R/fastqQuality.R

In tgirke/systemPipeR: systemPipeR: Workflow Environment for Data Analysis and Report Generation

#########################
## FASTQ Quality Plots ##
#########################
## Author: Thomas Girke
## Last update: 05-Dec-13
## Old version of this file is available under the name fastqQuality_old.R
## Utility:
##      Plots quality report for set of FASTQ files including
##              (A) Per cycle box plot of quality
##              (B) Per cycle base proportion
##              (C) Per cycle mean base quality
##              (D) Relative k-mer diversity: unique_k-mers / all_possible_k-mers
##              (E) Number of reads where all Phred scores are above a minimum cutoff
##              (F) Distribution of mean quality of reads
##              (G) Read length distribution
#               (H) Read occurrence distribution

## (A) Compute quality stats and store them in list
seeFastq <- function(fastq, batchsize, klength = 8) {
    ## Processing of single fastq file
    seeFastqSingle <- function(fastq, batchsize, klength) {
        ## Random sample N reads from fastq file (N=batchsize)
        f <- ShortRead::FastqSampler(fastq, batchsize)
        fq <- ShortRead::yield(f)
        nReads <- f$status()[["total"]] # Total number of reads in fastq file
        close(f)
        ## If reads are not of constant width then inject them into a matrix pre-populated with
        ## N/NA values and of dimensions N_rows = number_of_reads and N_columns = length_of_longest_read.
        if (length(unique(width(fq))) == 1) {
            q <- as.matrix(Biostrings::PhredQuality(Biostrings::quality(fq)))
            s <- as.matrix(ShortRead::sread(fq))
        } else {
            mymin <- min(width(fq))
            mymax <- max(width(fq))
            s <- matrix("N", length(fq), mymax)
            q <- matrix(NA, length(fq), mymax)
            for (i in mymin:mymax) {
                index <- width(fq) == i
                if (any(index)) {
                    s[index, 1:i] <- as.matrix(Biostrings::DNAStringSet(ShortRead::sread(fq)[index], start = 1, end = i))
                    q[index, 1:i] <- as.matrix(Biostrings::PhredQuality(Biostrings::quality(fq))[index])
                }
            }
        }
        s[s == "N"] <- NA
        row.names(q) <- paste("s", 1:length(q[, 1]), sep = "")
        colnames(q) <- 1:length(q[1, ])
        ## (A) Per cycle quality box plot
        ## Generate box plot from precomputed stats
        bpl <- graphics::boxplot(q, plot = FALSE)
        astats <- data.frame(bpl$names, t(matrix(bpl$stats, dim(bpl$stats))))
        colnames(astats) <- c("Cycle", "min", "low", "mid", "top", "max")
        astats[, 1] <- factor(astats[, 1], levels = unique(astats[, 1]), ordered = TRUE)
        ## (B) Per cycle base proportion
        bstats <- apply(s, 2, function(x) table(factor(x, levels = c("A", "C", "G", "T"))))
        colnames(bstats) <- 1:length(bstats[1, ])
        bstats <- t(apply(bstats, 1, function(x) x / colSums(bstats)))
        bstats <- data.frame(Nuc = rownames(bstats), bstats)
        convertDF <- function(df = df, mycolnames) {
            myfactor <- rep(colnames(df)[-1], each = length(df[, 1]))
            mydata <- as.vector(as.matrix(df[, -1]))
            df <- data.frame(df[, 1], mydata, myfactor)
            colnames(df) <- mycolnames
            return(df)
        }
