view edgeR.pl @ 8:734516a21b52 draft

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author fcaramia
date Thu, 13 Sep 2012 00:51:13 -0400
parents 674c75219f15
children e5fcbabbdea7
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#/bin/perl

use strict;
use warnings;
use Getopt::Std;
use File::Basename;
use File::Path qw(make_path remove_tree);
$| = 1;

# Grab and set all options
my %OPTIONS = (a => "glm", d => "tag", f => "BH", p => 0.3, r => 5, u => "movingave");

getopts('a:d:e:f:h:lmn:o:p:r:tu:', \%OPTIONS);

die qq(
Usage:   edgeR.pl [OPTIONS] factor::factor1::levels [factor::factor2::levels ...] cp::cont_pred1::values [cp::cont_pred2::values ...] cnt::contrast1 [cnt::contrast2] matrix

OPTIONS:	-a	STR	Type Of Analysis [glm, pw, limma] (default: $OPTIONS{a})
			-d	STR	The dispersion estimate to use for GLM analysis [tag, trend, common] (default: $OPTIONS{d})
			-e	STR	Path to place additional output files
			-f	STR	False discovery rate adjustment method [BH, holm, hochberg, hommel, BY, none] (default: $OPTIONS{f})
			-h	STR	Name of html file for additional files
			-l		Output the normalised digital gene expression matrix in log2 format (only applicable when using limma and -n is also specified)
			-m		Perform all pairwise comparisons
			-n	STR	File name to output the normalised digital gene expression matrix (only applicable when usinf glm or limma model)
			-o	STR	File name to output csv file with results
			-p	FLT	The proportion of all tags/genes to be used for the locally weighted estimation of the tagwise dispersion, ony applicable when 1 factor analysis selected (default: $OPTIONS{p})
			-r	INT	Common Dispersion Rowsum Filter, ony applicable when 1 factor analysis selected (default: $OPTIONS{r})
			-t		Estimate Tagwise Disp when performing 1 factor analysis
			-u	STR	Method for allowing the prior distribution for the dispersion to be abundance- dependent ["movingave", "tricube", "none"] (default: $OPTIONS{u})

) if(!@ARGV);

my $matrix = pop @ARGV;

make_path($OPTIONS{e});
open(Rcmd,">$OPTIONS{e}/r_script.R") or die "Cannot open $OPTIONS{e}/r_script.R\n\n"; 
print Rcmd "
	zz <- file(\"$OPTIONS{e}/r_script.err\", open=\"wt\")
	sink(zz)
	sink(zz, type=\"message\")
	
	library(edgeR)
	library(limma)

	# read in matrix and groups
	toc <- read.table(\"$matrix\", sep=\"\\t\", comment=\"\", as.is=T)
	groups <- sapply(toc[1, -1], strsplit, \":\")
	for(i in 1:length(groups)) { g <- make.names(groups[[i]][2]); names(groups)[i] <- g; groups[[i]] <- groups[[i]][-2] }
	colnames(toc) <- make.names(toc[2,])
	toc[,1] <- gsub(\",\", \".\", toc[,1])
	tagnames <- toc[-(1:2), 1]
	rownames(toc) <- toc[,1]
	toc <- toc[-(1:2), -1]
	for(i in colnames(toc)) toc[, i] <- as.numeric(toc[,i])
	norm_factors <- calcNormFactors(as.matrix(toc))

	pw_tests <- list()
	uniq_groups <- unique(names(groups))
	for(i in 1:(length(uniq_groups)-1)) for(j in (i+1):length(uniq_groups)) pw_tests[[length(pw_tests)+1]] <- c(uniq_groups[i], uniq_groups[j])
	DGE <- DGEList(toc, lib.size=norm_factors*colSums(toc), group=names(groups))
	pdf(\"$OPTIONS{e}/MA_plots_normalisation.pdf\", width=14)
	for(i in 1:length(pw_tests)) {
		j <- c(which(names(groups) == pw_tests[[i]][1])[1], which(names(groups) == pw_tests[[i]][2])[1])
		par(mfrow = c(1, 2))
		maPlot(toc[, j[1]], toc[, j[2]], normalize = TRUE, pch = 19, cex = 0.2, ylim = c(-10, 10), main=paste(\"MA Plot\", colnames(toc)[j[1]], \"vs\", colnames(toc)[j[2]]))
		grid(col = \"blue\")
		abline(h = log2(norm_factors[j[2]]), col = \"red\", lwd = 4)
		maPlot(DGE\$counts[, j[1]]/DGE\$samples\$lib.size[j[1]], DGE\$counts[, j[2]]/DGE\$samples\$lib.size[j[2]], normalize = FALSE, pch = 19, cex = 0.2, ylim = c(-8, 8), main=paste(\"MA Plot\", colnames(toc)[j[1]], \"vs\", colnames(toc)[j[2]], \"Normalised\"))
		grid(col = \"blue\")
	}
	dev.off()
	pdf(file=\"$OPTIONS{e}/MDSplot.pdf\")
	plotMDS(DGE, main=\"MDS Plot\", col=as.numeric(factor(names(groups)))+1, xlim=c(-3,3))
	dev.off()
	tested <- list()
";

