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predictPCRartifacts.R
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#!/usr/bin/Rscript
# AAVengeR/predictPCRartifacts.R
# John K. Everett, Ph.D.
#
# This script predicts potential PCR artifacts for AAV analyses.
# Two types of predictions are provided.
# 1. alignment between the ends of ITR remnants and gDNA preceding integration sites.
# 2. alignment between the gDNA following integration sites and vector sequences.
suppressPackageStartupMessages(library(dplyr))
suppressPackageStartupMessages(library(lubridate))
suppressPackageStartupMessages(library(rtracklayer))
suppressPackageStartupMessages(library(Biostrings))
suppressPackageStartupMessages(library(parallel))
# Read in the configuration file and perform basic sanity checks.
args <- commandArgs(trailingOnly=TRUE)
if(length(args) == 0) stop('Expected at least one command line argument')
source(file.path(yaml::read_yaml(args[1])$softwareDir, 'lib.R'))
opt <- startModule(args)
createOuputDir()
dir.create(file.path(opt$outputDir, opt$predictPCRartifacts_outputDir))
dir.create(file.path(opt$outputDir, opt$predictPCRartifacts_outputDir, 'dbs'))
# Start log.
opt$defaultLogFile <- file.path(opt$outputDir, opt$predictPCRartifacts_outputDir, 'log')
logo <- readLines(file.path(opt$softwareDir, 'figures', 'ASCII_logo.txt'))
write(logo, opt$defaultLogFile, append = FALSE)
write(paste0('version: ', readLines(file.path(opt$softwareDir, 'version', 'version')), "\n"), opt$defaultLogFile, append = TRUE)
quitOnErorr <- function(msg){
updateLog(msg)
message(msg)
message(paste0('See log for more details: ', opt$defaultLogFile))
q(save = 'no', status = 1, runLast = FALSE)
}
sites <- readRDS(file.path(opt$outputDir, opt$predictPCRartifacts_inputFile))
if(! opt$predictPCRartifacts_addAfter %in% names(sites)){
write(c(paste(now(), paste0(' Error - ', opt$predictPCRartifacts_addAfter, ' is not a column in your input data frame.'))), file = file.path(opt$outputDir, 'log'), append = TRUE)
q(save = 'no', status = 1, runLast = FALSE)
}
sites$uniqueSite <- paste0(sites$trial, '~', sites$subject, '~', sites$sample, '~', sites$posid)
cluster <- makeCluster(opt$predictPCRartifacts_CPUs)
clusterExport(cluster, c('opt', 'tmpFile'))
sites$refGenome <- file.path(opt$softwareDir, 'data', 'referenceGenomes', 'blat', paste0(sites$refGenome, '.2bit'))
sites$vectorFastaFile <- file.path(opt$softwareDir, 'data', 'vectors', sites$vector)
sites$vectorFastaFile <- gsub('^\\s*|\\s*$', '', sites$vectorFastaFile)
sites <- bind_rows(lapply(split(sites, sites$refGenome), function(x){
x$n <- ntile(1:nrow(x), opt$predictPCRartifacts_CPUs)
r <- bind_rows(parLapply(cluster, split(x, x$n), function(a){
library(dplyr)
library(Biostrings)
library(rtracklayer)
g <- import(TwoBitFile(a$refGenome[1]))
bind_rows(lapply(split(a, a$uniqueSite), function(x2){
us <- x2$uniqueSite
o <- unlist(strsplit(us, '~'))
x2 <- o[length(o)]
o <- unlist(strsplit(x2, '[\\+\\-]'))
strand <- stringr::str_extract(x2, '[\\-\\+]')
o[2] <- sub('\\.\\S+$', '', o[2])
pos <- as.integer(o[2])
if(! o[1] %in% names(g)) return(tibble(uniqueSite = us, PCRartifact1 = NA))
s <- g[names(g) == o[1]]
leaderSeq <- subset(x, posid == x2)$repLeaderSeq
i <- ifelse(nchar(leaderSeq) < opt$predictPCRartifacts_adjacentSeqLength, nchar(leaderSeq),opt$predictPCRartifacts_adjacentSeqLength)
