Nothing
R/FunctionInCommon.R
In r3Cseq: Analysis of Chromosome Conformation Capture and Next-generation Sequencing (3C-seq)
Defines functions
fishersMethod
makeInteractionMatrixNearCisPerWindow
get3CseqRefGene
assign3CseqSigContact
powerLawFittedRPM
getPowerLawFittedCoeficient
# TODO: These following functions were implemented to facilitate r3Cseq and r3CseqWithReplocates classes.
# 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
#####
getViewpoint<-function (obj){
stopifnot( is( obj, "r3Cseq" ) |is( obj, "r3CseqInBatch" ) )
#######get organism name ############
#######Fixed genome to genome.assembly
genome.assembly<-organismName(obj)
#######get forward primer############
primer_f<-viewpoint_primer_forward(obj)
primer_r<-viewpoint_primer_reverse(obj)
#######get viewpoint chromosome######
viewpoint_chr<-viewpoint_chromosome(obj)
#####################################
if('BSgenome.Hsapiens.UCSC.hg19.masked' %in% loadedNamespaces()==TRUE){
detach(package:BSgenome.Hsapiens.UCSC.hg19.masked,unload=TRUE)
}
if('BSgenome.Hsapiens.UCSC.hg18.masked' %in% loadedNamespaces()==TRUE){
detach(package:BSgenome.Hsapiens.UCSC.hg18.masked,unload=TRUE)
}
if('BSgenome.Mmusculus.UCSC.mm9.masked' %in% loadedNamespaces()==TRUE){
detach(package:BSgenome.Mmusculus.UCSC.mm9.masked,unload=TRUE)
}
if('BSgenome.Mmusculus.UCSC.mm10.masked' %in% loadedNamespaces()==TRUE){
detach(package:BSgenome.Mmusculus.UCSC.mm10.masked,unload=TRUE)
}
if('BSgenome.Rnorvegicus.UCSC.rn5.masked' %in% loadedNamespaces()==TRUE){
detach(package:BSgenome.Rnorvegicus.UCSC.rn5.masked,unload=TRUE)
}
if(genome.assembly=="hg18"){
library(BSgenome.Hsapiens.UCSC.hg18.masked)
genome <- BSgenome.Hsapiens.UCSC.hg18.masked
hits_f_f<-matchPattern(DNAString(primer_f),genome[[viewpoint_chr]],fixed=FALSE)
hits_f_r<-matchPattern(reverseComplement(DNAString(primer_f)),genome[[viewpoint_chr]],fixed=FALSE)
hits_f_positions<-c(start(hits_f_f),end(hits_f_f),start(hits_f_r),end(hits_f_r))
if(length(hits_f_positions)==0){
stop(paste("Could not find the forward primer position in",genome.assembly," :",viewpoint_chr, ". Please check the input forward primer"))
}
hits_r_f<-matchPattern(DNAString(primer_r),genome[[viewpoint_chr]],fixed=FALSE)
hits_r_r<-matchPattern(reverseComplement(DNAString(primer_r)),genome[[viewpoint_chr]],fixed=FALSE)
hits_r_positions<-c(start(hits_r_f),end(hits_r_f),start(hits_r_r),end(hits_r_r))
if(length(hits_r_positions)==0){
stop(paste("Could not find the reverse primer position in",genome.assembly," :",viewpoint_chr, ". Please check the input reverse primer"))
}
hit_positions<-c(hits_f_positions,hits_r_positions)
if(length(hit_positions)==4){
start_p<-min(hit_positions)
end_p<-max(hit_positions)
viewpoint<-GRanges(seqnames =as.character(viewpoint_chr),IRanges(start=start_p,end=end_p))
return(viewpoint)
}
if(length(hit_positions) > 4){
if(length(hits_r_positions) > 2 & length(hits_f_positions)==2){
rank.id<-order(abs(hits_r_positions-hits_f_positions))
hit_positions<-c(hits_f_positions,hits_r_positions[rank.id==1],hits_r_positions[rank.id==2])
start_p<-min(hit_positions)
end_p<-max(hit_positions)
viewpoint<-GRanges(seqnames=as.character(viewpoint_chr),IRanges(start=start_p,end=end_p))
return(viewpoint)
}
if(length(hits_f_positions) > 2 & length(hits_r_positions)==2){
rank.id<-order(abs(hits_f_positions-hits_r_positions))
hit_positions<-c(hits_f_positions[rank.id==1],hits_f_positions[rank.id==2],hits_r_positions)
start_p<-min(hit_positions)
end_p<-max(hit_positions)
viewpoint<-GRanges(seqnames=as.character(viewpoint_chr),IRanges(start=start_p,end=end_p))
return(viewpoint)
}
if(length(hits_f_positions) > 2 & length(hits_r_positions) > 2){
stop(paste("The position of your input primers are not unique. We detected multiple positions of your input primers!!!"))
}
}
}else if(genome.assembly=="hg19"){
library(BSgenome.Hsapiens.UCSC.hg19.masked)
genome <- BSgenome.Hsapiens.UCSC.hg19.masked
hits_f_f<-matchPattern(DNAString(primer_f),genome[[viewpoint_chr]],fixed=FALSE)
hits_f_r<-matchPattern(reverseComplement(DNAString(primer_f)),genome[[viewpoint_chr]],fixed=FALSE)
hits_f_positions<-c(start(hits_f_f),end(hits_f_f),start(hits_f_r),end(hits_f_r))
if(length(hits_f_positions)==0){
stop(paste("Could not find the forward primer position in",genome.assembly," :",viewpoint_chr, ". Please check the input forward primer"))
}
hits_r_f<-matchPattern(DNAString(primer_r),genome[[viewpoint_chr]],fixed=FALSE)
hits_r_r<-matchPattern(reverseComplement(DNAString(primer_r)),genome[[viewpoint_chr]],fixed=FALSE)
hits_r_positions<-c(start(hits_r_f),end(hits_r_f),start(hits_r_r),end(hits_r_r))
if(length(hits_r_positions)==0){
stop(paste("Could not find the reverse primer position in",genome.assembly," :",viewpoint_chr, ". Please check the input reverse primer"))
}
hit_positions<-c(hits_f_positions,hits_r_positions)
if(length(hit_positions)==4){
start_p<-min(hit_positions)
end_p<-max(hit_positions)
viewpoint<-GRanges(seqnames=as.character(viewpoint_chr),IRanges(start=start_p,end=end_p))
return(viewpoint)
}
if(length(hit_positions) > 4){
if(length(hits_r_positions) > 2 & length(hits_f_positions)==2){
rank.id<-order(abs(hits_r_positions-hits_f_positions))
hit_positions<-c(hits_f_positions,hits_r_positions[rank.id==1],hits_r_positions[rank.id==2])
start_p<-min(hit_positions)
end_p<-max(hit_positions)
viewpoint<-GRanges(seqnames=as.character(viewpoint_chr),IRanges(start=start_p,end=end_p))
return(viewpoint)
}
if(length(hits_f_positions) > 2 & length(hits_r_positions)==2){
rank.id<-order(abs(hits_f_positions-hits_r_positions))
hit_positions<-c(hits_f_positions[rank.id==1],hits_f_positions[rank.id==2],hits_r_positions)
start_p<-min(hit_positions)
end_p<-max(hit_positions)
viewpoint<-GRanges(seqnames=as.character(viewpoint_chr),IRanges(start=start_p,end=end_p))
return(viewpoint)
}
if(length(hits_f_positions) > 2 & length(hits_r_positions) > 2){
stop(paste("The position of your input primers are not unique. We detected multiple positions of your input primers!!!"))
}
}
}else if(genome.assembly =="mm9"){
library(BSgenome.Mmusculus.UCSC.mm9.masked)
genome <- BSgenome.Mmusculus.UCSC.mm9.masked
hits_f_f<-matchPattern(DNAString(primer_f),genome[[viewpoint_chr]],fixed=FALSE)
hits_f_r<-matchPattern(reverseComplement(DNAString(primer_f)),genome[[viewpoint_chr]],fixed=FALSE)
hits_f_positions<-c(start(hits_f_f),end(hits_f_f),start(hits_f_r),end(hits_f_r))
if(length(hits_f_positions)==0){
stop(paste("Could not find the forward primer position in",genome.assembly," :",viewpoint_chr, ". Please check the input forward primer"))
}
hits_r_f<-matchPattern(DNAString(primer_r),genome[[viewpoint_chr]],fixed=FALSE)
hits_r_r<-matchPattern(reverseComplement(DNAString(primer_r)),genome[[viewpoint_chr]],fixed=FALSE)
hits_r_positions<-c(start(hits_r_f),end(hits_r_f),start(hits_r_r),end(hits_r_r))
if(length(hits_r_positions)==0){
stop(paste("Could not find the reverse primer position in",genome.assembly," :",viewpoint_chr, ". Please check the input reverse primer"))
}
hit_positions<-c(hits_f_positions,hits_r_positions)
if(length(hit_positions)==4){
start_p<-min(hit_positions)
end_p<-max(hit_positions)
viewpoint<-GRanges(seqnames=as.character(viewpoint_chr),IRanges(start=start_p,end=end_p))
return(viewpoint)
}
if(length(hit_positions) > 4){
if(length(hits_r_positions) > 2 & length(hits_f_positions)==2){
rank.id<-order(abs(hits_r_positions-hits_f_positions))
hit_positions<-c(hits_f_positions,hits_r_positions[rank.id==1],hits_r_positions[rank.id==2])
start_p<-min(hit_positions)
end_p<-max(hit_positions)
viewpoint<-GRanges(seqnames=as.character(viewpoint_chr),IRanges(start=start_p,end=end_p))
return(viewpoint)
}
if(length(hits_f_positions) > 2 & length(hits_r_positions)==2){
rank.id<-order(abs(hits_f_positions-hits_r_positions))
hit_positions<-c(hits_f_positions[rank.id==1],hits_f_positions[rank.id==2],hits_r_positions)
start_p<-min(hit_positions)
end_p<-max(hit_positions)
viewpoint<-GRanges(seqnames=as.character(viewpoint_chr),IRanges(start=start_p,end=end_p))
return(viewpoint)
}
if(length(hits_f_positions) > 2 & length(hits_r_positions) > 2){
stop(paste("The position of your input primers are not unique. We detected multiple positions of your input primers!!!"))
}
}
}else if(genome.assembly =="mm10"){
library(BSgenome.Mmusculus.UCSC.mm10.masked)
genome <- BSgenome.Mmusculus.UCSC.mm10.masked
hits_f_f<-matchPattern(DNAString(primer_f),genome[[viewpoint_chr]],fixed=FALSE)
hits_f_r<-matchPattern(reverseComplement(DNAString(primer_f)),genome[[viewpoint_chr]],fixed=FALSE)
hits_f_positions<-c(start(hits_f_f),end(hits_f_f),start(hits_f_r),end(hits_f_r))
if(length(hits_f_positions)==0){
stop(paste("Could not find the forward primer position in",genome.assembly," :",viewpoint_chr, ". Please check the input forward primer"))
}
hits_r_f<-matchPattern(DNAString(primer_r),genome[[viewpoint_chr]],fixed=FALSE)
hits_r_r<-matchPattern(reverseComplement(DNAString(primer_r)),genome[[viewpoint_chr]],fixed=FALSE)
hits_r_positions<-c(start(hits_r_f),end(hits_r_f),start(hits_r_r),end(hits_r_r))
if(length(hits_r_positions)==0){
stop(paste("Could not find the reverse primer position in",genome.assembly," :",viewpoint_chr, ". Please check the input reverse primer"))
}
hit_positions<-c(hits_f_positions,hits_r_positions)
if(length(hit_positions)==4){
start_p<-min(hit_positions)
end_p<-max(hit_positions)
viewpoint<-GRanges(seqnames=as.character(viewpoint_chr),IRanges(start=start_p,end=end_p))
return(viewpoint)
}
if(length(hit_positions) > 4){
if(length(hits_r_positions) > 2 & length(hits_f_positions)==2){
rank.id<-order(abs(hits_r_positions-hits_f_positions))
hit_positions<-c(hits_f_positions,hits_r_positions[rank.id==1],hits_r_positions[rank.id==2])
start_p<-min(hit_positions)
end_p<-max(hit_positions)
viewpoint<-GRanges(seqnames=as.character(viewpoint_chr),IRanges(start=start_p,end=end_p))
return(viewpoint)
}
if(length(hits_f_positions) > 2 & length(hits_r_positions)==2){
rank.id<-order(abs(hits_f_positions-hits_r_positions))
hit_positions<-c(hits_f_positions[rank.id==1],hits_f_positions[rank.id==2],hits_r_positions)
start_p<-min(hit_positions)
end_p<-max(hit_positions)
viewpoint<-GRanges(seqnames=as.character(viewpoint_chr),IRanges(start=start_p,end=end_p))
return(viewpoint)
}
if(length(hits_f_positions) > 2 & length(hits_r_positions) > 2){
stop(paste("The position of your input primers are not unique. We detected multiple positions of your input primers!!!"))
