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main.R
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# get initial values
system("sed 's/: /=/g' config.txt | sed 's:{::' | sed 's:}::' | sed 's/, /; /g' > config.R")
source("config.R")
## create working dir for the panel
paneldir = paste(pjdir, panel, sep='/')
dir.create(paneldir, showWarnings = FALSE, recursive = TRUE)
system(paste("cp config.txt ", paneldir, sep=''))
system(paste("cp config.R ", paneldir, sep=''))
setwd(paneldir)
## get gene list
gene.list = read.table(genelist, sep='\t', header=T, fill=T)
## remove blank space, heading 0s
gene.list$NM = gsub(' ', '', gene.list$NM)
gene.list$exon = tolower(gsub(' ', '', gene.list$exon))
gene.list$exon = gsub('^0+', '', gene.list$exon)
if (assaytype == 'fusion'){
gene.list$sense = tolower(gsub(' ', '', gene.list$sense))
} else {
## for compatability with mutation assay, assign sense 'both'
gene.list$sense = 'both'
}
panel.NM = sort(unique(gene.list$NM))
writeLines(panel.NM, 'panel.NM')
system(paste("join panel.NM ", depdir, "/hg19.RefGene.NM > target.refseq.0", sep=''))
system(paste("sort -k1,1 -u target.refseq.0 > target.refseq", sep=''))
######################################################################
# step 1. retreive sequence from genome
cat("step 1. retreive sequence from genome...\n")
source(paste(ampdir, '/step.1.retreive.sequence.R', sep=''))
######################################################################
# step 2. call primer3 - several iteration rounds
cat("step 2. call primer3 - several iteration rounds...\n")
source(paste(ampdir, '/step.2.call.primer3.R', sep=''))
######################################################################
# setp 3. BLAT candidate primers against genome
cat("setp 3. BLAT candidate primers against genome...\n")
t = 11
s = 4
n.srv = ceiling(ncpu/4)
ClnPsrv = 3
n.fa=n.srv*ClnPsrv
repMatch=1024
start.port=9000
faDir = 'split'
depdir
pjdir
db = 'hg19.2bit'
minIden = 93
minScore = 12
maxIntron = 700001
NAtype = 'dna'
source(paste(ampdir, '/step.3.blat.candidate.primer.R', sep=''))
# organize balt results
system(paste('bash ', ampdir, '/scripts/afterBlat.sh', sep=''))
######################################################################
# step 4. pair GSP1-GPS2
cat("step 4. pair GSP1-GPS2...\n")
source(paste(ampdir, '/step.4.pairing.GSPs.R', sep=''))
## top candidates
system('head -n1 ranked.all.candidate.pairs > ranked.1st.pairs')
system("grep ' 1$' ranked.all.candidate.pairs >> ranked.1st.pairs")
######################################################################
# step 5. check uniqueness of 12 bases at 3' of all primers.
# - If not unique, candidate pairs from 'ranked.all.candidate.pairs'
# will be retreived, ranked 2/3/.., until all tail 12 bases
# are unique.
#
# - If all unique, proceed.
#
# - Save final tail 12 bases of all primers for future use.
# e.g, to check uniqueness when adding new primers
# to an existing panel.
#
cat("step 5. check uniqueness of 12 bases at 3' of all primers
and final output...\n")
pairs1 = read.table('ranked.1st.pairs', header=T, stringsAsFactors=F)
pairs1$target = sub(":.*", "", pairs1$r1.qName)
source(paste(ampdir, '/step.5.check.uniqueness.R', sep=''))
writeLines(all.t12, paste(panel, '.tail.12bases.'