t <- substr(leaderSeq, nchar(leaderSeq)-i+1, nchar(leaderSeq))
if(strand == '-'){
endPos <- pos + opt$predictPCRartifacts_adjacentSeqLength
startPos <- pos + 1
if(startPos > width(s)) startPos <- width(s)
if(endPos > width(s)) endPos <- width(s)
if(startPos == endPos) startPos <- startPos - 1
r <- tibble(uniqueSite = us, leaderSeqSeg = t, seq = as.character(reverseComplement( subseq(s, start = startPos, end = endPos))))
} else {
startPos <- pos - opt$predictPCRartifacts_adjacentSeqLength
endPos <- pos-1
if(startPos < 1) startPos <- 1
if(endPos < 1) endPos <- 1
if(startPos == endPos) endPos <- endPos + 1
r <- tibble(uniqueSite = us, leaderSeqSeg = t, seq = as.character(subseq(s, start = startPos, end = endPos)))
}
f <- tmpFile()
aln <- Biostrings::pairwiseAlignment(r$leaderSeqSeg, r$seq, gapOpening = opt$predictPCRartifacts_gapOpeningPenalty, gapExtension = opt$predictPCRartifacts_gapExtensionPenalty)
writePairwiseAlignments(aln, f)
aln <- readLines(f)
invisible(file.remove(f))
a <- sub('^\\S+\\s+\\d\\s+', '', aln[23])
a <- sub('\\s+\\d+$', '', a)
b <- sub('^\\S+\\s+\\d\\s+', '', aln[25])
b <- sub('\\s+\\d+$', '', b)
o <- strsplit(c(a, b), '')
m <- paste0(unlist(lapply(1:nchar(a), function(n){
b <- '.'
n1 <- o[[1]][n]
n2 <- o[[2]][n]
if( n1 == n2) b <- '*'
if(n1 == '-' | n2 == '-') b <- '-'
b
})), collapse = '')
# Look at the right half of the alignment and report it if there are >= opt$predictPCRartifacts_minReportHalfMatches matches.
t <- substr(m, ceiling(opt$predictPCRartifacts_adjacentSeqLength / 2)+1, nchar(m))
if((stringr::str_count(t, '\\*') - stringr::str_count(t, '\\-')) < opt$predictPCRartifacts_minReportHalfMatches){
o <- NA
} else if (stringr::str_count(m, '\\*') < opt$predictPCRartifacts_minReportMatches) {
o <- NA
} else {
o <- paste0(stringr::str_count(m, '\\*'), ' | ', m)
}
tibble(uniqueSite = us, PCRartifact1 = o)
}))
}))
left_join(x, r, by = 'uniqueSite') %>% relocate(PCRartifact1, .after = opt$predictPCRartifacts_addAfter)
}))
o <- bind_rows(lapply(split(sites, sites$refGenome), function(x){
g <- import(rtracklayer::TwoBitFile(x$refGenome[1]))
r <- bind_rows(lapply(x$uniqueSite, function(x2){
us <- x2
o <- unlist(strsplit(x2, '~'))
x2 <- o[length(o)]
o <- unlist(strsplit(x2, '[\\+\\-]'))
strand <- stringr::str_extract(x2, '[\\-\\+]')
o[2] <- sub('\\.\\S+$', '', o[2])
pos <- as.integer(o[2])
if(! o[1] %in% names(g)) return(tibble(uniqueSite = us, strand = strand, seq = paste0(rep('N', opt$predictPCRartifacts_adjacentSeqLength), collapse = '')))
s <- g[names(g) == o[1]]
if(strand == '-'){
startPos <- pos+1-opt$predictPCRartifacts_adjacentSeqLength
endPos <- pos
if(startPos < 1) startPos <- 1
if(endPos < 1) endPos <- 1
if(startPos == endPos) endPos <- endPos + 1
r <- tibble(uniqueSite = us, strand = strand, seq = as.character(reverseComplement( subseq(s, start = startPos, end = endPos) )))
} else {
startPos <- pos
endPos <- pos + opt$predictPCRartifacts_adjacentSeqLength - 1
if(startPos > width(s)) startPos <- width(s)
if(endPos > width(s)) endPos <- width(s)
if(startPos == endPos) startPos <- startPos - 1
r <- tibble(uniqueSite = us, strand = strand, seq = as.character(subseq(s, start = startPos, end = endPos)))
}
r
}))
left_join(x, r, by = 'uniqueSite')
}))
o2 <- bind_rows(lapply(split(o, o$vectorFastaFile), function(a){