}
}
}else if(genome.assembly =="rn5"){
library(BSgenome.Rnorvegicus.UCSC.rn5.masked)
genome <- BSgenome.Rnorvegicus.UCSC.rn5.masked
hits_f_f<-matchPattern(DNAString(primer_f),genome[[viewpoint_chr]],fixed=FALSE)
hits_f_r<-matchPattern(reverseComplement(DNAString(primer_f)),genome[[viewpoint_chr]],fixed=FALSE)
hits_f_positions<-c(start(hits_f_f),end(hits_f_f),start(hits_f_r),end(hits_f_r))
if(length(hits_f_positions)==0){
stop(paste("Could not find the forward primer position in",genome.assembly," :",viewpoint_chr, ". Please check the input forward primer"))
}
hits_r_f<-matchPattern(DNAString(primer_r),genome[[viewpoint_chr]],fixed=FALSE)
hits_r_r<-matchPattern(reverseComplement(DNAString(primer_r)),genome[[viewpoint_chr]],fixed=FALSE)
hits_r_positions<-c(start(hits_r_f),end(hits_r_f),start(hits_r_r),end(hits_r_r))
if(length(hits_r_positions)==0){
stop(paste("Could not find the reverse primer position in",genome.assembly," :",viewpoint_chr, ". Please check the input reverse primer"))
}
hit_positions<-c(hits_f_positions,hits_r_positions)
if(length(hit_positions)==4){
start_p<-min(hit_positions)
end_p<-max(hit_positions)
viewpoint<-GRanges(seqnames=as.character(viewpoint_chr),IRanges(start=start_p,end=end_p))
return(viewpoint)
}
if(length(hit_positions) > 4){
if(length(hits_r_positions) > 2 & length(hits_f_positions)==2){
rank.id<-order(abs(hits_r_positions-hits_f_positions))
hit_positions<-c(hits_f_positions,hits_r_positions[rank.id==1],hits_r_positions[rank.id==2])
start_p<-min(hit_positions)
end_p<-max(hit_positions)
viewpoint<-GRanges(seqnames=as.character(viewpoint_chr),IRanges(start=start_p,end=end_p))
return(viewpoint)
}
if(length(hits_f_positions) > 2 & length(hits_r_positions)==2){
rank.id<-order(abs(hits_f_positions-hits_r_positions))
hit_positions<-c(hits_f_positions[rank.id==1],hits_f_positions[rank.id==2],hits_r_positions)
start_p<-min(hit_positions)
end_p<-max(hit_positions)
viewpoint<-GRanges(seqnames=as.character(viewpoint_chr),IRanges(start=start_p,end=end_p))
return(viewpoint)
}
if(length(hits_f_positions) > 2 & length(hits_r_positions) > 2){
stop(paste("The position of your input primers are not unique. We detected multiple positions of your input primers!!!"))
}
}
}else{
stop("Require the selected genome: hg18, hg19, mm9, mm10 or rn5.")
}
}
excludeReadsNearViewpoint<-function (object,rawReadsGRanges,span_fragment=1){
if(span_fragment <0 | span_fragment >5){
stop("Span fragment number from the viewpoint must be in the range of 0-5 fragment.")
}
if(length(rawReadsGRanges)==0){
stop("No reads can be found!!!, please run 'getRawReads' function in order to get raw reads.")
}
#####get viewpoint##############
viewpoint<-getViewpoint(object)
######get restriction fragment##
orgName <- organismName(object)
####Initialized Restriction Enzyme Object##
enzymeDb <-new("repbaseEnzyme")
resEnzyme <-restrictionEnzyme(object)
####Build GRanges for restriction site#####
fragments<-getRestrictionFragments(enzymeDb,resEnzyme,orgName,as.character(seqnames(viewpoint)))
fragments$distance2viewpoint<-abs(fragments$end-start(viewpoint))
v.index<-which(fragments$distance2viewpoint==min(fragments$distance2viewpoint))
v.index<-v.index[1]
f.index<-c((v.index-span_fragment):(v.index+span_fragment))
selected.f<-fragments[f.index,]
expected.regions<-GRanges(seqnames =viewpoint_chromosome(object),IRanges(start=min(selected.f$start),end=max(selected.f$end)))
excluded.index <- subjectHits(findOverlaps(expected.regions,subject=rawReadsGRanges))
new.GRanges<-rawReadsGRanges[-(excluded.index)]
return(new.GRanges)
}
getFragmentsPerWindow<-function (obj,windowSize=5e3,mode){
stopifnot( is( obj, "r3Cseq" ) |is( obj, "r3CseqInBatch" ) )
if(windowSize <2000 | windowSize >100000){
stop("Please select window size between 2Kb - 100Kb")
}
if(mode==""){
mode<-"non-overlapping"
}
#######get organism name ############
genome<-organismName(obj)
#####################################
viewpoint<-getViewpoint(obj)
viewpoint.chr<-viewpoint_chromosome(obj)
#####################################
if('BSgenome.Hsapiens.UCSC.hg19.masked' %in% loadedNamespaces()==TRUE){
detach(package:BSgenome.Hsapiens.UCSC.hg19.masked,unload=TRUE)
}
if('BSgenome.Hsapiens.UCSC.hg18.masked' %in% loadedNamespaces()==TRUE){
detach(package:BSgenome.Hsapiens.UCSC.hg18.masked,unload=TRUE)
}
if('BSgenome.Mmusculus.UCSC.mm9.masked' %in% loadedNamespaces()==TRUE){
detach(package:BSgenome.Mmusculus.UCSC.mm9.masked,unload=TRUE)
}
if('BSgenome.Mmusculus.UCSC.mm10.masked' %in% loadedNamespaces()==TRUE){
detach(package:BSgenome.Mmusculus.UCSC.mm10.masked,unload=TRUE)
}
if('BSgenome.Rnorvegicus.UCSC.rn5.masked' %in% loadedNamespaces()==TRUE){
detach(package:BSgenome.Rnorvegicus.UCSC.rn5.masked,unload=TRUE)
}
if(genome=="hg18"){
library(BSgenome.Hsapiens.UCSC.hg18.masked)
genome <- BSgenome.Hsapiens.UCSC.hg18.masked
fragment<-data.frame()
for (chr in paste('chr',c(seq(1,22),'X','Y'),sep='')){
chr.size<-seqlengths(genome)[chr]
#if(chr==viewpoint.chr){
# if(mode=="non-overlapping"){
# ##Now getting the fragment#####
# window.size=windowSize
# step.size = window.size
# start.p<-start(viewpoint)
# end.p<-end(viewpoint)
#
# f.ends<-(rev(seq(-start.p, -window.size,by=step.size)))*-1
# f.starts <- c(1,(rev(seq(-start.p+window.size,1,by=step.size)))*-1)
# f.starts<-f.starts[-(2)]
#
# s.ends <- c(seq(from=end.p+window.size,to=chr.size,by=step.size),as.integer(chr.size))
# s.starts <- seq(from=end.p,to=chr.size,by=step.size)
#
# f.fragment<-data.frame(chromosome=chr,start=f.starts,end=f.ends)
# s.fragment<-data.frame(chromosome=chr,start=s.starts,end=s.ends)
# fragment.chr<-rbind(f.fragment,s.fragment)
# fragment<-rbind(fragment,fragment.chr)
# }
# if(mode=="overlapping"){
# ##Now getting the fragment#####
# window.size=windowSize
# step.size = window.size/2
# start.p<-start(viewpoint)
# end.p<-end(viewpoint)
#
# f.ends<-(rev(seq(-start.p, -window.size,by=step.size)))*-1
# f.starts <- c(1,(rev(seq(-start.p+window.size,1,by=step.size)))*-1)
# f.starts<-f.starts[-(2)]
#
# s.ends <- c(seq(from=end.p+window.size,to=chr.size,by=step.size),as.integer(chr.size))
# s.starts <- seq(from=end.p,to=chr.size,by=step.size)
# s.starts<-s.starts[-(length(s.starts))]
#
# f.fragment<-data.frame(chromosome=chr,start=f.starts,end=f.ends)
# s.fragment<-data.frame(chromosome=chr,start=s.starts,end=s.ends)
#
#
# fragment.chr<-rbind(f.fragment,s.fragment)
# fragment<-rbind(fragment,fragment.chr)
# }
#}else{
if(mode=="non-overlapping"){
##Now getting the fragment#####
window.size=windowSize
step.size = window.size
ends <- c(seq(from=window.size,to=chr.size,by=step.size),as.integer(chr.size))
starts <- seq(from=1,to=chr.size,by=step.size)
fragment.chr<-data.frame(chromosome=chr,start=starts,end=ends)
fragment<-rbind(fragment,fragment.chr)
}
if(mode=="overlapping"){
##Now getting the fragment#####
window.size=windowSize
step.size = window.size/2
ends <- c(seq(from=window.size,to=chr.size,by=step.size),as.integer(chr.size))
starts <- seq(from=window.size-step.size+1,to=chr.size,by=step.size)
fragment.chr<-data.frame(chromosome=chr,start=starts,end=ends)
fragment<-rbind(fragment,fragment.chr)
}
#}
}
fragment.GRanges<-GRanges(seqnames =fragment$chromosome,IRanges(start=fragment$start,end=fragment$end))
return(fragment.GRanges)
}else if(genome=="hg19"){
library(BSgenome.Hsapiens.UCSC.hg19.masked)
genome <- BSgenome.Hsapiens.UCSC.hg19.masked
fragment<-data.frame()
for (chr in paste('chr',c(seq(1,22),'X','Y'),sep='')){
chr.size<-seqlengths(genome)[chr]
#if(chr==viewpoint.chr){
# if(mode=="non-overlapping"){
# ##Now getting the fragment#####
# window.size=windowSize
# step.size = window.size
# start.p<-start(viewpoint)
# end.p<-end(viewpoint)
#
# f.ends<-(rev(seq(-start.p, -window.size,by=step.size)))*-1
# f.starts <- c(1,(rev(seq(-start.p+window.size,1,by=step.size)))*-1)
# f.starts<-f.starts[-(2)]
#
# s.ends <- c(seq(from=end.p+window.size,to=chr.size,by=step.size),as.integer(chr.size))
# s.starts <- seq(from=end.p,to=chr.size,by=step.size)
#
# f.fragment<-data.frame(chromosome=chr,start=f.starts,end=f.ends)
# s.fragment<-data.frame(chromosome=chr,start=s.starts,end=s.ends)
# fragment.chr<-rbind(f.fragment,s.fragment)
# fragment<-rbind(fragment,fragment.chr)
# }
# if(mode=="overlapping"){
# ##Now getting the fragment#####
# window.size=windowSize
# step.size = window.size/2
# start.p<-start(viewpoint)
# end.p<-end(viewpoint)
#
# f.ends<-(rev(seq(-start.p, -window.size,by=step.size)))*-1
# f.starts <- c(1,(rev(seq(-start.p+window.size,1,by=step.size)))*-1)
# f.starts<-f.starts[-(2)]
#
# s.ends <- c(seq(from=end.p+window.size,to=chr.size,by=step.size),as.integer(chr.size))
# s.starts <- seq(from=end.p,to=chr.size,by=step.size)
# s.starts<-s.starts[-(length(s.starts))]
#
# f.fragment<-data.frame(chromosome=chr,start=f.starts,end=f.ends)
# s.fragment<-data.frame(chromosome=chr,start=s.starts,end=s.ends)
#
#
# fragment.chr<-rbind(f.fragment,s.fragment)
# fragment<-rbind(fragment,fragment.chr)
# }
#}else{
