--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/scpipe.R	Wed Aug 15 13:54:40 2018 -0400
@@ -0,0 +1,149 @@
+options( show.error.messages=F, error = function () { cat( geterrmessage(), file=stderr() ); q( "no", 1, F ) } )
+
+# we need that to not crash galaxy with an UTF8 error on German LC settings.
+loc <- Sys.setlocale("LC_MESSAGES", "en_US.UTF-8")
+
+suppressPackageStartupMessages({
+    library(scPipe)
+    library(SingleCellExperiment)
+    library(optparse)
+    library(readr)
+    library(ggplot2)
+    library(plotly)
+    library(DT)
+    library(scater)
+    library(scran)
+    library(scales)
+    library(Rtsne)
+})
+
+option_list <- list(
+    make_option(c("-fasta","--fasta"), type="character", help="Genome fasta file"),
+    make_option(c("-exons","--exons"), type="character", help="Exon annotation gff3 file"),
+    make_option(c("-barcodes","--barcodes"), type="character", help="Cell barcodes csv file"),
+    make_option(c("-read1","--read1"), type="character", help="Read 1 fastq.gz"),
+    make_option(c("-read2","--read2"), type="character", help="Read 2 fastq.gz"),
+    make_option(c("-samplename","--samplename"), type="character", help="Name to use for sample"),
+    make_option(c("-bs1","--bs1"), type="integer", help="Barcode start in Read 1"),
+    make_option(c("-bl1","--bl1"), type="integer", help="Barcode length in Read 1"),
+    make_option(c("-bs2","--bs2"), type="integer", help="Barcode start in Read 2"),
+    make_option(c("-bl2","--bl2"), type="integer", help="Barcode length in Read 2"),
+    make_option(c("-us","--us"), type="integer", help="UMI start in Read 2"),
+    make_option(c("-ul","--ul"), type="integer", help="UMI length in Read 2"),
+    make_option(c("-rmlow","--rmlow"), type="logical", help="Remove reads with N in barcode or UMI"),
+    make_option(c("-rmN","--rmN"), type="logical", help="Remove reads with low quality"),
+    make_option(c("-minq","--minq"), type="integer", help="Minimum read quality"),
+    make_option(c("-numbq","--numbq"), type="integer", help="Maximum number of bases below minq"),
+    make_option(c("-stnd","--stnd"), type="logical", help="Perform strand-specific mapping"),
+    make_option(c("-max_mis","--max_mis"), type="integer", help="Maximum mismatch allowed in barcode"),
+    make_option(c("-UMI_cor","--UMI_cor"), type="integer", help="Correct UMI sequence error"),
+    make_option(c("-gene_fl","--gene_fl"), type="logical", help="Remove low abundant genes"),
+    make_option(c("-max_reads","--max_reads"), type="integer", help="Maximum reads processed"),
+    make_option(c("-min_count","--min_count"), type="integer", help="Minimum count to keep"),
+    make_option(c("-report","--report"), type="logical", help="HTML report of plots"),
+    make_option(c("-rdata","--rdata"), type="logical", help="Output RData file"),
+    make_option(c("-nthreads","--nthreads"), type="integer", help="Number of threads")
+  )
+
+parser <- OptionParser(usage = "%prog [options] file", option_list=option_list)
+args = parse_args(parser)
+
+fa_fn = args$fasta
+anno_fn = args$exons
+fq_R1 = args$read1
+fq_R2 = args$read2
+read_structure = list(
+    bs1 = args$bs1,   # barcode start position in fq_R1, -1 indicates no barcode
+    bl1 = args$bl1,    # barcode length in fq_R1, 0 since no barcode present
+    bs2 = args$bs2,    # barcode start position in fq_R2
+    bl2 = args$bl2,   # barcode length in fq_R2
+    us = args$us,    # UMI start position in fq_R2
+    ul = args$ul     # UMI length
+)
+
+if (args$us == -1) {
+  has_umi = FALSE
+} else {
+  has_umi = TRUE
+}
+
+filter_settings=list(rmlow=args$rmlow, rmN=args$rmN, minq=args$minq, numbq=args$numbq)
+
+# Outputs
+out_dir = "."
+fasta_index = file.path(out_dir, paste0(fa_fn, ".fasta_index"))
+combined_fastq = file.path(out_dir, "combined.fastq")
+aligned_bam = file.path(out_dir, "aligned.bam")
+mapped_bam = file.path(out_dir, "aligned.mapped.bam")
+
+
+print("Trimming barcodes")
+sc_trim_barcode(combined_fastq,
+                fq_R1,
+                fq_R2,
+                read_structure=read_structure,
+                filter_settings=filter_settings)
+
+print("Building genome index")
+Rsubread::buildindex(basename=fasta_index, reference=fa_fn)
+
+print("Aligning reads to genome")
+Rsubread::align(index=fasta_index,
+    readfile1=combined_fastq,
+    output_file=aligned_bam,
+    nthreads=args$nthreads)
+
+if (!is.null(args$barcodes)) {
+  barcode_anno=args$barcodes
+} else {
+  print("Detecting barcodes")
+  # detect 10X barcodes and generate sample_index.csv file
+  barcode_anno = "sample_index.csv"
+  sc_detect_bc(
+      infq=combined_fastq,
+      outcsv=barcode_anno,
+      bc_len=read_structure$bl2,
+      max_reads=args$max_reads,
+      min_count=args$min_count,
+      max_mismatch=args$max_mis
+  )
+}
+
+print("Assigning reads to exons")
+sc_exon_mapping(aligned_bam, mapped_bam, anno_fn, bc_len=read_structure$bl2, UMI_len=read_structure$ul, stnd=args$stnd)
+
+print("De-multiplexing data")
+sc_demultiplex(mapped_bam, out_dir, barcode_anno, has_UMI=has_umi)
+
+print("Counting genes")
+sc_gene_counting(out_dir, barcode_anno, UMI_cor=args$UMI_cor, gene_fl=args$gene_fl)
+
+print("Creating SingleCellExperiment object")
+sce <- create_sce_by_dir(out_dir)
+
+if (!is.null(args$report)) {
+print("Creating report")
+create_report(sample_name=args$samplename,
+   outdir=out_dir,
+   r1=fq_R1,
+   r2=fq_R2,
+   outfq=combined_fastq,
+   read_structure=read_structure,
+   filter_settings=filter_settings,
+   align_bam=aligned_bam,
+   genome_index=fasta_index,
+   map_bam=mapped_bam,
+   exon_anno=anno_fn,
+   stnd=args$stnd,
+   fix_chr=FALSE,
+   barcode_anno=barcode_anno,
+   max_mis=args$max_mis,
+   UMI_cor=args$UMI_cor,
+   gene_fl=args$gene_fl)
+}
+
+if (!is.null(args$rdata) ) {
+    save(sce, file = file.path(out_dir,"scPipe_analysis.RData"))
+}
+
+sessionInfo()