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RAD_Frequency.R
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RAD_Frequency.R
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## Load packages
list.of.packages <- c("JuliaCall", "reticulate", "optparse", "ggplot2",
"reshape2", "optparse", "dada2", "ShortRead")
lapply(list.of.packages,library,character.only = TRUE)
##Specifying Illumina vs. PacBio files, and what the sample name is.
option_list <- list(make_option(c("-s", "--script_path"), type="character", default=NULL, help="Directory where scripts are located.",
metavar="character"),
make_option(c("-c", "--cpus"), type="character", default=FALSE, help="task.cpus", metavar="character"),
make_option(c("-d", "--directory"), type="character", default=NULL, help="Specify working directory", metavar="character"),
make_option(c("-m", "--metadata"), type="character", default=NULL, help="Specify metadata", metavar="character"),
make_option(c("-a", "--pacbio_sample"), type="character", default=NULL, help="Specify PacBio file to run."),
make_option(c("-n", "--sample_name"), type="character", default=NULL, help="Specify sample name."),
make_option(c("-p", "--pacbio"), type="character", default=FALSE, help="Specify if these files are only PacBio.",
metavar="character", action="store_true"),
make_option(c("-i", "--illumina"), type="character", default=FALSE, help="Specify if these files are only Illumina.",
metavar="character", action="store_true"));
opt_parser <- OptionParser(option_list=option_list);
opt <- parse_args(opt_parser)
path <- opt$directory
script.dir <- opt$script_path
#####
## To run this script manually in R, uncomment the following lines. You do not need to change the preceding lines of path and script.dir,
## but remember to recomment the lines if you want to run the script automatically in the pipeline.
## path refers to the folder your metadata.csv and sequencing files (.fastq) are. (This is the -directory option).
## script.dir refers to the folder where all the script files are located. This should point to where you saved the cloned GitHub.
#path <- "/Users/uwvirongs/Documents/Michelle/tprk_pipeline/AS_files_redo3"
#script.dir <- "/Users/uwvirongs/Documents/tprK-master/"
## This script can also be run from the command line.
## Usage: rscript \path\to\og_files_to_all_reads.R -s [script_path] -d [directory]
#####
PacBio_fns <- c(as.character(opt$pacbio_sample))
## Identify primers to go from ATG to stop in tprK
tprKF <- "GGAAAGAAAAGAACCATACATCC"
tprKR <- "CGCAGTTCCGGATTCTGA"
rc <- dada2:::rc
noprimer_filenames <- paste(substr(basename(PacBio_fns),1,nchar(basename(PacBio_fns))-5),"noprimers.fastq",sep ='')
nop <- file.path(noprimer_filenames)
if(opt$illumina == FALSE) {
## Points to Julia install in docker "quay.io/greninger-lab/tprk"
julia <- julia_setup(JULIA_HOME = "/usr/local/julia/bin")
## Remove primers
for (count in c(1:length(nop))) {
if(file.exists(nop[count])) {
print(paste(noprimer_filenames[count], " already exists. Skipping removing primers step...", sep=""))
} else {
print("Removing primers from PacBio...")
print(opt$metadata)
print(PacBio_fns)
prim <- removePrimers(PacBio_fns, nop, primer.fwd=tprKF, primer.rev=rc(tprKR), orient=TRUE, verbose=TRUE)
}
}
print("Filtering PacBio reads...")
## Setting up file names to filter.
filter_filenames <- paste(substr(basename(PacBio_fns),1,nchar(basename(PacBio_fns))-5),"noprimers.filtered.fastq",sep ='')
filterEE1_filenames <- paste(substr(basename(PacBio_fns),1,nchar(basename(PacBio_fns))-5),"noprimers.filtered.EE1.fastq",sep ='')
filt <- file.path(filter_filenames)
filtEE1 <- file.path(filterEE1_filenames)
## Filter reads for tprK length and do not worry about expected errors.
for (count in c(1:length(filt))) {
if (file.exists(filt[count])) {
print(paste(filter_filenames[count]," already exists. Skipping filtering step..."), sep="")
} else {
print(paste("Filtering ",nop[count],"...",sep=""))
track <- fastqFilter(nop[count], filt[count], minLen=1400,maxLen=1800,
maxN=0,
compress=FALSE, multithread=TRUE)
}
}
##Consider: Filter reads for tprK length and allow only 1 expected error for the entire read.