if(mode=="non-overlapping"){
##Now getting the fragment#####
window.size=windowSize
step.size = window.size
ends <- c(seq(from=window.size,to=chr.size,by=step.size),as.integer(chr.size))
starts <- seq(from=1,to=chr.size,by=step.size)
fragment.chr<-data.frame(chromosome=chr,start=starts,end=ends)
fragment<-rbind(fragment,fragment.chr)
}
if(mode=="overlapping"){
##Now getting the fragment#####
window.size=windowSize
step.size = window.size/2
ends <- c(seq(from=window.size,to=chr.size,by=step.size),as.integer(chr.size))
starts <- seq(from=window.size-step.size+1,to=chr.size,by=step.size)
fragment.chr<-data.frame(chromosome=chr,start=starts,end=ends)
fragment<-rbind(fragment,fragment.chr)
}
#}
}
fragment.GRanges<-GRanges(seqnames =fragment$chromosome,IRanges(start=fragment$start,end=fragment$end))
return(fragment.GRanges)
}else if(genome =="mm9"){
library(BSgenome.Mmusculus.UCSC.mm9.masked)
genome <- BSgenome.Mmusculus.UCSC.mm9.masked
fragment<-data.frame()
for (chr in paste('chr',c(seq(1,19),'X','Y'),sep='')){
chr.size<-seqlengths(genome)[chr]
#if(chr==viewpoint.chr){
# if(mode=="non-overlapping"){
# ##Now getting the fragment#####
# window.size=windowSize
# step.size = window.size
# start.p<-start(viewpoint)
# end.p<-end(viewpoint)
#
# f.ends<-(rev(seq(-start.p, -window.size,by=step.size)))*-1
# f.starts <- c(1,(rev(seq(-start.p+window.size,1,by=step.size)))*-1)
# f.starts<-f.starts[-(2)]
#
# s.ends <- c(seq(from=end.p+window.size,to=chr.size,by=step.size),as.integer(chr.size))
# s.starts <- seq(from=end.p,to=chr.size,by=step.size)
#
# f.fragment<-data.frame(chromosome=chr,start=f.starts,end=f.ends)
# s.fragment<-data.frame(chromosome=chr,start=s.starts,end=s.ends)
# fragment.chr<-rbind(f.fragment,s.fragment)
# fragment<-rbind(fragment,fragment.chr)
# }
# if(mode=="overlapping"){
# ##Now getting the fragment#####
# window.size=windowSize
# step.size = window.size/2
# start.p<-start(viewpoint)
# end.p<-end(viewpoint)
#
# f.ends<-(rev(seq(-start.p, -window.size,by=step.size)))*-1
# f.starts <- c(1,(rev(seq(-start.p+window.size,1,by=step.size)))*-1)
# f.starts<-f.starts[-(2)]
#
# s.ends <- c(seq(from=end.p+window.size,to=chr.size,by=step.size),as.integer(chr.size))
# s.starts <- seq(from=end.p,to=chr.size,by=step.size)
# s.starts<-s.starts[-(length(s.starts))]
#
# f.fragment<-data.frame(chromosome=chr,start=f.starts,end=f.ends)
# s.fragment<-data.frame(chromosome=chr,start=s.starts,end=s.ends)
# fragment.chr<-rbind(f.fragment,s.fragment)
# fragment<-rbind(fragment,fragment.chr)
# }
#}else{
if(mode=="non-overlapping"){
##Now getting the fragment#####
window.size=windowSize
step.size = window.size
ends <- c(seq(from=window.size,to=chr.size,by=step.size),as.integer(chr.size))
starts <- seq(from=1,to=chr.size,by=step.size)
fragment.chr<-data.frame(chromosome=chr,start=starts,end=ends)
fragment<-rbind(fragment,fragment.chr)
}
if(mode=="overlapping"){
##Now getting the fragment#####
window.size=windowSize
step.size = window.size/2
ends <- c(seq(from=window.size,to=chr.size,by=step.size),as.integer(chr.size))
starts <- seq(from=window.size-step.size+1,to=chr.size,by=step.size)
fragment.chr<-data.frame(chromosome=chr,start=starts,end=ends)
fragment<-rbind(fragment,fragment.chr)
}
#}
}
fragment.GRanges<-GRanges(seqnames = fragment$chromosome,IRanges(start=fragment$start,end=fragment$end))
return(fragment.GRanges)
}else if (genome =="mm10"){
library(BSgenome.Mmusculus.UCSC.mm10.masked)
genome <- BSgenome.Mmusculus.UCSC.mm10.masked
fragment<-data.frame()
for (chr in paste('chr',c(seq(1,19),'X','Y'),sep='')){
chr.size<-seqlengths(genome)[chr]
#if(chr==viewpoint.chr){
# if(mode=="non-overlapping"){
# ##Now getting the fragment#####
# window.size=windowSize
# step.size = window.size
# start.p<-start(viewpoint)
# end.p<-end(viewpoint)
#
# f.ends<-(rev(seq(-start.p, -window.size,by=step.size)))*-1
# f.starts <- c(1,(rev(seq(-start.p+window.size,1,by=step.size)))*-1)
# f.starts<-f.starts[-(2)]
#
# s.ends <- c(seq(from=end.p+window.size,to=chr.size,by=step.size),as.integer(chr.size))
# s.starts <- seq(from=end.p,to=chr.size,by=step.size)
#
# f.fragment<-data.frame(chromosome=chr,start=f.starts,end=f.ends)
# s.fragment<-data.frame(chromosome=chr,start=s.starts,end=s.ends)
# fragment.chr<-rbind(f.fragment,s.fragment)
# fragment<-rbind(fragment,fragment.chr)
# }
# if(mode=="overlapping"){
# ##Now getting the fragment#####
# window.size=windowSize
# step.size = window.size/2
# start.p<-start(viewpoint)
# end.p<-end(viewpoint)
#
# f.ends<-(rev(seq(-start.p, -window.size,by=step.size)))*-1
# f.starts <- c(1,(rev(seq(-start.p+window.size,1,by=step.size)))*-1)
# f.starts<-f.starts[-(2)]
#
# s.ends <- c(seq(from=end.p+window.size,to=chr.size,by=step.size),as.integer(chr.size))
# s.starts <- seq(from=end.p,to=chr.size,by=step.size)
# s.starts<-s.starts[-(length(s.starts))]
#
# f.fragment<-data.frame(chromosome=chr,start=f.starts,end=f.ends)
# s.fragment<-data.frame(chromosome=chr,start=s.starts,end=s.ends)
# fragment.chr<-rbind(f.fragment,s.fragment)
# fragment<-rbind(fragment,fragment.chr)
# }
#}else{
if(mode=="non-overlapping"){
##Now getting the fragment#####
window.size=windowSize
step.size = window.size
ends <- c(seq(from=window.size,to=chr.size,by=step.size),as.integer(chr.size))
starts <- seq(from=1,to=chr.size,by=step.size)
fragment.chr<-data.frame(chromosome=chr,start=starts,end=ends)
fragment<-rbind(fragment,fragment.chr)
}
if(mode=="overlapping"){
##Now getting the fragment#####
window.size=windowSize
step.size = window.size/2
ends <- c(seq(from=window.size,to=chr.size,by=step.size),as.integer(chr.size))
starts <- seq(from=window.size-step.size+1,to=chr.size,by=step.size)
fragment.chr<-data.frame(chromosome=chr,start=starts,end=ends)
fragment<-rbind(fragment,fragment.chr)
}
#}
}
fragment.GRanges<-GRanges(seqnames =fragment$chromosome,IRanges(start=fragment$start,end=fragment$end))
return(fragment.GRanges)
}else if (genome =="rn5"){
library(BSgenome.Rnorvegicus.UCSC.rn5.masked)
genome <- BSgenome.Rnorvegicus.UCSC.rn5.masked
fragment<-data.frame()
for (chr in paste('chr',c(seq(1,20),'X'),sep='')){
chr.size<-seqlengths(genome)[chr]
#if(chr==viewpoint.chr){
# if(mode=="non-overlapping"){
# ##Now getting the fragment#####
# window.size=windowSize
# step.size = window.size
# start.p<-start(viewpoint)
# end.p<-end(viewpoint)
#
# f.ends<-(rev(seq(-start.p, -window.size,by=step.size)))*-1
# f.starts <- c(1,(rev(seq(-start.p+window.size,1,by=step.size)))*-1)
# f.starts<-f.starts[-(2)]
#
# s.ends <- c(seq(from=end.p+window.size,to=chr.size,by=step.size),as.integer(chr.size))
# s.starts <- seq(from=end.p,to=chr.size,by=step.size)
#
# f.fragment<-data.frame(chromosome=chr,start=f.starts,end=f.ends)
# s.fragment<-data.frame(chromosome=chr,start=s.starts,end=s.ends)
# fragment.chr<-rbind(f.fragment,s.fragment)
# fragment<-rbind(fragment,fragment.chr)
# }
# if(mode=="overlapping"){
# ##Now getting the fragment#####
# window.size=windowSize
# step.size = window.size/2
# start.p<-start(viewpoint)
# end.p<-end(viewpoint)
#
# f.ends<-(rev(seq(-start.p, -window.size,by=step.size)))*-1
# f.starts <- c(1,(rev(seq(-start.p+window.size,1,by=step.size)))*-1)
# f.starts<-f.starts[-(2)]
#
# s.ends <- c(seq(from=end.p+window.size,to=chr.size,by=step.size),as.integer(chr.size))
# s.starts <- seq(from=end.p,to=chr.size,by=step.size)
# s.starts<-s.starts[-(length(s.starts))]
#
# f.fragment<-data.frame(chromosome=chr,start=f.starts,end=f.ends)
# s.fragment<-data.frame(chromosome=chr,start=s.starts,end=s.ends)
# fragment.chr<-rbind(f.fragment,s.fragment)
# fragment<-rbind(fragment,fragment.chr)
# }
#}else{
if(mode=="non-overlapping"){
##Now getting the fragment#####
window.size=windowSize
step.size = window.size
ends <- c(seq(from=window.size,to=chr.size,by=step.size),as.integer(chr.size))
starts <- seq(from=1,to=chr.size,by=step.size)
fragment.chr<-data.frame(chromosome=chr,start=starts,end=ends)
fragment<-rbind(fragment,fragment.chr)
}
if(mode=="overlapping"){
##Now getting the fragment#####
window.size=windowSize
step.size = window.size/2
ends <- c(seq(from=window.size,to=chr.size,by=step.size),as.integer(chr.size))
starts <- seq(from=window.size-step.size+1,to=chr.size,by=step.size)
fragment.chr<-data.frame(chromosome=chr,start=starts,end=ends)
fragment<-rbind(fragment,fragment.chr)
}
#}
}
fragment.GRanges<-GRanges(seqnames =fragment$chromosome,IRanges(start=fragment$start,end=fragment$end))
return(fragment.GRanges)
}else{
stop("Require the selected genome: hg18, hg19, mm9, mm10 or rn5.")
}
}
normalcalRPM<-function (x, lib.size){
seqDepth <-10^6
if(lib.size >=10^6){
seqDepth <- 10^6
}else if(lib.size >=10^5 && lib.size< 10^6){
seqDepth <- 10^5
}else if(lib.size >=10^4 && lib.size< 10^5 ){
seqDepth <- 10^4
}else if(lib.size >=10^3 && lib.size < 10^4){
seqDepth <-10^3
}else{
stop("This is too low sequencing depth!!!!!. We have to stop the analysis.")