vectorFastaFile <- a$vectorFastaFile[1]
#browser()
invisible(file.remove(list.files(file.path(opt$outputDir, opt$predictPCRartifacts_outputDir, 'dbs'), full.names = TRUE)))
system(paste0('makeblastdb -in ', vectorFastaFile, ' -dbtype nucl -out ', file.path(opt$outputDir, opt$predictPCRartifacts_outputDir, 'dbs', 'd')), ignore.stderr = TRUE)
cluster <- makeCluster(opt$predictPCRartifacts_CPUs)
clusterExport(cluster, c('opt'))
r <- bind_rows(parLapply(cluster, split(a, 1:nrow(a)), function(x){
#r <- bind_rows(lapply(split(a, 1:nrow(a)), function(x){
library(dplyr)
library(Biostrings)
library(stringr)
source(file.path(opt$softwareDir, 'lib.R'))
f <- tmpFile()
write(c('>seq', x$seq), file = file.path(opt$outputDir, opt$predictPCRartifacts_outputDir, 'dbs', f), append = FALSE)
system(paste0('blastn -dust no -soft_masking false -evalue 50 -outfmt 6 -word_size ', opt$predictPCRartifacts_wordSize,
' -gapopen ', opt$predictPCRartifacts_gapOpeningPenalty, ' -gapextend ', opt$predictPCRartifacts_gapExtensionPenalty, ' -query ',
file.path(opt$outputDir, opt$predictPCRartifacts_outputDir, 'dbs', f), ' -db ',
file.path(opt$outputDir, opt$predictPCRartifacts_outputDir, 'dbs', 'd'),
' -out ', file.path(opt$outputDir, opt$predictPCRartifacts_outputDir, 'dbs', paste0(f, '.blast'))), ignore.stdout = TRUE, ignore.stderr = TRUE)
if(file.info(file.path(opt$outputDir, opt$predictPCRartifacts_outputDir, 'dbs', paste0(f, '.blast')))$size > 0){
b <- read.table(file.path(opt$outputDir, opt$predictPCRartifacts_outputDir, 'dbs', paste0(f, '.blast')), sep = '\t', header = FALSE)
names(b) <- c('qname', 'sseqid', 'pident', 'length', 'mismatch', 'gapopen', 'qstart', 'qend', 'sstart', 'send', 'evalue', 'bitscore')
b$matches = b$length - b$mismatch
b <- dplyr::filter(b, matches >= opt$predictPCRartifacts_minReportHalfMatches) %>% dplyr::slice_max(bitscore, with_ties = TRUE)
if(nrow(b) > 0){
b <- dplyr::slice_max(b, matches, with_ties = FALSE)
f2 <- tmpFile()
v <- readDNAStringSet(x$vectorFastaFile)
b$sseqid <- gsub('^\\s*|\\s*$', '', b$sseqid)
names(v) <- gsub('^\\s*|\\s*$', '', names(v))
v <- v[names(v) == b$sseqid]
### if(grepl('mm9', a$refGenome[1])) browser()
writeXStringSet(v, file.path(opt$outputDir, opt$predictPCRartifacts_outputDir, 'dbs', paste0(f2, '.fasta')))
system(paste0('makeblastdb -in ', file.path(opt$outputDir, opt$predictPCRartifacts_outputDir, 'dbs', paste0(f2, '.fasta')), ' -dbtype nucl -out ', file.path(opt$outputDir, opt$predictPCRartifacts_outputDir, 'dbs', f2)), ignore.stderr = TRUE)
system(paste0('blastn -dust no -soft_masking false -evalue 50 -outfmt 1 -word_size ', opt$predictPCRartifacts_wordSize,
' -gapopen ', opt$predictPCRartifacts_gapOpeningPenalty, ' -gapextend ', opt$predictPCRartifacts_gapExtensionPenalty, ' -query ',
file.path(opt$outputDir, opt$predictPCRartifacts_outputDir, 'dbs', f), ' -db ',
file.path(opt$outputDir, opt$predictPCRartifacts_outputDir, 'dbs', f2),
' -out ', file.path(opt$outputDir, opt$predictPCRartifacts_outputDir, 'dbs', paste0(f, '.blast2'))), ignore.stdout = TRUE, ignore.stderr = TRUE)
if(file.info(file.path(opt$outputDir, opt$predictPCRartifacts_outputDir, 'dbs', paste0(f, '.blast2')))$size > 0){
p <- readLines(file.path(opt$outputDir, opt$predictPCRartifacts_outputDir, 'dbs', paste0(f, '.blast2')))