my $all_cont;
my @add_cont;
my @fact;
my @fact_names;
my @cp;
my @cp_names;
if(@ARGV) {
	foreach my $input (@ARGV) {
		my @tmp = split "::", $input;
		if($tmp[0] eq "factor") {
			$tmp[1] =~ s/[ \?\(\)\[\]\/\\=+<>:;\"\',\*\^\|\&-]/./g;
			push @fact_names, $tmp[1];
			$tmp[2] =~ s/:/\", \"/g;
			$tmp[2] = "\"".$tmp[2]."\"";
			push @fact, $tmp[2];
		} elsif($tmp[0] eq "cp") {
			$tmp[1] =~ s/[ \?\(\)\[\]\/\\=+<>:;\"\',\*\^\|\&-]/./g;
			push @cp_names, $tmp[1];
			$tmp[2] =~ s/:/, /g;
			push @cp, $tmp[2];
		} elsif($tmp[0] eq "cnt") {
			push @add_cont, $tmp[1];
		} else {
			die("Unknown Input: $input\n");
		}
	}
}

if($OPTIONS{a} eq "pw") {
	print Rcmd "
		disp <- estimateCommonDisp(DGE, rowsum.filter=$OPTIONS{r})
	";
	if(defined $OPTIONS{t}) {
		print Rcmd "
			disp <- estimateTagwiseDisp(disp, trend=\"$OPTIONS{u}\", prop.used=$OPTIONS{p})
			pdf(file=\"$OPTIONS{e}/Tagwise_Dispersion_vs_Abundance.pdf\")
			plot(log2(1e06*disp\$conc\$conc.common), disp\$tagwise.dispersion, xlab=\"Counts per million (log2 scale)\", ylab=\"Tagwise dispersion\")
			abline(h=disp\$common.dispersion, col=\"firebrick\", lwd=3)
			dev.off()
		"
	}
	print Rcmd "
		for(i in 1:length(pw_tests)) {
			tested[[i]] <- exactTest(disp, pair=pw_tests[[i]])
			names(tested)[i] <- paste(pw_tests[[i]][2], \"-\", pw_tests[[i]][1], sep=\"\")
		}
		pdf(file=\"$OPTIONS{e}/Smear_Plots.pdf\")
		for(i in 1:length(pw_tests)) {
			if(nrow(decideTestsDGE(tested[[i]] , p.value=0.05)) > 0) {
				de_tags <- rownames(decideTestsDGE(tested[[i]] , p.value=0.05, adjust.method=\"$OPTIONS{f}\"))
				ttl <- \"(Diff. Exp. Genes With adj. Pvalue < 0.05 highlighted)\"
			} else {
				de_tags <- rownames(topTags(tested[[i]], n=100)\$table)
				ttl <- \"(Top 100 tags highlighted)\"
			}
			