}
RPM <- x / (lib.size/seqDepth)
return(RPM)
}
getPowerLawFittedCoeficient <- function(values) {
values<-values[values>=50]
v <- data.table(num = 1, nr_reads = as.integer(values))
v <- v[, sum(num), by = nr_reads]
setkey(v, nr_reads)
v$V1 <- rev(cumsum(rev(v$V1)))
setnames(v, c('nr_reads', 'reverse_cumulative'))
ranges<-quantile(v$nr_reads, c(.20,0.9998))
x<-as.vector(round(ranges))
v <- v[nr_reads >= min(x) & nr_reads <=max(x)]
lin.m <- lm(log(reverse_cumulative) ~ log(nr_reads), data = v)
a = coefficients(lin.m)[2]
b = coefficients(lin.m)[1]
return(c(a, b))
}
powerLawFittedRPM <- function(values,lin.reg.coef,alpha = 1.35, T = 10^6) {
a = lin.reg.coef[1]
b = lin.reg.coef[2]
lambda = (T/(zeta(alpha) * exp(b)))^(1/alpha)
beta = -1 * a/alpha
values.norm = lambda * (values)^beta
return(values.norm)
}
assign3CseqSigContact<-function(obj,readcount.ranges,smoothing.parameter,fdr){
stopifnot( is( obj, "r3Cseq" ) |is( obj, "r3CseqInBatch" ) )
if(length(readcount.ranges)>0){
viewpoint<-getViewpoint(obj)
viewpoint.chr<-viewpoint_chromosome(obj)
cis.ranges<-readcount.ranges[seqnames(readcount.ranges)==viewpoint.chr,]
cis<-data.frame(chromosome=seqnames(cis.ranges),
start=start(cis.ranges),end=end(cis.ranges),
relative.position=start(cis.ranges)-start(viewpoint),
observed=cis.ranges$nReads,rpm=cis.ranges$RPMs)
#######Smooth the data############
e.smooth<-vsmooth.spline(cis$relative.position,cis$observed,spar=smoothing.parameter)
e.fitted<-fitted(e.smooth)
cis$expected<-as.numeric(e.fitted)
######Calculate z-score and assign p-value in cis
cis$z<-(cis$observed-cis$expected)/sd(cis$observed-cis$expected)
cis$p.value<-2*pnorm(-abs(cis$z))
cis$p.value[cis$z < 0]<-1
cis$q.value<-qvalue(cis$p.value, fdr.level=fdr, pi0.method="bootstrap")$qvalues
cis.interaction<-data.frame(chromosome=cis$chromosome,start=cis$start,end=cis$end,nReads=cis$observed,RPMs=cis$rpm,z=cis$z,p.value=cis$p.value,q.value=cis$q.value)
########Calculate interaction in trans#####
ref1<-subset(cis,relative.position< -100000)
ref2<-subset(cis,relative.position> 100000)
ref_cis<-rbind(ref1,ref2)
ref_cis<-ref_cis[,c(1:3,5:6)]
ref_cis<-ref_cis[sample(1:nrow(ref_cis),0.5*nrow(ref_cis)),]
ref.trans.ranges<-readcount.ranges[seqnames(readcount.ranges)!=viewpoint.chr,]
ref.trans<-data.frame(chromosome=seqnames(ref.trans.ranges),start=start(ref.trans.ranges),end=end(ref.trans.ranges),observed=ref.trans.ranges$nReads,rpm=ref.trans.ranges$RPMs)
trans<-rbind(ref_cis,ref.trans)
trans$z<-(trans$observed-mean(trans$observed))/sd(trans$observed)
trans$p.value<-2*pnorm(-abs(trans$z))
trans$p.value[trans$z< 0]<-1
trans$q.value<-qvalue(trans$p.value, fdr.level=fdr, pi0.method="bootstrap")$qvalues
trans<-subset(trans,chromosome !=viewpoint.chr)
trans.interaction<-data.frame(chromosome=trans$chromosome,start=trans$start,end=trans$end,nReads=trans$observed,RPMs=trans$rpm,z=trans$z,p.value=trans$p.value,q.value=trans$q.value)
#######combined bothcis and trans results########
all.int<-rbind(cis.interaction,trans.interaction)
all.GRangedData<-GRanges(seqnames =all.int$chromosome,
IRanges(start=all.int$start,end=all.int$end),
nReads=all.int$nReads,
RPMs=all.int$RPMs,
z=all.int$z,
p.value=all.int$p.value,
q.value=all.int$q.value)
###Return RangedData object#######
return(all.GRangedData)
}else{
stop("No reads found, please reads the r3Cseq manual to create the read counts!!!.")
}
}
get3CseqRefGene<-function(obj){
stopifnot( is( obj, "r3Cseq" ) |is(obj, "r3CseqInBatch" ) )
####Get organism#######################
genome <- organismName(obj)
if(genome=="hg18"){
refGene.file<-system.file("data/hg18refGene.rdata", package="r3Cseq")
if(file.exists(refGene.file)==TRUE){
load(file=refGene.file)
refGene<-hg18refGene
return(refGene)
}else{
stop("Couldn't find hg18refGene.rdata")
}
}else if(genome=="hg19"){
refGene.file<-system.file("data/hg19refGene.rdata", package="r3Cseq")
if(file.exists(refGene.file)==TRUE){
load(file=refGene.file)
refGene<-hg19refGene
return(refGene)
}else{
stop("Couldn't find hg19refGene.rdata")
}
}else if(genome =="mm9"){
refGene.file<-system.file("data/mm9refGene.rdata", package="r3Cseq")
if(file.exists(refGene.file)==TRUE){
load(file=refGene.file)
refGene<-mm9refGene
return(refGene)
}else{
stop("Couldn't find mm9refGene.rdata")
}
}else if(genome =="mm10"){
refGene.file<-system.file("data/mm10refGene.rdata", package="r3Cseq")
if(file.exists(refGene.file)==TRUE){
load(file=refGene.file)
refGene<-mm10refGene
return(refGene)
}else{
stop("Couldn't find mm10refGene.rdata")
}
}else if(genome =="rn5"){
refGene.file<-system.file("data/rn5refGene.rdata", package="r3Cseq")
if(file.exists(refGene.file)==TRUE){
load(file=refGene.file)
refGene<-rn5refGene
return(refGene)
}else{
stop("Couldn't find rn5refGene.rdata")
}
}else{
stop("Require the selected genome: hg18, hg19, mm9, mm10 or rn5.")
}
}
makeInteractionMatrixNearCisPerWindow<-function(obj,smoothing.parameter,rawReads.Ranged,max.window=25e3,viewpoint,distanceFromViewpoint=5e5){
stopifnot( is( obj, "r3Cseq" ) |is( obj, "r3CseqInBatch" ) )
if('BSgenome.Hsapiens.UCSC.hg19.masked' %in% loadedNamespaces()==TRUE){
detach(package:BSgenome.Hsapiens.UCSC.hg19.masked,unload=TRUE)
}
if('BSgenome.Hsapiens.UCSC.hg18.masked' %in% loadedNamespaces()==TRUE){
detach(package:BSgenome.Hsapiens.UCSC.hg18.masked,unload=TRUE)
}
if('BSgenome.Mmusculus.UCSC.mm9.masked' %in% loadedNamespaces()==TRUE){
detach(package:BSgenome.Mmusculus.UCSC.mm9.masked,unload=TRUE)
}
if('BSgenome.Mmusculus.UCSC.mm10.masked' %in% loadedNamespaces()==TRUE){
detach(package:BSgenome.Mmusculus.UCSC.mm10.masked,unload=TRUE)
}
if('BSgenome.Rnorvegicus.UCSC.rn5.masked' %in% loadedNamespaces()==TRUE){
detach(package:BSgenome.Rnorvegicus.UCSC.rn5.masked,unload=TRUE)
}
###get raw reads
if(length(rawReads.Ranged)==0){
stop("No reads found, please run getRawReads function!!!.")
}
if(length(viewpoint)==0){
stop("No viewpoint, please run getVeiwpoint function to get the viewpoint !!!.")
}
viewpoint<-getViewpoint(obj)
viewpoint.chr <-as.character(seqnames(viewpoint))
rawReads.chr<-rawReads.Ranged[as.character(seqnames(rawReads.Ranged))==viewpoint.chr]
rawReads.filted<-excludeReadsNearViewpoint(obj,rawReads.chr,span_fragment=2)
#######get organism name ############
genome<-organismName(obj)
#####################################
chr.size<-0
if(genome=="hg19"){
library(BSgenome.Hsapiens.UCSC.hg19.masked)
genome <- BSgenome.Hsapiens.UCSC.hg19.masked
chr.size<-seqlengths(genome)[viewpoint.chr]
}else if(genome=="hg18"){
library(BSgenome.Hsapiens.UCSC.hg18.masked)
genome <- BSgenome.Hsapiens.UCSC.hg18.masked
chr.size<-seqlengths(genome)[viewpoint.chr]
}else if(genome =="mm9"){
library(BSgenome.Mmusculus.UCSC.mm9.masked)
genome <- BSgenome.Mmusculus.UCSC.mm9.masked
chr.size<-seqlengths(genome)[viewpoint.chr]
}else if(genome =="mm10"){
library(BSgenome.Mmusculus.UCSC.mm10.masked)
genome <- BSgenome.Mmusculus.UCSC.mm10.masked
chr.size<-seqlengths(genome)[viewpoint.chr]
}else if(genome =="rn5"){
library(BSgenome.Rnorvegicus.UCSC.rn5.masked)
genome <- BSgenome.Rnorvegicus.UCSC.rn5.masked
chr.size<-seqlengths(genome)[viewpoint.chr]
}else{
stop("Require the selected genome: hg18, hg19, mm9, mm10 or rn5")
}
r.start <-start(viewpoint)-distanceFromViewpoint
r.end <-end(viewpoint)+distanceFromViewpoint
r.size<-r.end-r.start+1
ref.position<-c(seq(from=1,to=r.size,by=1e3),r.size)
ref.v<-data.frame(position=ref.position)
print("Analyzing interaction regions for each window....")
for(window.size in rev(c(seq(from=2e3,to=max.window,by=1000)))){
fragment.GRanges<-GRanges()
step.size = window.size
ends <- c(seq(from=min(start(rawReads.filted))+window.size,to=chr.size,by=step.size),as.integer(chr.size))
starts <- seq(from=min(start(rawReads.filted)),to=chr.size,by=step.size)
fragment<-data.frame(chromosome=viewpoint.chr,start=starts,end=ends-1)
fragment.GRanges<-GRanges(seqnames =fragment$chromosome,IRanges(start=fragment$start,end=fragment$end))
readsPerFragment <- countOverlaps(fragment.GRanges,subject=rawReads.filted)
reads<-data.frame(nReads=readsPerFragment)
#####migrating the code to support the BioC 3.9#####
values(fragment.GRanges)<-reads
countTable<-fragment.GRanges
countTable<-countTable[countTable$nReads >0,]
cis<-data.frame(chromosome=as.character(seqnames(countTable)),
start=start(countTable),
end=end(countTable),
relative.position=start(countTable)-start(viewpoint),observed=countTable$nReads)
#######Smooth the data############
e.smooth<-vsmooth.spline(cis$relative.position,cis$observed,spar=smoothing.parameter)
e.fitted<-fitted(e.smooth)
cis$expected<-as.numeric(e.fitted)
######Calculate z-score and assign p-value in cis
cis$z<-(cis$observed-cis$expected)/sd(cis$observed-cis$expected)
cis$p.value<-2*pnorm(-abs(cis$z))
cis$p.value[cis$z < 0]<-1
cis$q.value<-qvalue(cis$p.value, fdr.level=0.05, pi0.method="bootstrap")$qvalues
cis.in<-GRanges(seqnames = as.character(cis$chromosome),
IRanges(start=cis$start,end=cis$end),
nReads=cis$observed,z=cis$z,p.value=cis$p.value,q.value=cis$q.value)
cis.in<-cis.in[start(cis.in) >=r.start & end(cis.in) <= r.end,]
cis.in$rel.start<-start(cis.in)-r.start
cis.in$rel.end<-end(cis.in)-r.start
##Creat vector of q-value##
my_vector<-rep(1,r.size)
my.IRanges<-IRanges(start=cis.in$rel.start,end=cis.in$rel.end)
#fix the IRanges problem here##########
#cis.p <-as.integer(my.IRanges)
cis.p <-as.integer(IPos(my.IRanges))
#######################################
my_vector[cis.p]<-c(rep(cis.in$q.value,each=window.size))
ref.value<-data.frame(q.value=my_vector[ref.position])
colnames(ref.value)<-paste("window.",window.size,sep="")
ref.v<-cbind(ref.v,ref.value)
}
return(ref.v)
print("The interaction matrix is created !!!.")