invisible(file.remove(list.files(file.path(opt$outputDir, opt$predictPCRartifacts_outputDir, 'dbs'), pattern = f2, full.names = TRUE)))
if(length(p) == 0){
invisible(file.remove(list.files(file.path(opt$outputDir, opt$predictPCRartifacts_outputDir, 'dbs'), pattern = f, full.names = TRUE)))
return(tibble(uniqueSite = x$uniqueSite, PCRartifact2 = NA))
}
q <- p[grepl('Query_1', p)]
if(length(q) == 0){
invisible(file.remove(list.files(file.path(opt$outputDir, opt$predictPCRartifacts_outputDir, 'dbs'), pattern = f, full.names = TRUE)))
return(tibble(uniqueSite = x$uniqueSite, PCRartifact2 = NA))
}
dashes <- stringr::str_count(str_extract(q, '[ATCGN\\-]+'), '-')
i <- stringr::str_locate(q, '[ATCGN\\-]+')
q <- as.integer(unlist(stringr::str_extract_all(sub('Query_1\\s+', '', q), '\\d+')))
p <- p[grepl(paste0('\\.\\s+', b$send, '$'), p)][1]
p <- substr(p, i[1], i[2])
p <- gsub('\\.', '*', p)
p <- gsub('\\s', '.', p)
p <- unlist(strsplit(p, ''))
names(p) <- q[1]:(q[2]+dashes)
p <- paste0(unlist(lapply(1:(opt$predictPCRartifacts_adjacentSeqLength+dashes), function(n){
n <- as.character(n)
if(n %in% names(p)) return(p[names(p) == n])
return('.')
})), collapse = '')
# Look at the right half of the alignment and report it if there are >= opt$predictPCRartifacts_minReportHalfMatches matches.
t <- substr(p, ceiling(opt$predictPCRartifacts_adjacentSeqLength / 2)+1, nchar(p))
if((stringr::str_count(t, '\\*') - stringr::str_count(t, '\\-')) < opt$predictPCRartifacts_minReportHalfMatches){
p <- NA
} else if (stringr::str_count(p, '\\*') < opt$predictPCRartifacts_minReportMatches) {
p <- NA
} else {
p <- paste0(stringr::str_count(p, '\\*'), ' | ', p)
}
invisible(file.remove(list.files(file.path(opt$outputDir, opt$predictPCRartifacts_outputDir, 'dbs'), pattern = f, full.names = TRUE)))
return(tibble(uniqueSite = x$uniqueSite, PCRartifact2 = p))
} else {
invisible(file.remove(list.files(file.path(opt$outputDir, opt$predictPCRartifacts_outputDir, 'dbs'), pattern = f, full.names = TRUE)))
return(tibble(uniqueSite = x$uniqueSite, PCRartifact2 = NA))
}
} else {
invisible(file.remove(list.files(file.path(opt$outputDir, opt$predictPCRartifacts_outputDir, 'dbs'), pattern = f, full.names = TRUE)))
return(tibble(uniqueSite = x$uniqueSite, PCRartifact2 = NA))
}
} else {
invisible(file.remove(list.files(file.path(opt$outputDir, opt$predictPCRartifacts_outputDir, 'dbs'), pattern = f, full.names = TRUE)))
return(tibble(uniqueSite = x$uniqueSite, PCRartifact2 = NA))
}
}))
stopCluster(cluster)
r
}))
sites <- left_join(sites, o2, by = 'uniqueSite') %>% relocate(PCRartifact2, .after = PCRartifact1) %>% select(-uniqueSite)
sites <- arrange(sites, desc(sonicLengths))
sites$vectorFastaFile <- sapply(sites$vectorFastaFile, lpe)
sites$refGenome <- sapply(sites$refGenome, lpe)
sites$refGenome <- sub('\\.2bit$', '', sites$refGenome)
if(tolower(opt$databaseConfigGroup) != 'none'){
suppressPackageStartupMessages(library(RMariaDB))
uploadSitesToDB(sites)
}
saveRDS(sites, file = file.path(opt$outputDir, opt$predictPCRartifacts_outputDir, 'sites.rds'))
openxlsx::write.xlsx(sites, file = file.path(opt$outputDir, opt$predictPCRartifacts_outputDir, 'sites.xlsx'))
readr::write_tsv(sites, file.path(opt$outputDir, opt$predictPCRartifacts_outputDir, 'sites.tsv.gz'))
q(save = 'no', status = 0, runLast = FALSE)