initialize repository

ascii_alignment_pileup.R

+#!/usr/bin/env Rscript
+
+#==================#
+#   HEADER START   #
+#==================#
+### Created: Sep 29, 2019
+### Author: Alexander Kanitz
+### Affiliation: Zavolan Group, Biozentrum, University of Basel
+### Email: alexander.kanitz@alumni.ethz.ch
+#==================#
+#    HEADER END    #
+#==================#
+
+
+#========================#
+#   SESSION INFO START   #
+#========================#
+# TESTED WITH:
+#
+# R version 3.6.0 (2019-04-26)
+# Platform: x86_64-pc-linux-gnu (64-bit)
+# Running under: CentOS Linux 7 (Core)
+#
+# attached base packages:
+#  [1] tools     stats4    parallel  stats     graphics  grDevices utils
+#  [8] datasets  methods   base
+#
+# other attached packages:
+#  [1] GenomicAlignments_1.20.0    Rsamtools_2.0.0
+#  [3] SummarizedExperiment_1.14.0 DelayedArray_0.10.0
+#  [5] BiocParallel_1.18.0         matrixStats_0.54.0
+#  [7] Biobase_2.44.0              rtracklayer_1.44.0
+#  [9] GenomicRanges_1.36.0        GenomeInfoDb_1.20.0
+# [11] Biostrings_2.52.0           XVector_0.24.0
+# [13] IRanges_2.18.0              S4Vectors_0.22.0
+# [15] BiocGenerics_0.30.0         optparse_1.6.2
+#========================#
+#    SESSION INFO END    #
+#========================#
+
+
+#==========================#
+#   PRE-REQUISITES START   #
+#==========================#
+#---> LOAD OPTION PARSER <---#
+if (suppressWarnings(suppressPackageStartupMessages(require("optparse"))) == FALSE) { stop("Package 'optparse' required!\nExecution aborted.") }
+
+#---> GET SCRIPT NAME <---#
+args.all <- commandArgs(trailingOnly = FALSE)
+name.field <- "--file="
+script <- basename(sub(name.field, "", args.all[grep(name.field, args.all)]))
+
+#---> DESCRIPTION <---#
+description <- "Generates an ASCII-style pileup of read alignments in one or more BAM files
+against one or more regions specified in a BED file.\n"
+author <- "Author: Alexander Kanitz"
+affiliation <- "Affiliation: Biozentrum, University of Basel"
+email <- "Email: alexander.kanitz@alumni.ethz.ch"
+version <- "Version: 1.0.1 (29-SEP-2019)"
+requirements <- c("optparse", "rtracklayer", "GenomicAlignments", "tools")
+requirements_txt <- paste("Requires:", paste(requirements, collapse=", "), sep=" ")
+msg <- paste(description, author, affiliation, email, version, requirements_txt, sep="\n")
+notes <- "Notes:
+- For the input queries, consider the `--maximum-region-width` parameter, which
+  is provided for safety. While it is possible to increase it, wide regions may
+  require excessive memory.
+"
+
+#---> COMMAND-LINE ARGUMENTS <---#
+## List of allowed/recognized arguments
+option_list <- list(
+        make_option(
+            "--reference",
+            action="store",
+            type="character",
+            default=NULL,
+            help="Reference genome sequence in FASTA format. The file *MUST* be compressed with
+            BGZIP. If supplied, the reference sequence for the query region(s) will be added to
+            the output. Note that on the first run with a specific reference genome file, an FAI
+            index is generated which will take some time.",
+            metavar="file"
+        ),
+        make_option(
+            "--annotations",
+            action="store",
+            type="character",
+            default=NULL,
+            help="Annotation file in GFF/GTF format used to annotate sequences. If supplied,
+            features overlapping the query region(s) will be visualized in the output. Ensure that
+            the argument to option `annotation-name-field` corresponds to a field in the
+            annotations, otherwise the script will fail.",
+            metavar="file"
+        ),
+        make_option(
+            "--output-directory",
+            action="store",
+            type="character",
+            default=getwd(),
+            help="Output directory. One output file will be created for each region in `--bed` and
+            the filenames will be generated from the basenames of the supplied BAM file(s) and the
+            name field (4th column) of the BED file. [default \"%default\"]",
+            metavar="dir"
+        ),
+        make_option(
+            "--maximum-region-width",
+            action="store",
+            type="integer",
+            default=200,
+            help="Maximum input region width. Use with care as wide regions will use excessive
+            resources. [default %default]",
+            metavar="int"
+        ),
+        make_option(
+            "--do-not-collapse-alignments",
+            action="store_true",
+            type="logical",
+            default=FALSE,
+            help="Show alignments of reads with identical sequences individually."
+        ),
+        make_option(
+            "--minimum-count",
+            action="store",
+            type="integer",
+            default=1,
+            help="Alignments of reads with less copies than the specified number will not be
+            printed. Option is not considered if `do-not-collapse-alignments` is set.