			plotSmear(disp, pair=pw_tests[[i]], de.tags = de_tags, main = paste(\"FC plot\", ttl))
			abline(h = c(-2, 2), col = \"dodgerblue\")
		}
		dev.off()
	";
} elsif($OPTIONS{a} eq "glm") {
	for(my $fct = 0; $fct <= $#fact_names; $fct++) {
		print Rcmd "
			$fact_names[$fct] <- c($fact[$fct])
		";
	}
	for(my $fct = 0; $fct <= $#cp_names; $fct++) {
		print Rcmd "
			$cp_names[$fct] <- c($cp[$fct])
		";
	}
	my $all_fact = "";
	if(@fact_names) {
		foreach (@fact_names) {
			$all_fact .= " + factor($_)";
		}
    	}
	my $all_cp = "";
	if(@cp_names) {
		$all_cp = " + ".join(" + ", @cp_names);
	}
	print Rcmd "
		group_fact <- factor(names(groups))
		design <- model.matrix(~ -1 + group_fact${all_fact}${all_cp})
		colnames(design) <- sub(\"group_fact\", \"\", colnames(design))
	";
	foreach my $fct (@fact_names) {
		print Rcmd "
			colnames(design) <- make.names(sub(\"factor.$fct.\", \"\", colnames(design)))
		";
	}
	print Rcmd "
		disp <- estimateGLMCommonDisp(DGE, design)
	";
	if($OPTIONS{d} eq "tag" || $OPTIONS{d} eq "trend") {
		print Rcmd "
			disp <- estimateGLMTrendedDisp(disp, design)
		";
	}
	if($OPTIONS{d} eq "tag") {
		print Rcmd "
			disp <- estimateGLMTagwiseDisp(disp, design)
			fit <- glmFit(disp, design)
			pdf(file=\"$OPTIONS{e}/Tagwise_Dispersion_vs_Abundance.pdf\")
			plot(fit\$abund+log(1e06), sqrt(disp\$tagwise.dispersion), xlab=\"Counts per million (log2 scale)\", ylab=\"Tagwise dispersion\")
			oo <- order(disp\$abundance)
			lines(fit\$abundance[oo]+log(1e06), sqrt(disp\$trended.dispersion[oo]), col=\"dodgerblue\", lwd=3)
			abline(h=sqrt(disp\$common.dispersion), col=\"firebrick\", lwd=3)
			dev.off()
		";
	}
	if(@add_cont) {
		$all_cont = "\"".join("\", \"", @add_cont)."\"";
		print Rcmd "
			cont <- c(${all_cont})
			for(i in uniq_groups)  cont <- gsub(paste(groups[[i]], \"([^0-9])\", sep=\"\"), paste(i, \"\\\\1\", sep=\"\"), cont)
			for(i in uniq_groups)  cont <- gsub(paste(groups[[i]], \"\$\", sep=\"\"), i, cont)
		";
	} else {
		print Rcmd "
			cont <- NULL
		";
	}
	if(defined $OPTIONS{m}) {
		print Rcmd "
			for(i in 1:length(pw_tests)) cont <- c(cont, paste(pw_tests[[i]][2], \"-\", pw_tests[[i]][1], sep=\"\"))
		";
	}
	if(!defined $OPTIONS{m} && !@add_cont){
		die("No Contrasts have been specified, you must at least either select multiple pairwise comparisons or specify a custom contrast\n");
	}
	print Rcmd "
		fit <- glmFit(disp, design)
		cont <- makeContrasts(contrasts=cont, levels=design)
		for(i in colnames(cont)) tested[[i]] <- glmLRT(disp, fit, contrast=cont[,i])
	";
	if(defined $OPTIONS{n}) {
		print Rcmd "
			tab <- data.frame(ID=rownames(fit\$fitted.values), fit\$fitted.values, stringsAsFactors=F)
			write.table(tab, \"$OPTIONS{n}\", quote=F, sep=\"\\t\", row.names=F)
		";
	}			
} elsif($OPTIONS{a} eq "limma") {
	for(my $fct = 0; $fct <= $#fact_names; $fct++) {
		print Rcmd "
			$fact_names[$fct] <- c($fact[$fct])
		";
	}
	for(my $fct = 0; $fct <= $#cp_names; $fct++) {
		print Rcmd "
			$cp_names[$fct] <- c($cp[$fct])
		";
	}
	my $all_fact = "";
	if(@fact_names) {
		foreach (@fact_names) {
			$all_fact .= " + factor($_)";
		}
	}
	my $all_cp = "";
	if(@cp_names) {
		$all_cp = " + ".join(" + ", @cp_names);
	}
	print Rcmd "
		group_fact <- factor(names(groups))
		design <- model.matrix(~ -1 + group_fact${all_fact}${all_cp})
		colnames(design) <- sub(\"group_fact\", \"\", colnames(design))
	";
	foreach my $fct (@fact_names) {
		print Rcmd "
			colnames(design) <- make.names(sub(\"factor.$fct.\", \"\", colnames(design)))
		";
	}
	print Rcmd "
		isexpr <- rowSums(cpm(toc)>1) >= 2
		toc <- toc[isexpr, ]
		pdf(file=\"$OPTIONS{e}/LIMMA_voom.pdf\")
		y <- voom(toc, design, plot=TRUE, lib.size=colSums(toc)*norm_factors)
		dev.off()