        bstats <- convertDF(bstats, mycolnames = c("Base", "Frequency", "Cycle"))
        bstats[, 3] <- as.numeric(gsub("X", "", bstats[, 3]))
        bstats[, 3] <- factor(bstats[, 3], levels = unique(bstats[, 3]), ordered = TRUE)
        ## (C) Per cycle average quality of each base type
        A <- q
        A[s %in% c("T", "G", "C")] <- NA
        A <- colMeans(A, na.rm = TRUE)
        T <- q
        T[s %in% c("A", "G", "C")] <- NA
        T <- colMeans(T, na.rm = TRUE)
        G <- q
        G[s %in% c("T", "A", "C")] <- NA
        G <- colMeans(G, na.rm = TRUE)
        C <- q
        C[s %in% c("T", "G", "A")] <- NA
        C <- colMeans(C, na.rm = TRUE)
        cstats <- data.frame(Quality = c(A, C, G, T), Base = rep(c("A", "C", "G", "T"), each = length(A)), Cycle = c(names(A), names(C), names(G), names(T)))
        cstats[, 3] <- factor(cstats[, 3], levels = unique(cstats[, 3]), ordered = TRUE)
        ## (D) Relative K-mer Diversity
        dna <- ShortRead::sread(fq)
        loopv <- 1:(min(width(dna)) - (klength - 1))
        kcount <- sapply(loopv, function(x) length(unique(Biostrings::DNAStringSet(start = x, end = x + klength - 1, dna))))
        reldiv <- kcount / (5^klength) # 5 instead of 4 because of Ns
        reldiv <- c(rep(NA, klength - 1), reldiv) # Adds dummy NAs to align with sequencing cycles
        names(reldiv) <- 1:length(reldiv)
        dstats <- data.frame(RelDiv = reldiv, Method = rep(c(1), each = length(reldiv)), Cycle = names(reldiv))
        dstats[, 3] <- factor(dstats[, 3], levels = unique(dstats[, 3]), ordered = TRUE)
        ## (E) Number of reads where all Phred scores are above a minimum cutoff
        ev <- c("0" = 0, "1" = 10, "2" = 20, "3" = 30, "4" = 40)
        edf <- sapply(ev, function(x) sapply(as.numeric(names(ev)), function(y) sum(rowSums(q >= x, na.rm = TRUE) >= (rowSums(!is.na(q)) - y))))
        rownames(edf) <- names(ev)
        colnames(edf) <- ev
        edf <- edf / max(edf) * 100
        edf <- data.frame(Percent = paste(">", colnames(edf), sep = ""), t(edf), check.names = FALSE)
        estats <- convertDF(edf, mycolnames = c("minQuality", "Percent", "Outliers"))
        estats[, 1] <- factor(estats[, 1], levels = unique(estats[, 1]), ordered = TRUE)
        estats[, 3] <- factor(estats[, 3], levels = unique(estats[, 3]), ordered = TRUE)
        ## (F) Distribution of mean quality of reads
        qv <- table(round(rowMeans(q)))[as.character(0:max(q, na.rm = TRUE))]
        qv[is.na(qv)] <- 0
        names(qv) <- 0:max(q, na.rm = TRUE)
        fstats <- data.frame(Quality = names(qv), Percent = as.numeric(qv))
        fstats[, 2] <- as.numeric(as.vector(fstats[, 2])) / length(q[, 1]) * 100
        fstats[, 1] <- factor(fstats[, 1], levels = unique(fstats[, 1]), ordered = TRUE)
        ## (G) Read length distribution
        l <- rep(0, max(width(fq)))
        names(l) <- 1:length(l)
        lv <- table(width(fq))
        l[names(lv)] <- lv
        gstats <- data.frame(Cycle = names(l), Percent = l)
        gstats[, 2] <- gstats[, 2] / sum(gstats[, 2]) * 100
        gstats[, 1] <- factor(gstats[, 1], levels = unique(gstats[, 1]), ordered = TRUE)
        ## (H) Read occurrence distribution
        qa1 <- ShortRead::qa(fq, basename(fastq))
        hstats <- qa1[["sequenceDistribution"]][, 1:2]
        hstats <- data.frame(nOccurrences = hstats[, 1], Percent = hstats[, 1] * hstats[, 2] / batchsize * 100)