}
fishersMethod <-function(x) pchisq(-2 * sum(log(x)),df=2*length(x),lower.tail=FALSE)
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Defines functions fishersMethod makeInteractionMatrixNearCisPerWindow get3CseqRefGene assign3CseqSigContact powerLawFittedRPM getPowerLawFittedCoeficient
# TODO: These following functions were implemented to facilitate r3Cseq and r3CseqWithReplocates classes.
# 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
#####
getViewpoint<-function (obj){
stopifnot( is( obj, "r3Cseq" ) |is( obj, "r3CseqInBatch" ) )
#######get organism name ############
#######Fixed genome to genome.assembly
genome.assembly<-organismName(obj)
#######get forward primer############
primer_f<-viewpoint_primer_forward(obj)
primer_r<-viewpoint_primer_reverse(obj)
#######get viewpoint chromosome######
viewpoint_chr<-viewpoint_chromosome(obj)
#####################################
if('BSgenome.Hsapiens.UCSC.hg19.masked' %in% loadedNamespaces()==TRUE){
detach(package:BSgenome.Hsapiens.UCSC.hg19.masked,unload=TRUE)
}
if('BSgenome.Hsapiens.UCSC.hg18.masked' %in% loadedNamespaces()==TRUE){
detach(package:BSgenome.Hsapiens.UCSC.hg18.masked,unload=TRUE)
}
if('BSgenome.Mmusculus.UCSC.mm9.masked' %in% loadedNamespaces()==TRUE){
detach(package:BSgenome.Mmusculus.UCSC.mm9.masked,unload=TRUE)
}
if('BSgenome.Mmusculus.UCSC.mm10.masked' %in% loadedNamespaces()==TRUE){
detach(package:BSgenome.Mmusculus.UCSC.mm10.masked,unload=TRUE)
}
if('BSgenome.Rnorvegicus.UCSC.rn5.masked' %in% loadedNamespaces()==TRUE){
detach(package:BSgenome.Rnorvegicus.UCSC.rn5.masked,unload=TRUE)
}
if(genome.assembly=="hg18"){
library(BSgenome.Hsapiens.UCSC.hg18.masked)
genome <- BSgenome.Hsapiens.UCSC.hg18.masked
hits_f_f<-matchPattern(DNAString(primer_f),genome[[viewpoint_chr]],fixed=FALSE)
hits_f_r<-matchPattern(reverseComplement(DNAString(primer_f)),genome[[viewpoint_chr]],fixed=FALSE)
hits_f_positions<-c(start(hits_f_f),end(hits_f_f),start(hits_f_r),end(hits_f_r))
if(length(hits_f_positions)==0){
stop(paste("Could not find the forward primer position in",genome.assembly," :",viewpoint_chr, ". Please check the input forward primer"))
}
hits_r_f<-matchPattern(DNAString(primer_r),genome[[viewpoint_chr]],fixed=FALSE)
hits_r_r<-matchPattern(reverseComplement(DNAString(primer_r)),genome[[viewpoint_chr]],fixed=FALSE)
hits_r_positions<-c(start(hits_r_f),end(hits_r_f),start(hits_r_r),end(hits_r_r))
if(length(hits_r_positions)==0){
stop(paste("Could not find the reverse primer position in",genome.assembly," :",viewpoint_chr, ". Please check the input reverse primer"))
}
hit_positions<-c(hits_f_positions,hits_r_positions)
if(length(hit_positions)==4){
start_p<-min(hit_positions)
end_p<-max(hit_positions)
viewpoint<-GRanges(seqnames =as.character(viewpoint_chr),IRanges(start=start_p,end=end_p))
return(viewpoint)
}
if(length(hit_positions) > 4){
if(length(hits_r_positions) > 2 & length(hits_f_positions)==2){
rank.id<-order(abs(hits_r_positions-hits_f_positions))
hit_positions<-c(hits_f_positions,hits_r_positions[rank.id==1],hits_r_positions[rank.id==2])
start_p<-min(hit_positions)
end_p<-max(hit_positions)
viewpoint<-GRanges(seqnames=as.character(viewpoint_chr),IRanges(start=start_p,end=end_p))
return(viewpoint)
}
if(length(hits_f_positions) > 2 & length(hits_r_positions)==2){
rank.id<-order(abs(hits_f_positions-hits_r_positions))
hit_positions<-c(hits_f_positions[rank.id==1],hits_f_positions[rank.id==2],hits_r_positions)
start_p<-min(hit_positions)
end_p<-max(hit_positions)
viewpoint<-GRanges(seqnames=as.character(viewpoint_chr),IRanges(start=start_p,end=end_p))
return(viewpoint)
}
if(length(hits_f_positions) > 2 & length(hits_r_positions) > 2){
stop(paste("The position of your input primers are not unique. We detected multiple positions of your input primers!!!"))
}
}
}else if(genome.assembly=="hg19"){
library(BSgenome.Hsapiens.UCSC.hg19.masked)
genome <- BSgenome.Hsapiens.UCSC.hg19.masked
hits_f_f<-matchPattern(DNAString(primer_f),genome[[viewpoint_chr]],fixed=FALSE)
hits_f_r<-matchPattern(reverseComplement(DNAString(primer_f)),genome[[viewpoint_chr]],fixed=FALSE)
hits_f_positions<-c(start(hits_f_f),end(hits_f_f),start(hits_f_r),end(hits_f_r))
if(length(hits_f_positions)==0){
stop(paste("Could not find the forward primer position in",genome.assembly," :",viewpoint_chr, ". Please check the input forward primer"))
}
hits_r_f<-matchPattern(DNAString(primer_r),genome[[viewpoint_chr]],fixed=FALSE)
hits_r_r<-matchPattern(reverseComplement(DNAString(primer_r)),genome[[viewpoint_chr]],fixed=FALSE)
hits_r_positions<-c(start(hits_r_f),end(hits_r_f),start(hits_r_r),end(hits_r_r))
if(length(hits_r_positions)==0){
stop(paste("Could not find the reverse primer position in",genome.assembly," :",viewpoint_chr, ". Please check the input reverse primer"))
}
hit_positions<-c(hits_f_positions,hits_r_positions)
if(length(hit_positions)==4){
start_p<-min(hit_positions)
end_p<-max(hit_positions)
viewpoint<-GRanges(seqnames=as.character(viewpoint_chr),IRanges(start=start_p,end=end_p))
return(viewpoint)
}
if(length(hit_positions) > 4){
if(length(hits_r_positions) > 2 & length(hits_f_positions)==2){
rank.id<-order(abs(hits_r_positions-hits_f_positions))
hit_positions<-c(hits_f_positions,hits_r_positions[rank.id==1],hits_r_positions[rank.id==2])
start_p<-min(hit_positions)
end_p<-max(hit_positions)
viewpoint<-GRanges(seqnames=as.character(viewpoint_chr),IRanges(start=start_p,end=end_p))
return(viewpoint)
}
if(length(hits_f_positions) > 2 & length(hits_r_positions)==2){
rank.id<-order(abs(hits_f_positions-hits_r_positions))
hit_positions<-c(hits_f_positions[rank.id==1],hits_f_positions[rank.id==2],hits_r_positions)
start_p<-min(hit_positions)
end_p<-max(hit_positions)
viewpoint<-GRanges(seqnames=as.character(viewpoint_chr),IRanges(start=start_p,end=end_p))
return(viewpoint)
}
if(length(hits_f_positions) > 2 & length(hits_r_positions) > 2){
stop(paste("The position of your input primers are not unique. We detected multiple positions of your input primers!!!"))
}
}
}else if(genome.assembly =="mm9"){
library(BSgenome.Mmusculus.UCSC.mm9.masked)
genome <- BSgenome.Mmusculus.UCSC.mm9.masked
hits_f_f<-matchPattern(DNAString(primer_f),genome[[viewpoint_chr]],fixed=FALSE)
hits_f_r<-matchPattern(reverseComplement(DNAString(primer_f)),genome[[viewpoint_chr]],fixed=FALSE)
hits_f_positions<-c(start(hits_f_f),end(hits_f_f),start(hits_f_r),end(hits_f_r))
if(length(hits_f_positions)==0){
stop(paste("Could not find the forward primer position in",genome.assembly," :",viewpoint_chr, ". Please check the input forward primer"))
}
hits_r_f<-matchPattern(DNAString(primer_r),genome[[viewpoint_chr]],fixed=FALSE)
hits_r_r<-matchPattern(reverseComplement(DNAString(primer_r)),genome[[viewpoint_chr]],fixed=FALSE)
hits_r_positions<-c(start(hits_r_f),end(hits_r_f),start(hits_r_r),end(hits_r_r))
if(length(hits_r_positions)==0){
stop(paste("Could not find the reverse primer position in",genome.assembly," :",viewpoint_chr, ". Please check the input reverse primer"))
}
hit_positions<-c(hits_f_positions,hits_r_positions)
if(length(hit_positions)==4){
start_p<-min(hit_positions)
end_p<-max(hit_positions)
viewpoint<-GRanges(seqnames=as.character(viewpoint_chr),IRanges(start=start_p,end=end_p))
return(viewpoint)
}
if(length(hit_positions) > 4){
if(length(hits_r_positions) > 2 & length(hits_f_positions)==2){
rank.id<-order(abs(hits_r_positions-hits_f_positions))
hit_positions<-c(hits_f_positions,hits_r_positions[rank.id==1],hits_r_positions[rank.id==2])
start_p<-min(hit_positions)
end_p<-max(hit_positions)
viewpoint<-GRanges(seqnames=as.character(viewpoint_chr),IRanges(start=start_p,end=end_p))
return(viewpoint)
}
if(length(hits_f_positions) > 2 & length(hits_r_positions)==2){
rank.id<-order(abs(hits_f_positions-hits_r_positions))
hit_positions<-c(hits_f_positions[rank.id==1],hits_f_positions[rank.id==2],hits_r_positions)
start_p<-min(hit_positions)
end_p<-max(hit_positions)
viewpoint<-GRanges(seqnames=as.character(viewpoint_chr),IRanges(start=start_p,end=end_p))
return(viewpoint)
}
if(length(hits_f_positions) > 2 & length(hits_r_positions) > 2){
stop(paste("The position of your input primers are not unique. We detected multiple positions of your input primers!!!"))
}
}
}else if(genome.assembly =="mm10"){
library(BSgenome.Mmusculus.UCSC.mm10.masked)
genome <- BSgenome.Mmusculus.UCSC.mm10.masked
hits_f_f<-matchPattern(DNAString(primer_f),genome[[viewpoint_chr]],fixed=FALSE)
hits_f_r<-matchPattern(reverseComplement(DNAString(primer_f)),genome[[viewpoint_chr]],fixed=FALSE)
hits_f_positions<-c(start(hits_f_f),end(hits_f_f),start(hits_f_r),end(hits_f_r))
if(length(hits_f_positions)==0){
stop(paste("Could not find the forward primer position in",genome.assembly," :",viewpoint_chr, ". Please check the input forward primer"))
}
hits_r_f<-matchPattern(DNAString(primer_r),genome[[viewpoint_chr]],fixed=FALSE)
hits_r_r<-matchPattern(reverseComplement(DNAString(primer_r)),genome[[viewpoint_chr]],fixed=FALSE)
hits_r_positions<-c(start(hits_r_f),end(hits_r_f),start(hits_r_r),end(hits_r_r))
if(length(hits_r_positions)==0){
stop(paste("Could not find the reverse primer position in",genome.assembly," :",viewpoint_chr, ". Please check the input reverse primer"))
}
hit_positions<-c(hits_f_positions,hits_r_positions)
if(length(hit_positions)==4){
start_p<-min(hit_positions)
end_p<-max(hit_positions)
viewpoint<-GRanges(seqnames=as.character(viewpoint_chr),IRanges(start=start_p,end=end_p))
return(viewpoint)
}
if(length(hit_positions) > 4){
if(length(hits_r_positions) > 2 & length(hits_f_positions)==2){
rank.id<-order(abs(hits_r_positions-hits_f_positions))
hit_positions<-c(hits_f_positions,hits_r_positions[rank.id==1],hits_r_positions[rank.id==2])
start_p<-min(hit_positions)
end_p<-max(hit_positions)
viewpoint<-GRanges(seqnames=as.character(viewpoint_chr),IRanges(start=start_p,end=end_p))
return(viewpoint)
}
if(length(hits_f_positions) > 2 & length(hits_r_positions)==2){
rank.id<-order(abs(hits_f_positions-hits_r_positions))
hit_positions<-c(hits_f_positions[rank.id==1],hits_f_positions[rank.id==2],hits_r_positions)
start_p<-min(hit_positions)
end_p<-max(hit_positions)
viewpoint<-GRanges(seqnames=as.character(viewpoint_chr),IRanges(start=start_p,end=end_p))
return(viewpoint)
}
if(length(hits_f_positions) > 2 & length(hits_r_positions) > 2){
stop(paste("The position of your input primers are not unique. We detected multiple positions of your input primers!!!"))
