRUE){
expReadCounts <-expReadCount(object)
contrReadCounts <-contrReadCount(object)
if(length(expReadCounts)>0){
objName <- deparse(substitute(object))
expLibSize <- expLibrarySize(object)
contrLibSize <- contrLibrarySize(object)
expReadCounts<-expReadCount(object)
contrReadCounts<-contrReadCount(object)
expRPMs <- normalcalRPM(expReadCounts$nReads,expLibSize)
contrRPMs <- normalcalRPM(contrReadCounts$nReads,contrLibSize)
expReadCounts$RPMs <- expRPMs
contrReadCounts$RPMs <-contrRPMs
expRPM(object) <-expReadCounts
contrRPM(object) <-contrReadCounts
assign(objName,object,envir=parent.frame())
invisible(1)
print(paste("Normal RPM calculation is done."))
}else{
stop("No reads count per regions found in the r3Cseq object.")
}
}
}
}
)
##################
setGeneric(
name="getInteractions",
def=function(object,smoothing.parameter=0.1,fdr=0.05){
standardGeneric("getInteractions")
}
)
setMethod("getInteractions",
signature(object = "r3Cseq"),
function (object,smoothing.parameter,fdr){
if(!is(object,"r3Cseq")){
stop("Need the r3Cseq object")
}
if(smoothing.parameter <0.06 | smoothing.parameter>0.4){
stop("The smoothing parameter must be 0.06-0.4 (default=0.1).")
}
if(isControlInvolved(object)==FALSE){
expRPM.RangedData <-expRPM(object)
if(length(expRPM.RangedData)>0){
objName <- deparse(substitute(object))
expInteraction<-assign3CseqSigContact(object,expRPM.RangedData,smoothing.parameter,fdr)
expInteractionRegions(object) <-expInteraction
assign(objName,object,envir=parent.frame())
invisible(1)
print("Calculation is done. Use function 'expInteractionRegions' or 'contrInteractionRegions' to get the result.")
}else{
stop("No RPM found in the r3Cseq object, you have to run the function 'getReadCountPerRestrictionFragment' following by the function 'calculateRPM'.")
}
}
if(isControlInvolved(object)==TRUE){
expRPM.RangedData <-expRPM(object)
contrRPM.RangedData <-contrRPM(object)
if(length(expRPM.RangedData)>0){
objName <- deparse(substitute(object))
expInteraction<-assign3CseqSigContact(object,expRPM.RangedData,smoothing.parameter,fdr)
expInteractionRegions(object) <-expInteraction
##calculate in the control##
contrInteraction<-assign3CseqSigContact(object,contrRPM.RangedData,smoothing.parameter,fdr)
contrInteractionRegions(object) <-contrInteraction
assign(objName,object,envir=parent.frame())
invisible(1)
print("Calculation is done. Use function 'expInteractionRegions' or 'contrInteractionRegions' to get the result.")
}else{
stop("No RPM found in the r3Cseq object, you have to run the function 'getReadCountPerRestrictionFragment' following by the function 'calculateRPM'.")
}
}
}
)
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