        hstats[, 1] <- factor(hstats[, 1], levels = unique(hstats[, 1]), ordered = TRUE)
        ## Assemble results in list
        return(list(fqstats = c(batchsize = batchsize, nReads = nReads, klength = klength), astats = astats, bstats = bstats, cstats = cstats, dstats = dstats, estats = estats, fstats = fstats, gstats = gstats, hstats = hstats))
    }
    ## Loop to run seeFastqSingle on one or many fastq files
    fqlist <- lapply(names(fastq), function(x) seeFastqSingle(fastq = fastq[x], batchsize = batchsize, klength = klength))
    names(fqlist) <- names(fastq)
    return(fqlist)
}
## Alias
# fastqQuality <- seeFastq

## (B) Plot seeFastq results
seeFastqPlot <- function(fqlist, arrange = c(1, 2, 3, 4, 5, 8, 6, 7), ...) {
    ## Create plotting instances from fqlist
    fastqPlot <- function(x = fqlist) {
        Cycle <- low <- mid <- top <- Frequency <- Base <- Quality <- RelDiv <- Method <- minQuality <- Percent <- Outliers <- NULL
        ## (A) Per cycle quality box plot
        astats <- x[[1]][["astats"]]
        a <- ggplot2::ggplot(astats, ggplot2::aes(x = Cycle, ymin = min, lower = low, middle = mid, upper = top, ymax = max)) +
            ggplot2::geom_boxplot(stat = "identity", color = "#606060", fill = "#56B4E9") +
            ggplot2::scale_x_discrete(breaks = c(1, seq(0, length(astats[, 1]), by = 10)[-1])) +
            ggplot2::ylab("Quality") +
            ggplot2::theme(legend.position = "none", plot.title = ggplot2::element_text(size = 12)) +
            ggplot2::ggtitle(names(x))
        ## (B) Per cycle base proportion
        bstats <- x[[1]][["bstats"]]
        b <- ggplot2::ggplot(bstats, ggplot2::aes(x = Cycle, y = Frequency, fill = Base), color = "black") +
            ggplot2::scale_x_discrete(breaks = c(1, seq(0, length(unique(bstats$Cycle)), by = 10)[-1])) +
            ggplot2::geom_bar(stat = "identity") +
            ggplot2::theme(legend.position = "top", legend.key.size = ggplot2::unit(0.2, "cm")) +
            ggplot2::ylab("Proportion")
        ## (C) Per cycle average quality of each base type
        cstats <- x[[1]][["cstats"]]
        c <- ggplot2::ggplot(cstats, ggplot2::aes(x = Cycle, y = Quality, group = Base, color = Base)) +
            ggplot2::geom_line() +
            ggplot2::scale_x_discrete(breaks = c(1, seq(0, length(unique(bstats$Cycle)), by = 10)[-1])) +
            ggplot2::theme(legend.position = "none")
        ## (D) Relative K-mer Diversity
        dstats <- x[[1]][["dstats"]]
        d <- ggplot2::ggplot(dstats, ggplot2::aes(x = Cycle, y = RelDiv, group = Method, color = Method)) +
            ggplot2::geom_line() +
            ggplot2::scale_x_discrete(breaks = c(1, seq(0, length(unique(bstats$Cycle)), by = 10)[-1])) +
            ggplot2::ylab(paste("k", x[[1]][["fqstats"]][["klength"]], "-mer Div", sep = "")) +
            ggplot2::theme(legend.position = "none")
        ## (E) Number of reads where all Phred scores are above a minimum cutoff
        estats <- x[[1]][["estats"]]
        e <- ggplot2::ggplot(estats, ggplot2::aes(x = minQuality, y = Percent, fill = Outliers)) +
            ggplot2::geom_bar(position = "dodge", stat = "identity") +
            ggplot2::theme(legend.position = "top", legend.key.size = ggplot2::unit(0.2, "cm")) +
            ggplot2::xlab("All Bases Above Min Quality") +
            ggplot2::ylab("% Reads")
        ## (F) Distribution of mean quality of reads