}
}
}else if(genome.assembly =="rn5"){
library(BSgenome.Rnorvegicus.UCSC.rn5.masked)
genome <- BSgenome.Rnorvegicus.UCSC.rn5.masked
hits_f_f<-matchPattern(DNAString(primer_f),genome[[viewpoint_chr]],fixed=FALSE)
hits_f_r<-matchPattern(reverseComplement(DNAString(primer_f)),genome[[viewpoint_chr]],fixed=FALSE)
hits_f_positions<-c(start(hits_f_f),end(hits_f_f),start(hits_f_r),end(hits_f_r))
if(length(hits_f_positions)==0){
stop(paste("Could not find the forward primer position in",genome.assembly," :",viewpoint_chr, ". Please check the input forward primer"))
}
hits_r_f<-matchPattern(DNAString(primer_r),genome[[viewpoint_chr]],fixed=FALSE)
hits_r_r<-matchPattern(reverseComplement(DNAString(primer_r)),genome[[viewpoint_chr]],fixed=FALSE)
hits_r_positions<-c(start(hits_r_f),end(hits_r_f),start(hits_r_r),end(hits_r_r))
if(length(hits_r_positions)==0){
stop(paste("Could not find the reverse primer position in",genome.assembly," :",viewpoint_chr, ". Please check the input reverse primer"))
}
hit_positions<-c(hits_f_positions,hits_r_positions)
if(length(hit_positions)==4){
start_p<-min(hit_positions)
end_p<-max(hit_positions)
viewpoint<-GRanges(seqnames=as.character(viewpoint_chr),IRanges(start=start_p,end=end_p))
return(viewpoint)
}
if(length(hit_positions) > 4){
if(length(hits_r_positions) > 2 & length(hits_f_positions)==2){
rank.id<-order(abs(hits_r_positions-hits_f_positions))
hit_positions<-c(hits_f_positions,hits_r_positions[rank.id==1],hits_r_positions[rank.id==2])
start_p<-min(hit_positions)
end_p<-max(hit_positions)
viewpoint<-GRanges(seqnames=as.character(viewpoint_chr),IRanges(start=start_p,end=end_p))
return(viewpoint)
}
if(length(hits_f_positions) > 2 & length(hits_r_positions)==2){
rank.id<-order(abs(hits_f_positions-hits_r_positions))
hit_positions<-c(hits_f_positions[rank.id==1],hits_f_positions[rank.id==2],hits_r_positions)
start_p<-min(hit_positions)
end_p<-max(hit_positions)
viewpoint<-GRanges(seqnames=as.character(viewpoint_chr),IRanges(start=start_p,end=end_p))
return(viewpoint)
}
if(length(hits_f_positions) > 2 & length(hits_r_positions) > 2){
stop(paste("The position of your input primers are not unique. We detected multiple positions of your input primers!!!"))
}
}
}else{
stop("Require the selected genome: hg18, hg19, mm9, mm10 or rn5.")
}
}
excludeReadsNearViewpoint<-function (object,rawReadsGRanges,span_fragment=1){
if(span_fragment <0 | span_fragment >5){
stop("Span fragment number from the viewpoint must be in the range of 0-5 fragment.")
}
if(length(rawReadsGRanges)==0){
stop("No reads can be found!!!, please run 'getRawReads' function in order to get raw reads.")
}
#####get viewpoint##############
viewpoint<-getViewpoint(object)
######get restriction fragment##
orgName <- organismName(object)
####Initialized Restriction Enzyme Object##
enzymeDb <-new("repbaseEnzyme")
resEnzyme <-restrictionEnzyme(object)
####Build GRanges for restriction site#####
fragments<-getRestrictionFragments(enzymeDb,resEnzyme,orgName,as.character(seqnames(viewpoint)))
fragments$distance2viewpoint<-abs(fragments$end-start(viewpoint))
v.index<-which(fragments$distance2viewpoint==min(fragments$distance2viewpoint))
v.index<-v.index[1]
f.index<-c((v.index-span_fragment):(v.index+span_fragment))
selected.f<-fragments[f.index,]
expected.regions<-GRanges(seqnames =viewpoint_chromosome(object),IRanges(start=min(selected.f$start),end=max(selected.f$end)))
excluded.index <- subjectHits(findOverlaps(expected.regions,subject=rawReadsGRanges))
new.GRanges<-rawReadsGRanges[-(excluded.index)]
return(new.GRanges)
}
getFragmentsPerWindow<-function (obj,windowSize=5e3,mode){
stopifnot( is( obj, "r3Cseq" ) |is( obj, "r3CseqInBatch" ) )
if(windowSize <2000 | windowSize >100000){
stop("Please select window size between 2Kb - 100Kb")
}
if(mode==""){
mode<-"non-overlapping"
}
#######get organism name ############
genome<-organismName(obj)
#####################################
viewpoint<-getViewpoint(obj)
viewpoint.chr<-viewpoint_chromosome(obj)
#####################################
if('BSgenome.Hsapiens.UCSC.hg19.masked' %in% loadedNamespaces()==TRUE){
detach(package:BSgenome.Hsapiens.UCSC.hg19.masked,unload=TRUE)
}
if('BSgenome.Hsapiens.UCSC.hg18.masked' %in% loadedNamespaces()==TRUE){
detach(package:BSgenome.Hsapiens.UCSC.hg18.masked,unload=TRUE)
}
if('BSgenome.Mmusculus.UCSC.mm9.masked' %in% loadedNamespaces()==TRUE){
detach(package:BSgenome.Mmusculus.UCSC.mm9.masked,unload=TRUE)
}
if('BSgenome.Mmusculus.UCSC.mm10.masked' %in% loadedNamespaces()==TRUE){
detach(package:BSgenome.Mmusculus.UCSC.mm10.masked,unload=TRUE)
}
if('BSgenome.Rnorvegicus.UCSC.rn5.masked' %in% loadedNamespaces()==TRUE){
detach(package:BSgenome.Rnorvegicus.UCSC.rn5.masked,unload=TRUE)
}
if(genome=="hg18"){
library(BSgenome.Hsapiens.UCSC.hg18.masked)
genome <- BSgenome.Hsapiens.UCSC.hg18.masked
fragment<-data.frame()
for (chr in paste('chr',c(seq(1,22),'X','Y'),sep='')){
chr.size<-seqlengths(genome)[chr]
#if(chr==viewpoint.chr){
# if(mode=="non-overlapping"){
# ##Now getting the fragment#####
# window.size=windowSize
# step.size = window.size
# start.p<-start(viewpoint)
# end.p<-end(viewpoint)
#
# f.ends<-(rev(seq(-start.p, -window.size,by=step.size)))*-1
# f.starts <- c(1,(rev(seq(-start.p+window.size,1,by=step.size)))*-1)
# f.starts<-f.starts[-(2)]
#
# s.ends <- c(seq(from=end.p+window.size,to=chr.size,by=step.size),as.integer(chr.size))
# s.starts <- seq(from=end.p,to=chr.size,by=step.size)
#
# f.fragment<-data.frame(chromosome=chr,start=f.starts,end=f.ends)
# s.fragment<-data.frame(chromosome=chr,start=s.starts,end=s.ends)
# fragment.chr<-rbind(f.fragment,s.fragment)
# fragment<-rbind(fragment,fragment.chr)
# }
# if(mode=="overlapping"){
# ##Now getting the fragment#####
# window.size=windowSize
# step.size = window.size/2
# start.p<-start(viewpoint)
# end.p<-end(viewpoint)
#
# f.ends<-(rev(seq(-start.p, -window.size,by=step.size)))*-1
# f.starts <- c(1,(rev(seq(-start.p+window.size,1,by=step.size)))*-1)
# f.starts<-f.starts[-(2)]
#
# s.ends <- c(seq(from=end.p+window.size,to=chr.size,by=step.size),as.integer(chr.size))
# s.starts <- seq(from=end.p,to=chr.size,by=step.size)
# s.starts<-s.starts[-(length(s.starts))]
#
# f.fragment<-data.frame(chromosome=chr,start=f.starts,end=f.ends)
# s.fragment<-data.frame(chromosome=chr,start=s.starts,end=s.ends)
#
#
# fragment.chr<-rbind(f.fragment,s.fragment)
# fragment<-rbind(fragment,fragment.chr)
# }
#}else{
if(mode=="non-overlapping"){
##Now getting the fragment#####
window.size=windowSize
step.size = window.size
ends <- c(seq(from=window.size,to=chr.size,by=step.size),as.integer(chr.size))
starts <- seq(from=1,to=chr.size,by=step.size)
fragment.chr<-data.frame(chromosome=chr,start=starts,end=ends)
fragment<-rbind(fragment,fragment.chr)
}
if(mode=="overlapping"){
##Now getting the fragment#####
window.size=windowSize
step.size = window.size/2
ends <- c(seq(from=window.size,to=chr.size,by=step.size),as.integer(chr.size))
starts <- seq(from=window.size-step.size+1,to=chr.size,by=step.size)
fragment.chr<-data.frame(chromosome=chr,start=starts,end=ends)
fragment<-rbind(fragment,fragment.chr)
}
#}
}
fragment.GRanges<-GRanges(seqnames =fragment$chromosome,IRanges(start=fragment$start,end=fragment$end))
return(fragment.GRanges)
}else if(genome=="hg19"){
library(BSgenome.Hsapiens.UCSC.hg19.masked)
genome <- BSgenome.Hsapiens.UCSC.hg19.masked
fragment<-data.frame()
for (chr in paste('chr',c(seq(1,22),'X','Y'),sep='')){
chr.size<-seqlengths(genome)[chr]
#if(chr==viewpoint.chr){
# if(mode=="non-overlapping"){
# ##Now getting the fragment#####
# window.size=windowSize
# step.size = window.size
# start.p<-start(viewpoint)
# end.p<-end(viewpoint)
#
# f.ends<-(rev(seq(-start.p, -window.size,by=step.size)))*-1
# f.starts <- c(1,(rev(seq(-start.p+window.size,1,by=step.size)))*-1)
# f.starts<-f.starts[-(2)]
#
# s.ends <- c(seq(from=end.p+window.size,to=chr.size,by=step.size),as.integer(chr.size))
# s.starts <- seq(from=end.p,to=chr.size,by=step.size)
#
# f.fragment<-data.frame(chromosome=chr,start=f.starts,end=f.ends)
# s.fragment<-data.frame(chromosome=chr,start=s.starts,end=s.ends)
# fragment.chr<-rbind(f.fragment,s.fragment)
# fragment<-rbind(fragment,fragment.chr)
# }
# if(mode=="overlapping"){
# ##Now getting the fragment#####
# window.size=windowSize
# step.size = window.size/2
# start.p<-start(viewpoint)
# end.p<-end(viewpoint)
#
# f.ends<-(rev(seq(-start.p, -window.size,by=step.size)))*-1
# f.starts <- c(1,(rev(seq(-start.p+window.size,1,by=step.size)))*-1)
# f.starts<-f.starts[-(2)]
#
# s.ends <- c(seq(from=end.p+window.size,to=chr.size,by=step.size),as.integer(chr.size))
# s.starts <- seq(from=end.p,to=chr.size,by=step.size)
# s.starts<-s.starts[-(length(s.starts))]
#
# f.fragment<-data.frame(chromosome=chr,start=f.starts,end=f.ends)
# s.fragment<-data.frame(chromosome=chr,start=s.starts,end=s.ends)
#
#
# fragment.chr<-rbind(f.fragment,s.fragment)
# fragment<-rbind(fragment,fragment.chr)
# }
#}else{