		pdf(file=\"$OPTIONS{e}/LIMMA_MDS_plot.pdf\")
		plotMDS(y, labels=colnames(toc), col=as.numeric(factor(names(groups)))+1, gene.selection=\"common\")
		dev.off()
		fit <- lmFit(y, design)
	";
	if(defined $OPTIONS{n}) {
		if(defined $OPTIONS{l}) {
			print Rcmd "
				tab <- data.frame(ID=rownames(y\$E), y\$E, stringsAsFactors=F)
			";
		} else {
			print Rcmd "
				tab <- data.frame(ID=rownames(y\$E), 2^y\$E, stringsAsFactors=F)
			";
		}
		print Rcmd "
			write.table(tab, \"$OPTIONS{n}\", quote=F, sep=\"\\t\", row.names=F)
		";
	}		
	if(@add_cont) {
		$all_cont = "\"".join("\", \"", @add_cont)."\"";
		print Rcmd "
			cont <- c(${all_cont})
			for(i in uniq_groups)  cont <- gsub(paste(groups[[i]], \"([^0-9])\", sep=\"\"), paste(i, \"\\\\1\", sep=\"\"), cont)
			for(i in uniq_groups)  cont <- gsub(paste(groups[[i]], \"\$\", sep=\"\"), i, cont)
		";
	} else {
		print Rcmd "
			cont <- NULL
		";
	}
	if(defined $OPTIONS{m}) {
		print Rcmd "
			for(i in 1:length(pw_tests)) cont <- c(cont, paste(pw_tests[[i]][2], \"-\", pw_tests[[i]][1], sep=\"\"))
		";
	}
	if(!defined $OPTIONS{m} && !@add_cont){
		die("No Contrasts have been specified, you must at least either select multiple pairwise comparisons or specify a custom contrast\n");
	}
	print Rcmd "
		cont <- makeContrasts(contrasts=cont, levels=design)
		fit2 <- contrasts.fit(fit, cont)
		fit2 <- eBayes(fit2)
	";
} else {
	die("Anaysis type $OPTIONS{a} not found\n");
	
}

if($OPTIONS{a} ne "limma") {
	print Rcmd "
		options(digits = 6)
		tab <- NULL
		for(i in names(tested)) {
			tab_tmp <- topTags(tested[[i]], n=Inf, adjust.method=\"$OPTIONS{f}\")[[1]]
			colnames(tab_tmp) <- paste(i, colnames(tab_tmp), sep=\":\")
			tab_tmp <- tab_tmp[tagnames,]
			if(is.null(tab)) {
				tab <- tab_tmp
			} else tab <- cbind(tab, tab_tmp)
		}
		tab <- cbind(Feature=rownames(tab), tab)
	";
} else {
	print Rcmd "
		tab <- NULL
		options(digits = 6)
		for(i in colnames(fit2)) {
			tab_tmp <- topTable(fit2, coef=i, n=Inf, sort.by=\"none\", adjust.method=\"$OPTIONS{f}\")
			colnames(tab_tmp)[-1] <- paste(i, colnames(tab_tmp)[-1], sep=\":\")
			if(is.null(tab)) {
				tab <- tab_tmp
			} else tab <- cbind(tab, tab_tmp[,-1])
		}
	";
}
print Rcmd "
	write.table(tab, \"$OPTIONS{o}\", quote=F, sep=\"\\t\", row.names=F)
	sink(type=\"message\")
	sink()
";
close(Rcmd);
system("R --no-restore --no-save --no-readline < $OPTIONS{e}/r_script.R > $OPTIONS{e}/r_script.out");

open(HTML, ">$OPTIONS{h}");
print HTML "<html><head><title>EdgeR: Empirical analysis of digital gene expression data</title></head><body><h3>EdgeR Additional Files:</h3><p><ul>\n";
print HTML "<li><a href=MA_plots_normalisation.pdf>MA_plots_normalisation.pdf</a></li>\n";
print HTML "<li><a href=MDSplot.pdf>MDSplot.pdf</a></li>\n";
if($OPTIONS{a} eq "pw") {
	if(defined $OPTIONS{t}) {
		print HTML "<li><a href=Tagwise_Dispersion_vs_Abundance.pdf>Tagwise_Dispersion_vs_Abundance.pdf</a></li>\n";
	}
	print HTML "<li><a href=Smear_Plots.pdf>Smear_Plots.pdf</a></li>\n";
} elsif($OPTIONS{a} eq "glm" && $OPTIONS{d} eq "tag") {
	print HTML "<li><a href=Tagwise_Dispersion_vs_Abundance.pdf>Tagwise_Dispersion_vs_Abundance.pdf</a></li>\n";
} elsif($OPTIONS{a} eq "limma") {
	print HTML "<li><a href=LIMMA_MDS_plot.pdf>LIMMA_MDS_plot.pdf</a></li>\n";
	print HTML "<li><a href=LIMMA_voom.pdf>LIMMA_voom.pdf</a></li>\n";
}
print HTML "<li><a href=r_script.R>r_script.R</a></li>\n";
print HTML "<li><a href=r_script.out>r_script.out</a></li>\n";
print HTML "<li><a href=r_script.err>r_script.err</a></li>\n";
print HTML "</ul></p>\n";
close(HTML);