        fstats <- x[[1]][["fstats"]]
        f <- ggplot2::ggplot(fstats, ggplot2::aes(x = Quality, y = Percent)) +
            ggplot2::geom_bar(fill = "#0072B2", stat = "identity") +
            ggplot2::theme(legend.position = "none", plot.title = ggplot2::element_text(size = 9)) +
            ggplot2::ggtitle(paste(formatC(x[[1]][["fqstats"]][["batchsize"]], big.mark = ",", format = "f", digits = 0), "of", formatC(x[[1]][["fqstats"]][["nReads"]], big.mark = ",", format = "f", digits = 0), "Reads")) +
            ggplot2::scale_x_discrete(breaks = c(0, seq(0, length(unique(fstats$Quality)), by = 5)[-1])) +
            ggplot2::xlab("Mean Quality") +
            ggplot2::ylab("% Reads")
        ## (G) Read length distribution
        gstats <- x[[1]][["gstats"]]
        g <- ggplot2::ggplot(gstats, ggplot2::aes(x = Cycle, y = Percent)) +
            ggplot2::geom_bar(fill = "#0072B2", stat = "identity") +
            ggplot2::theme(legend.position = "none") +
            ggplot2::scale_x_discrete(breaks = c(1, seq(0, length(unique(gstats$Cycle)), by = 10)[-1])) +
            ggplot2::xlab("Read Length") +
            ggplot2::ylab("% Reads")
        ## (H) Read occurrence distribution
        hstats <- x[[1]][["hstats"]]
        myintervals <- data.frame(labels = c("1", "2-10", "11-100", "101-1k", "1k-10k", ">10k"), lower = c(1, 2, 11, 101, 1001, 10001), upper = c(2, 11, 101, 1001, 10001, Inf))
        iv <- sapply(seq(along = myintervals[, 1]), function(x) sum(hstats[as.numeric(as.vector(hstats$nOccurrences)) >= myintervals[x, 2] & as.numeric(as.vector(hstats$nOccurrences)) < myintervals[x, 3], "Percent"]))
        hstats <- data.frame(labels = myintervals[, 1], Percent = iv)
        hstats[, 1] <- factor(hstats[, 1], levels = unique(hstats[, 1]), ordered = TRUE)
        h <- ggplot2::ggplot(hstats, ggplot2::aes(x = labels, y = Percent)) +
            ggplot2::geom_bar(fill = "#0072B2", stat = "identity") +
            ggplot2::theme(legend.position = "none") +
            ggplot2::xlab("Read Occurrence") +
            ggplot2::ylab("% Reads")
        ## Assemble results in list
        return(list(a = a, b = b, c = c, d = d, g = g, e = e, f = f, h = h))
    }
    ## Loop to run fastqPlot and store instances in list
    fqplot <- lapply(names(fqlist), function(z) fastqPlot(x = fqlist[z]))
    names(fqplot) <- names(fqlist)
    ## Final plot
    grid::grid.newpage() # Open a new page on grid device
    grid::pushViewport(grid::viewport(layout = grid::grid.layout(length(arrange), length(fqplot))))
    for (i in seq(along = fqplot)) {
        for (j in seq(along = arrange)) {
            suppressWarnings(print(fqplot[[i]][[arrange[j]]], vp = grid::viewport(layout.pos.row = j, layout.pos.col = i)))
        }
    }
}
## Alias
# plotFQ <- seeFastqPlot

## Usage:
## Download some sample fastq files
# system("wget http://biocluster.ucr.edu/~tgirke/HTML_Presentations/Manuals/Rngsapps/chipseqBioc2012/data.zip")
# system("unzip data.zip")

## Generate FASTQ quality plots
# source("http://faculty.ucr.edu/~tgirke/Documents/R_BioCond/My_R_Scripts/fastqQuality.R")
# fastq <- list.files("data", "*.fastq$"); fastq <- paste("data/", fastq, sep="")
# names(fastq) <- paste("flowcell_6_lane", 1:4, sep="_")
# fqlist <- seeFastq(fastq=fastq, batchsize=100000, klength=8)
# pdf("fastqQuality.pdf", height=16, width=4*length(fastq))
# seeFastqPlot(fqlist, arrange=seq(along=fqlist))
# dev.off()