if(mode=="non-overlapping"){
##Now getting the fragment#####
window.size=windowSize
step.size = window.size
ends <- c(seq(from=window.size,to=chr.size,by=step.size),as.integer(chr.size))
starts <- seq(from=1,to=chr.size,by=step.size)
fragment.chr<-data.frame(chromosome=chr,start=starts,end=ends)
fragment<-rbind(fragment,fragment.chr)
}
if(mode=="overlapping"){
##Now getting the fragment#####
window.size=windowSize
step.size = window.size/2
ends <- c(seq(from=window.size,to=chr.size,by=step.size),as.integer(chr.size))
starts <- seq(from=window.size-step.size+1,to=chr.size,by=step.size)
fragment.chr<-data.frame(chromosome=chr,start=starts,end=ends)
fragment<-rbind(fragment,fragment.chr)
}
#}
}
fragment.GRanges<-GRanges(seqnames =fragment$chromosome,IRanges(start=fragment$start,end=fragment$end))
return(fragment.GRanges)
}else if(genome =="mm9"){
library(BSgenome.Mmusculus.UCSC.mm9.masked)
genome <- BSgenome.Mmusculus.UCSC.mm9.masked
fragment<-data.frame()
for (chr in paste('chr',c(seq(1,19),'X','Y'),sep='')){
chr.size<-seqlengths(genome)[chr]
#if(chr==viewpoint.chr){
# if(mode=="non-overlapping"){
# ##Now getting the fragment#####
# window.size=windowSize
# step.size = window.size
# start.p<-start(viewpoint)
# end.p<-end(viewpoint)
#
# f.ends<-(rev(seq(-start.p, -window.size,by=step.size)))*-1
# f.starts <- c(1,(rev(seq(-start.p+window.size,1,by=step.size)))*-1)
# f.starts<-f.starts[-(2)]
#
# s.ends <- c(seq(from=end.p+window.size,to=chr.size,by=step.size),as.integer(chr.size))
# s.starts <- seq(from=end.p,to=chr.size,by=step.size)
#
# f.fragment<-data.frame(chromosome=chr,start=f.starts,end=f.ends)
# s.fragment<-data.frame(chromosome=chr,start=s.starts,end=s.ends)
# fragment.chr<-rbind(f.fragment,s.fragment)
# fragment<-rbind(fragment,fragment.chr)
# }
# if(mode=="overlapping"){
# ##Now getting the fragment#####
# window.size=windowSize
# step.size = window.size/2
# start.p<-start(viewpoint)
# end.p<-end(viewpoint)
#
# f.ends<-(rev(seq(-start.p, -window.size,by=step.size)))*-1
# f.starts <- c(1,(rev(seq(-start.p+window.size,1,by=step.size)))*-1)
# f.starts<-f.starts[-(2)]
#
# s.ends <- c(seq(from=end.p+window.size,to=chr.size,by=step.size),as.integer(chr.size))
# s.starts <- seq(from=end.p,to=chr.size,by=step.size)
# s.starts<-s.starts[-(length(s.starts))]
#
# f.fragment<-data.frame(chromosome=chr,start=f.starts,end=f.ends)
# s.fragment<-data.frame(chromosome=chr,start=s.starts,end=s.ends)
# fragment.chr<-rbind(f.fragment,s.fragment)
# fragment<-rbind(fragment,fragment.chr)
# }
#}else{
if(mode=="non-overlapping"){
##Now getting the fragment#####
window.size=windowSize
step.size = window.size
ends <- c(seq(from=window.size,to=chr.size,by=step.size),as.integer(chr.size))
starts <- seq(from=1,to=chr.size,by=step.size)
fragment.chr<-data.frame(chromosome=chr,start=starts,end=ends)
fragment<-rbind(fragment,fragment.chr)
}
if(mode=="overlapping"){
##Now getting the fragment#####
window.size=windowSize
step.size = window.size/2
ends <- c(seq(from=window.size,to=chr.size,by=step.size),as.integer(chr.size))
starts <- seq(from=window.size-step.size+1,to=chr.size,by=step.size)
fragment.chr<-data.frame(chromosome=chr,start=starts,end=ends)
fragment<-rbind(fragment,fragment.chr)
}
#}
}
fragment.GRanges<-GRanges(seqnames = fragment$chromosome,IRanges(start=fragment$start,end=fragment$end))
return(fragment.GRanges)
}else if (genome =="mm10"){
library(BSgenome.Mmusculus.UCSC.mm10.masked)
genome <- BSgenome.Mmusculus.UCSC.mm10.masked
fragment<-data.frame()
for (chr in paste('chr',c(seq(1,19),'X','Y'),sep='')){
chr.size<-seqlengths(genome)[chr]
#if(chr==viewpoint.chr){
# if(mode=="non-overlapping"){
# ##Now getting the fragment#####
# window.size=windowSize
# step.size = window.size
# start.p<-start(viewpoint)
# end.p<-end(viewpoint)
#
# f.ends<-(rev(seq(-start.p, -window.size,by=step.size)))*-1
# f.starts <- c(1,(rev(seq(-start.p+window.size,1,by=step.size)))*-1)
# f.starts<-f.starts[-(2)]
#
# s.ends <- c(seq(from=end.p+window.size,to=chr.size,by=step.size),as.integer(chr.size))
# s.starts <- seq(from=end.p,to=chr.size,by=step.size)
#
# f.fragment<-data.frame(chromosome=chr,start=f.starts,end=f.ends)
# s.fragment<-data.frame(chromosome=chr,start=s.starts,end=s.ends)
# fragment.chr<-rbind(f.fragment,s.fragment)
# fragment<-rbind(fragment,fragment.chr)
# }
# if(mode=="overlapping"){
# ##Now getting the fragment#####
# window.size=windowSize
# step.size = window.size/2
# start.p<-start(viewpoint)
# end.p<-end(viewpoint)
#
# f.ends<-(rev(seq(-start.p, -window.size,by=step.size)))*-1
# f.starts <- c(1,(rev(seq(-start.p+window.size,1,by=step.size)))*-1)
# f.starts<-f.starts[-(2)]
#
# s.ends <- c(seq(from=end.p+window.size,to=chr.size,by=step.size),as.integer(chr.size))
# s.starts <- seq(from=end.p,to=chr.size,by=step.size)
# s.starts<-s.starts[-(length(s.starts))]
#
# f.fragment<-data.frame(chromosome=chr,start=f.starts,end=f.ends)
# s.fragment<-data.frame(chromosome=chr,start=s.starts,end=s.ends)
# fragment.chr<-rbind(f.fragment,s.fragment)
# fragment<-rbind(fragment,fragment.chr)
# }
#}else{
if(mode=="non-overlapping"){
##Now getting the fragment#####
window.size=windowSize
step.size = window.size
ends <- c(seq(from=window.size,to=chr.size,by=step.size),as.integer(chr.size))
starts <- seq(from=1,to=chr.size,by=step.size)
fragment.chr<-data.frame(chromosome=chr,start=starts,end=ends)
fragment<-rbind(fragment,fragment.chr)
}
if(mode=="overlapping"){
##Now getting the fragment#####
window.size=windowSize
step.size = window.size/2
ends <- c(seq(from=window.size,to=chr.size,by=step.size),as.integer(chr.size))
starts <- seq(from=window.size-step.size+1,to=chr.size,by=step.size)
fragment.chr<-data.frame(chromosome=chr,start=starts,end=ends)
fragment<-rbind(fragment,fragment.chr)
}
#}
}
fragment.GRanges<-GRanges(seqnames =fragment$chromosome,IRanges(start=fragment$start,end=fragment$end))
return(fragment.GRanges)
}else if (genome =="rn5"){
library(BSgenome.Rnorvegicus.UCSC.rn5.masked)
genome <- BSgenome.Rnorvegicus.UCSC.rn5.masked
fragment<-data.frame()
for (chr in paste('chr',c(seq(1,20),'X'),sep='')){
chr.size<-seqlengths(genome)[chr]
#if(chr==viewpoint.chr){
# if(mode=="non-overlapping"){
# ##Now getting the fragment#####
# window.size=windowSize
# step.size = window.size
# start.p<-start(viewpoint)
# end.p<-end(viewpoint)
#
# f.ends<-(rev(seq(-start.p, -window.size,by=step.size)))*-1
# f.starts <- c(1,(rev(seq(-start.p+window.size,1,by=step.size)))*-1)
# f.starts<-f.starts[-(2)]
#
# s.ends <- c(seq(from=end.p+window.size,to=chr.size,by=step.size),as.integer(chr.size))
# s.starts <- seq(from=end.p,to=chr.size,by=step.size)
#
# f.fragment<-data.frame(chromosome=chr,start=f.starts,end=f.ends)
# s.fragment<-data.frame(chromosome=chr,start=s.starts,end=s.ends)
# fragment.chr<-rbind(f.fragment,s.fragment)
# fragment<-rbind(fragment,fragment.chr)
# }
# if(mode=="overlapping"){
# ##Now getting the fragment#####
# window.size=windowSize
# step.size = window.size/2
# start.p<-start(viewpoint)
# end.p<-end(viewpoint)
#
# f.ends<-(rev(seq(-start.p, -window.size,by=step.size)))*-1
# f.starts <- c(1,(rev(seq(-start.p+window.size,1,by=step.size)))*-1)
# f.starts<-f.starts[-(2)]
#
# s.ends <- c(seq(from=end.p+window.size,to=chr.size,by=step.size),as.integer(chr.size))
# s.starts <- seq(from=end.p,to=chr.size,by=step.size)
# s.starts<-s.starts[-(length(s.starts))]
#
# f.fragment<-data.frame(chromosome=chr,start=f.starts,end=f.ends)
# s.fragment<-data.frame(chromosome=chr,start=s.starts,end=s.ends)
# fragment.chr<-rbind(f.fragment,s.fragment)
# fragment<-rbind(fragment,fragment.chr)
# }
#}else{
if(mode=="non-overlapping"){
##Now getting the fragment#####
window.size=windowSize
step.size = window.size
ends <- c(seq(from=window.size,to=chr.size,by=step.size),as.integer(chr.size))
starts <- seq(from=1,to=chr.size,by=step.size)
fragment.chr<-data.frame(chromosome=chr,start=starts,end=ends)
fragment<-rbind(fragment,fragment.chr)
}
if(mode=="overlapping"){
##Now getting the fragment#####
window.size=windowSize
step.size = window.size/2
ends <- c(seq(from=window.size,to=chr.size,by=step.size),as.integer(chr.size))
starts <- seq(from=window.size-step.size+1,to=chr.size,by=step.size)
fragment.chr<-data.frame(chromosome=chr,start=starts,end=ends)
fragment<-rbind(fragment,fragment.chr)
}
#}
}
fragment.GRanges<-GRanges(seqnames =fragment$chromosome,IRanges(start=fragment$start,end=fragment$end))
return(fragment.GRanges)
}else{
stop("Require the selected genome: hg18, hg19, mm9, mm10 or rn5.")
}
}
normalcalRPM<-function (x, lib.size){
seqDepth <-10^6
if(lib.size >=10^6){
seqDepth <- 10^6
}else if(lib.size >=10^5 && lib.size< 10^6){
seqDepth <- 10^5
}else if(lib.size >=10^4 && lib.size< 10^5 ){
seqDepth <- 10^4
}else if(lib.size >=10^3 && lib.size < 10^4){
seqDepth <-10^3
}else{
stop("This is too low sequencing depth!!!!!. We have to stop the analysis.")