, format(Sys.time(), "%Y.%b.%d"), sep=''))
######################################################################
# Lastly,
# - add GSP tags
# - output
# - save primer.bed
######################################################################
pairs.keep$GSP1 = paste(GSP1tag, pairs.keep$r1.seq,sep='')
pairs.keep$GSP2 = paste(GSP2tag, pairs.keep$r2.seq,sep='')
pairs.keep$gene = sapply(strsplit(pairs.keep$target, '_'), "[[", 1)
pairs.keep$exon = sapply(strsplit(pairs.keep$target, '_'), "[[", 2)
pairs.keep$exonSize = sapply(strsplit(pairs.keep$target, '_'), "[[", 3)
if (assaytype == 'fusion'){
pairs.keep$sense = sapply(strsplit(pairs.keep$target, '_'), "[[", 4)
} else {
pairs.keep$sense = pairs.keep$exonSize
}
gene.NM = ref[,c('name2','name')]
names(gene.NM) = c('gene', 'NM')
final = merge(pairs.keep, gene.NM, by='gene')
final$gsp1.name = paste(final$gene, '_ex', final$exon, '_', final$sense
, '.1 (', final$NM, ')', sep='')
final$gsp2.name = paste(final$gene, '_ex', final$exon, '_', final$sense
, '.2 (', final$NM, ')', sep='')
# output all exons
gsp1 = final[, c('gsp1.name', 'GSP1')]
gsp2 = final[, c('gsp2.name', 'GSP2')]
# primer bed - only GSP2 is sequenced/relevant
primer.bed = final[, c('r1.chr', 'r2.tStart', 'r2.tEnd', 'gsp2.name')]
write.csv(gsp1, paste(panel, '_all.gsp1.csv', sep=''), quote=F, row.names=F)
write.csv(gsp2, paste(panel, '_all.gsp2.csv', sep=''), quote=F, row.names=F)
write.table(primer.bed, paste(panel, '_all.gsp2.primer.bed', sep=''), sep='\t'
, quote=F, row.names=F, col.names=F)
# selected exon/sense
if (assaytype == 'fusion'){
final$nm.sense = paste(final$NM, tolower(final$sense), sep='_')
gene.list$nm.sense = paste(gene.list$NM, gene.list$sense, sep='_')
nm.sense = unique(gene.list$nm.sense)
final$sense.select = 0
final$sense.select[final$nm.sense %in% nm.sense] = 1
}
final$nm.exon = paste(final$NM, as.numeric(final$exon), sep='_')
gene.list$nm.exon = paste(gene.list$NM, gene.list$exon, sep='_')
nm.exon = unique(gene.list$nm.exon)
final$exon.select = 0
final$exon.select[final$nm.exon %in% nm.exon] = 1
nm.all.exons = gene.list$NM[gene.list$exon=='all']
final$exon.select[final$NM %in% nm.all.exons] = 1
final$select = 0
if (assaytype == 'fusion'){
final$select[final$exon.select ==1 & final$sense.select ==1] = 1
} else {
final$select[final$exon.select ==1] = 1
}
## backup final data
write.table(final, paste(panel, '_intermediate.data.txt', sep=''), sep='\t'
, quote=F, row.names=F)
final.s = subset(final, select==1)
##############################
## final output
##############################
GSP1 = final.s[, c('gsp1.name', 'GSP1')]
GSP2 = final.s[, c('gsp2.name', 'GSP2')]
# primer bed - only GSP2 is sequenced/relevant
primer.bed.s = final.s[, c('r1.chr', 'r2.tStart', 'r2.tEnd', 'gsp2.name')]
write.csv(GSP1, paste(panel, '_GSP1.csv', sep=''), quote=F, row.names=F)
write.csv(GSP2, paste(panel, '_GSP2.csv', sep=''), quote=F, row.names=F)
write.table(primer.bed.s, paste(panel, '_GSP2.primer.bed', sep=''), sep='\t'
, quote=F, row.names=F, col.names=F)
## cleanup
system('mv unpaired.targets targets.failed.pairing.GSPs.txt')
if (file.exists('missed.seq_seq_primer3.exome.setting.3')){
system('mv missed.seq_seq_primer3.exome.setting.3 targets.failed.primer3.design.txt')
}
system('rm -rf split seq seq.noMask out all.psl.matt.sorted primer.pos.tm.sorted primer.psl missed.seq_* splitFa.Rout panel.NM primer.candidates.0 primer3.exome.setting*')
cat("AMP-primer-design completed successfully.\n")
## END