+            [default %default]",
+            metavar="int"
+        ),
+        make_option(
+            "--annotation-name-field",
+            action="store",
+            type="character",
+            default="Name",
+            help="Annotation field used to populate the `name` column in the output.
+            [default \"%default\"]",
+            metavar="str"
+        ),
+        make_option(
+            "--padding-character",
+            action="store",
+            default=".",
+            help="Character used for padding alignments. [default \"%default\"]",
+            metavar="char"
+        ),
+        make_option(
+            "--indel-character",
+            action="store",
+            default="-",
+            help="Character to denote insertions and deletions in alignments. [default \"%default\"]",
+            metavar="char"
+        ),
+        make_option(
+            c("-h", "--help"),
+            action="store_true",
+            default=FALSE,
+            help="Show this information and die."
+        ),
+        make_option(
+            c("-v", "--verbose"),
+            action="store_true",
+            default=FALSE,
+            help="Print log messages to STDOUT."
+        )
+)
+
+## Parse options
+opt_parser <- OptionParser(
+    usage=paste(script, "[--help] [--verbose] [OPTIONS] BED BAM [BAM2 ...]\n"),
+    option_list=option_list,
+    add_help_option=FALSE,
+    description=msg,
+    epilogue=notes
+)
+cli <- parse_args(opt_parser, positional_arguments=c(2, Inf))
+
+# Re-assign CLI arguments
+fl.query <- cli$args[[1]]
+fl.bam <- cli$args[2:length(cli$args)]
+fl.ref <- cli$options[["reference"]]
+fl.anno <- cli$options[["annotations"]]
+dir.out <- cli$options[["output-directory"]]
+width.max <- cli$options[["maximum-region-width"]]
+collapse <- ! cli$options[["do-not-collapse-alignments"]]
+count.min <- cli$options[["minimum-count"]]
+char.pad <- cli$options[["padding-character"]]
+char.indel <- cli$options[["indel-character"]]
+field.name.anno <- cli$options[["annotation-name-field"]]
+verb <- cli$options[["verbose"]]
+#==========================#
+#    PRE-REQUISITES END    #
+#==========================#
+
+
+#================#
+#   MAIN START   #
+#================#
+#---> START MESSAGE <---#
+if (verb) cat("Starting '", script, "'...\n", sep="")
+
+#---> LOAD REQUIRED LIBRARIES <---#
+if (verb) cat("Loading libraries...\n", sep="")
+for (req in requirements) {
+    if ( suppressWarnings(suppressPackageStartupMessages(require(req, character.only=TRUE))) == FALSE ) { stop("Package '", req, "' required!") }
+}
+
+#---> IMPORT FILES <---#
+# Print status message
+if (verb) cat("Importing input files...\n", sep="")
+# Load files
+bed <- import(con=fl.query)
+if (! is.null(fl.ref)) {ref <- FaFile(fl.ref)}
+if (! is.null(fl.anno)) {anno <- import(con=fl.anno)}
+
+# Get file prefix from BAM files
+fl.prefix <- paste(basename(file_path_sans_ext(fl.bam)), collapse=".")
+
+#--->   <---#
+# Print status message
+if (verb) cat("Iterating over regions in BED file...\n")
+# Iterate over input regions
+for(index in seq_along(bed)) {
+
+    #---> PREPARE STACKING OF ALIGNMENTS  <---#
+    # Assign current region
+    region <- bed[index]
+    # Print status message
+    if (verb) cat("Processing region '", mcols(region)[["name"]], "'...\n", sep="")
+    # Exit with error if region is too wide
+    if (width(region) > width.max) {stop("Supplied region too large. Consider increasing the `width.max` parameter, but note that for very large regions, the memory footprint may be excessive.")}
+    # Initialize DNAStringSet container object
+    seq.out <- DNAStringSet()
+
+    #---> ADD READ ALIGNMENTS  <---#
+    # Print status message
+    if (verb) cat("Iterating over BAM files...\n")
+    # Iterate over BAM files
+    for (bam in fl.bam) {
+        # Print status message
+        if (verb) cat("Adding read alignments for file '", bam,"'...\n", sep="")
+        # Get alignments for current BAM file
+        seq.stack <- stackStringsFromBam(
+            bam,
+            param=region,
+            D.letter=char.indel,
+            N.letter=char.indel,
+            Lpadding.letter=char.pad,
+            Rpadding.letter=char.pad
+        )
+        # Add to container
+        seq.out <- append(seq.out, seq.stack)
+    }
+    # Get complement if on minus strand
+    if (as.character(strand(region))[[1]] == "-") {
+        seq.out <- complement(seq.out)
+    }
+    # Convert to character
+    seq.out <- as.character(seq.out)
+    # Convert to dataframe for writing output
+    df <- data.frame(seq=seq.out, count=rep(NA, length(seq.out)), stringsAsFactors=FALSE)
+