}
RPM <- x / (lib.size/seqDepth)
return(RPM)
}
getPowerLawFittedCoeficient <- function(values) {
values<-values[values>=50]
v <- data.table(num = 1, nr_reads = as.integer(values))
v <- v[, sum(num), by = nr_reads]
setkey(v, nr_reads)
v$V1 <- rev(cumsum(rev(v$V1)))
setnames(v, c('nr_reads', 'reverse_cumulative'))
ranges<-quantile(v$nr_reads, c(.20,0.9998))
x<-as.vector(round(ranges))
v <- v[nr_reads >= min(x) & nr_reads <=max(x)]
lin.m <- lm(log(reverse_cumulative) ~ log(nr_reads), data = v)
a = coefficients(lin.m)[2]
b = coefficients(lin.m)[1]
return(c(a, b))
}
powerLawFittedRPM <- function(values,lin.reg.coef,alpha = 1.35, T = 10^6) {
a = lin.reg.coef[1]
b = lin.reg.coef[2]
lambda = (T/(zeta(alpha) * exp(b)))^(1/alpha)
beta = -1 * a/alpha
values.norm = lambda * (values)^beta
return(values.norm)
}
assign3CseqSigContact<-function(obj,readcount.ranges,smoothing.parameter,fdr){
stopifnot( is( obj, "r3Cseq" ) |is( obj, "r3CseqInBatch" ) )
if(length(readcount.ranges)>0){
viewpoint<-getViewpoint(obj)
viewpoint.chr<-viewpoint_chromosome(obj)
cis.ranges<-readcount.ranges[seqnames(readcount.ranges)==viewpoint.chr,]
cis<-data.frame(chromosome=seqnames(cis.ranges),
start=start(cis.ranges),end=end(cis.ranges),
relative.position=start(cis.ranges)-start(viewpoint),
observed=cis.ranges$nReads,rpm=cis.ranges$RPMs)
#######Smooth the data############
e.smooth<-vsmooth.spline(cis$relative.position,cis$observed,spar=smoothing.parameter)
e.fitted<-fitted(e.smooth)
cis$expected<-as.numeric(e.fitted)
######Calculate z-score and assign p-value in cis
cis$z<-(cis$observed-cis$expected)/sd(cis$observed-cis$expected)
cis$p.value<-2*pnorm(-abs(cis$z))
cis$p.value[cis$z < 0]<-1
cis$q.value<-qvalue(cis$p.value, fdr.level=fdr, pi0.method="bootstrap")$qvalues
cis.interaction<-data.frame(chromosome=cis$chromosome,start=cis$start,end=cis$end,nReads=cis$observed,RPMs=cis$rpm,z=cis$z,p.value=cis$p.value,q.value=cis$q.value)
########Calculate interaction in trans#####
ref1<-subset(cis,relative.position< -100000)
ref2<-subset(cis,relative.position> 100000)
ref_cis<-rbind(ref1,ref2)
ref_cis<-ref_cis[,c(1:3,5:6)]
ref_cis<-ref_cis[sample(1:nrow(ref_cis),0.5*nrow(ref_cis)),]
ref.trans.ranges<-readcount.ranges[seqnames(readcount.ranges)!=viewpoint.chr,]
ref.trans<-data.frame(chromosome=seqnames(ref.trans.ranges),start=start(ref.trans.ranges),end=end(ref.trans.ranges),observed=ref.trans.ranges$nReads,rpm=ref.trans.ranges$RPMs)
trans<-rbind(ref_cis,ref.trans)
trans$z<-(trans$observed-mean(trans$observed))/sd(trans$observed)
trans$p.value<-2*pnorm(-abs(trans$z))
trans$p.value[trans$z< 0]<-1
trans$q.value<-qvalue(trans$p.value, fdr.level=fdr, pi0.method="bootstrap")$qvalues
trans<-subset(trans,chromosome !=viewpoint.chr)
trans.interaction<-data.frame(chromosome=trans$chromosome,start=trans$start,end=trans$end,nReads=trans$observed,RPMs=trans$rpm,z=trans$z,p.value=trans$p.value,q.value=trans$q.value)
#######combined bothcis and trans results########
all.int<-rbind(cis.interaction,trans.interaction)
all.GRangedData<-GRanges(seqnames =all.int$chromosome,
IRanges(start=all.int$start,end=all.int$end),
nReads=all.int$nReads,
RPMs=all.int$RPMs,
z=all.int$z,
p.value=all.int$p.value,
q.value=all.int$q.value)
###Return RangedData object#######
return(all.GRangedData)
}else{
stop("No reads found, please reads the r3Cseq manual to create the read counts!!!.")
}
}
get3CseqRefGene<-function(obj){
stopifnot( is( obj, "r3Cseq" ) |is(obj, "r3CseqInBatch" ) )
####Get organism#######################
genome <- organismName(obj)
if(genome=="hg18"){
refGene.file<-system.file("data/hg18refGene.rdata", package="r3Cseq")
if(file.exists(refGene.file)==TRUE){
load(file=refGene.file)
refGene<-hg18refGene
return(refGene)
}else{
stop("Couldn't find hg18refGene.rdata")
}
}else if(genome=="hg19"){
refGene.file<-system.file("data/hg19refGene.rdata", package="r3Cseq")
if(file.exists(refGene.file)==TRUE){
load(file=refGene.file)
refGene<-hg19refGene
return(refGene)
}else{
stop("Couldn't find hg19refGene.rdata")
}
}else if(genome =="mm9"){
refGene.file<-system.file("data/mm9refGene.rdata", package="r3Cseq")
if(file.exists(refGene.file)==TRUE){
load(file=refGene.file)
refGene<-mm9refGene
return(refGene)
}else{
stop("Couldn't find mm9refGene.rdata")
}
}else if(genome =="mm10"){
refGene.file<-system.file("data/mm10refGene.rdata", package="r3Cseq")
if(file.exists(refGene.file)==TRUE){
load(file=refGene.file)
refGene<-mm10refGene
return(refGene)
}else{
stop("Couldn't find mm10refGene.rdata")
}
}else if(genome =="rn5"){
refGene.file<-system.file("data/rn5refGene.rdata", package="r3Cseq")
if(file.exists(refGene.file)==TRUE){
load(file=refGene.file)
refGene<-rn5refGene
return(refGene)
}else{
stop("Couldn't find rn5refGene.rdata")
}
}else{
stop("Require the selected genome: hg18, hg19, mm9, mm10 or rn5.")
}
}
makeInteractionMatrixNearCisPerWindow<-function(obj,smoothing.parameter,rawReads.Ranged,max.window=25e3,viewpoint,distanceFromViewpoint=5e5){
stopifnot( is( obj, "r3Cseq" ) |is( obj, "r3CseqInBatch" ) )
if('BSgenome.Hsapiens.UCSC.hg19.masked' %in% loadedNamespaces()==TRUE){
detach(package:BSgenome.Hsapiens.UCSC.hg19.masked,unload=TRUE)
}
if('BSgenome.Hsapiens.UCSC.hg18.masked' %in% loadedNamespaces()==TRUE){
detach(package:BSgenome.Hsapiens.UCSC.hg18.masked,unload=TRUE)
}
if('BSgenome.Mmusculus.UCSC.mm9.masked' %in% loadedNamespaces()==TRUE){
detach(package:BSgenome.Mmusculus.UCSC.mm9.masked,unload=TRUE)
}
if('BSgenome.Mmusculus.UCSC.mm10.masked' %in% loadedNamespaces()==TRUE){
detach(package:BSgenome.Mmusculus.UCSC.mm10.masked,unload=TRUE)
}
if('BSgenome.Rnorvegicus.UCSC.rn5.masked' %in% loadedNamespaces()==TRUE){
detach(package:BSgenome.Rnorvegicus.UCSC.rn5.masked,unload=TRUE)
}
###get raw reads
if(length(rawReads.Ranged)==0){
stop("No reads found, please run getRawReads function!!!.")
}
if(length(viewpoint)==0){
stop("No viewpoint, please run getVeiwpoint function to get the viewpoint !!!.")
}
viewpoint<-getViewpoint(obj)
viewpoint.chr <-as.character(seqnames(viewpoint))
rawReads.chr<-rawReads.Ranged[as.character(seqnames(rawReads.Ranged))==viewpoint.chr]
rawReads.filted<-excludeReadsNearViewpoint(obj,rawReads.chr,span_fragment=2)
#######get organism name ############
genome<-organismName(obj)
#####################################
chr.size<-0
if(genome=="hg19"){
library(BSgenome.Hsapiens.UCSC.hg19.masked)
genome <- BSgenome.Hsapiens.UCSC.hg19.masked
chr.size<-seqlengths(genome)[viewpoint.chr]
}else if(genome=="hg18"){
library(BSgenome.Hsapiens.UCSC.hg18.masked)
genome <- BSgenome.Hsapiens.UCSC.hg18.masked
chr.size<-seqlengths(genome)[viewpoint.chr]
}else if(genome =="mm9"){
library(BSgenome.Mmusculus.UCSC.mm9.masked)
genome <- BSgenome.Mmusculus.UCSC.mm9.masked
chr.size<-seqlengths(genome)[viewpoint.chr]
}else if(genome =="mm10"){
library(BSgenome.Mmusculus.UCSC.mm10.masked)
genome <- BSgenome.Mmusculus.UCSC.mm10.masked
chr.size<-seqlengths(genome)[viewpoint.chr]
}else if(genome =="rn5"){
library(BSgenome.Rnorvegicus.UCSC.rn5.masked)
genome <- BSgenome.Rnorvegicus.UCSC.rn5.masked
chr.size<-seqlengths(genome)[viewpoint.chr]
}else{
stop("Require the selected genome: hg18, hg19, mm9, mm10 or rn5")
}
r.start <-start(viewpoint)-distanceFromViewpoint
r.end <-end(viewpoint)+distanceFromViewpoint
r.size<-r.end-r.start+1
ref.position<-c(seq(from=1,to=r.size,by=1e3),r.size)
ref.v<-data.frame(position=ref.position)
print("Analyzing interaction regions for each window....")
for(window.size in rev(c(seq(from=2e3,to=max.window,by=1000)))){
fragment.GRanges<-GRanges()
step.size = window.size
ends <- c(seq(from=min(start(rawReads.filted))+window.size,to=chr.size,by=step.size),as.integer(chr.size))
starts <- seq(from=min(start(rawReads.filted)),to=chr.size,by=step.size)
fragment<-data.frame(chromosome=viewpoint.chr,start=starts,end=ends-1)
fragment.GRanges<-GRanges(seqnames =fragment$chromosome,IRanges(start=fragment$start,end=fragment$end))
readsPerFragment <- countOverlaps(fragment.GRanges,subject=rawReads.filted)
reads<-data.frame(nReads=readsPerFragment)
#####migrating the code to support the BioC 3.9#####
values(fragment.GRanges)<-reads
countTable<-fragment.GRanges
countTable<-countTable[countTable$nReads >0,]
cis<-data.frame(chromosome=as.character(seqnames(countTable)),
start=start(countTable),
end=end(countTable),
relative.position=start(countTable)-start(viewpoint),observed=countTable$nReads)
#######Smooth the data############
e.smooth<-vsmooth.spline(cis$relative.position,cis$observed,spar=smoothing.parameter)
e.fitted<-fitted(e.smooth)
cis$expected<-as.numeric(e.fitted)
######Calculate z-score and assign p-value in cis
cis$z<-(cis$observed-cis$expected)/sd(cis$observed-cis$expected)
cis$p.value<-2*pnorm(-abs(cis$z))
cis$p.value[cis$z < 0]<-1
cis$q.value<-qvalue(cis$p.value, fdr.level=0.05, pi0.method="bootstrap")$qvalues
cis.in<-GRanges(seqnames = as.character(cis$chromosome),
IRanges(start=cis$start,end=cis$end),
nReads=cis$observed,z=cis$z,p.value=cis$p.value,q.value=cis$q.value)
cis.in<-cis.in[start(cis.in) >=r.start & end(cis.in) <= r.end,]
cis.in$rel.start<-start(cis.in)-r.start
cis.in$rel.end<-end(cis.in)-r.start
##Creat vector of q-value##
my_vector<-rep(1,r.size)
my.IRanges<-IRanges(start=cis.in$rel.start,end=cis.in$rel.end)
#fix the IRanges problem here##########
#cis.p <-as.integer(my.IRanges)
cis.p <-as.integer(IPos(my.IRanges))
#######################################
my_vector[cis.p]<-c(rep(cis.in$q.value,each=window.size))
ref.value<-data.frame(q.value=my_vector[ref.position])
colnames(ref.value)<-paste("window.",window.size,sep="")
ref.v<-cbind(ref.v,ref.value)
}
return(ref.v)
print("The interaction matrix is created !!!.")
}
fishersMethod <-function(x) pchisq(-2 * sum(log(x)),df=2*length(x),lower.tail=FALSE)
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