# for (count in c(1:length(filtEE1))) {
# track <- fastqFilter(nop[count], filtEE1[count], minLen=1400,maxLen=1800,
# maxN=0, maxEE=1,
# compress=FALSE, multithread=TRUE)
# }
RAD_filenames <- paste(substr(basename(PacBio_fns),1,nchar(basename(PacBio_fns))-5),"noprimers.filtered.RAD.fasta",sep ='')
RAD_files <- file.path(RAD_filenames)
## Build RAD files for each PacBio sample. This step takes forever!!!
for (count in c(1:length(filt))) {
to_rad_name <- paste(RAD_filenames[count])
# Skips RAD step if files already exist, because it takes forever.
if(file.exists(to_rad_name)) {
print(paste(to_rad_name, " already exists. Skipping RAD step...", sep=""))
} else{
# Only want to set up Julia once, takes forever
if (count == 1) {
print("Setting up Julia...")
print("Constructing RAD files...")
julia_command("Pkg.init(); Pkg.update(); Pkg.clone(\"https://github.com/MurrellGroup/NextGenSeqUtils.jl\"); using NextGenSeqUtils")
julia_command("Pkg.clone(\"https://github.com/MurrellGroup/DPMeansClustering.jl.git\")")
julia_command("Pkg.clone(\"https://github.com/MurrellGroup/RobustAmpliconDenoising.jl.git\"); using RobustAmpliconDenoising")
}
# julia_command("using Pkg")
# julia_command("Pkg.build(\"SpecialFunctions\")")
# julia_command("Pkg.add(PackageSpec(name=\"NextGenSeqUtils\", rev= \"1.0\", url = \"https://github.com/MurrellGroup/NextGenSeqUtils.jl.git\"))")
# julia_command("Pkg.add(PackageSpec(name=\"DPMeansClustering\", rev=\"1.0\", url = \"https://github.com/MurrellGroup/DPMeansClustering.jl.git\"))")
# julia_command("Pkg.add(PackageSpec(name=\"RobustAmpliconDenoising\", rev=\"1.0\", url = \"https://github.com/MurrellGroup/RobustAmpliconDenoising.jl.git\"))")
# julia_command("using RobustAmpliconDenoising")
julia_readfastq <- paste("seqs, QVs, seq_names = read_fastq(\"",filt[count],'")',sep="")
julia_command(julia_readfastq)
julia_command("templates,template_sizes,template_indices = denoise(seqs)")
julia_writefasta <- paste("write_fasta(\"",RAD_files[count],'",templates,names = ["seqs$(j)_$(template_sizes[j])" for j in 1:length(template_sizes)])',sep="")
julia_command(julia_writefasta)
}
}
## RAD denoised files are written. Let's get some frequencies of different variable regions
RAD_files_nolines <- paste(substr(RAD_files,1,nchar(RAD_files)-5),"nolines.fasta",sep ='')
RAD_files_fix <- paste(substr(RAD_files,1,nchar(RAD_files)-5),"nolines.fix.fasta",sep ='')
# Fixes up the fastas so they wrap and don't have awkward new lines.
# TODO: fix this section so it works. For some reason the pipeline currently runs without it? But probably should fix this anyway.
awk_command <- paste("awk '/^>/ {printf(\"\\n%s\\n\",$0);next; } { printf(\"%s\",$0);} END {printf(\"\\n\");}' < ",RAD_files," > ",RAD_files_nolines," ;")
fix_firstline <- paste("tail -n+2 ",RAD_files_nolines," > ",RAD_files_fix)
for (count in c(1:length(awk_command))) {
system(awk_command[count])
system(fix_firstline[count])
}
} else {
print("Illumina option specified. Skipping making PacBio frequency files...")
}