+    #---> COLLAPSE READ ALIGNMENTS <---#
+    if (collapse) {
+        # Print status message
+        if (verb) cat("Collapsing identical reads/alignments...\n")
+        # Get unique alignments
+        df <- data.frame(table(df[["seq"]]), stringsAsFactors=FALSE)
+        if (! ncol(df) == 2) df <- data.frame(matrix(ncol = 2, nrow = 0))
+        colnames(df) <- c("seq", "count")
+        df[["seq"]] <- as.character(df[["seq"]])
+        # Filter out any alignments that do not make the specified minimum count cutoff
+        df <- df[df[["count"]] >= count.min, ]
+    }
+
+    #---> SORTING ALIGNMENTS  <---#
+    # Print status message
+    if (verb) cat("Sorting alignments...\n")
+    # Reverse sequence if on minus strand
+    if (as.character(strand(region))[[1]] == "-") {
+        df[["seq"]] <- reverse(df[["seq"]])
+    }
+    # Sort by position of first nucleotide, count and position of last nucleotide
+    if (nrow(df)) {
+        last_char <- nchar(df[["seq"]][[1]])
+        pos.nuc.first <- regexpr(paste0("[^", char.pad, "\\.]"), df[["seq"]])
+        pos.nuc.last <- last_char - regexpr(paste0("[^", char.pad, "\\.]"), unlist(lapply(df[["seq"]], reverse))) + 1
+        df <- df[order(
+            pos.nuc.first, df[["count"]], pos.nuc.last,
+            decreasing=c(FALSE, TRUE, FALSE)
+        ), ]
+    }
+    # Reverse sequence again if on minus strand
+    if (as.character(strand(region))[[1]] == "-") {
+        df[["seq"]] <- reverse(df[["seq"]])
+    }
+
+    #---> ADD REFERENCE SEQUENCE  <---#
+    if (! is.null(fl.ref)) {
+        # Print status message
+        if (verb) cat("Adding reference sequence...\n")
+        # Generate name for reference sequence
+        name.ref <- paste(
+            seqnames(region),
+            paste(start(region), end(region), sep="-"),
+            strand(region), sep=":"
+        )
+        # Get sequence
+        row.ref <- getSeq(ref, region)  # will create .fai index if not present
+        # Get reverse if on minus strand
+        if (as.character(strand(region))[[1]] == "-") {
+            row.ref <- reverse(row.ref)
+        }
+        # Compile row and add to dataframe
+        df.ref <- data.frame(seq=as.character(row.ref), count=name.ref, stringsAsFactors=FALSE)
+        df <- rbind(df.ref, df)
+    }
+
+    #---> ADD ANNOTATIONS FOR REGION <---#
+    # Add annotations
+    if (! is.null(fl.anno)) {
+        # Print status message
+        if (verb) cat("Adding overlapping features...\n")
+        # Find overlapping features
+        features <- anno[to(findOverlaps(region, anno))]
+        # Set order of addition from most upstream to most downstream
+        if (as.character(strand(region)) == "+") {
+            order.features <- order(start(features))
+        } else {
+            order.features <- rev(order(end(features)))
+        }
+        # Order features
+        features <- features[order.features]
+        # Iterate over features
+        seqs <- sapply(seq_along(features), function(index) {
+            feat <- features[index]
+            # Set markup character depending on strand
+            char.anno <- ifelse(strand(region) == "+", ">", "<")
+            # Calculate lengths of feature (in region) & left/right padding
+            diff.start <- start(feat) - start(region)
+            n.padl <- max(0, diff.start)
+            n.anno <- min(min(0, diff.start) + width(feat), width(region) - n.padl)
+            n.padr <- width(region) - n.padl - n.anno
+            # Generate string encompassing feature
+            str.padl <- paste(rep(char.pad, n.padl), collapse="")
+            str.anno <- paste(rep(char.anno, n.anno), collapse="")
+            str.padr <- paste(rep(char.pad, n.padr), collapse="")
+            str.final <- paste0(str.padl, str.anno, str.padr)
+        })
+        df.anno <- data.frame(seq=seqs, count=mcols(features)[[field.name.anno]], stringsAsFactors=FALSE)
+        df <- rbind(df.anno, df)
+    }
+
+    #---> WRITE OUTPUT  <---#
+    if (collapse) {
+        name.file <- paste(fl.prefix, mcols(region)[["name"]], "min", as.character(count.min), "pileup", "tab", sep=".")
+    } else {
+        name.file <- paste(fl.prefix, mcols(region)[["name"]], "uncollapsed", "pileup", "tab", sep=".")
+    }
+    fl.out <- file.path(dir.out, name.file)
+    # Print status message
+    if (verb) cat("Writing output to file '", fl.out, "'...\n", sep="")
+    # Write tab-separated output
+    write.table(df, fl.out, quote=FALSE, sep="\t", col.names=FALSE, row.names=FALSE)
+}
+
+#---> END MESSAGE <---#
+if (verb) cat("Done.\n\nSession info:\n")
+if (verb) print(sessionInfo())
+#================#
+#    MAIN END    #
+#================#