polii.gene.call.R

#!/usr/bin/env Rscript 

# polii.gene.call.r
# Copyright © 2014-15, Owen Marshall

# This program is free software; you can redistribute it and/or modify
# it under the terms of the GNU General Public License as published by
# the Free Software Foundation; either version 2 of the License, or (at
# your option) any later version. 
# 
# This program is distributed in the hope that it will be useful, but
# WITHOUT ANY WARRANTY; without even the implied warranty of
# MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE.  See the GNU
# General Public License for more details. 
# 
# You should have received a copy of the GNU General Public License
# along with this program; if not, write to the Free Software
# Foundation, Inc., 59 Temple Place, Suite 330, Boston, MA  02111-1307 
# USA

### FDR calcs ###
# Method based on original perl scripts by Tony Southall (TDS) as published in
# Southall et al. (2013). Dev Cell, 26(1), 101–12. doi:10.1016/j.devcel.2013.05.020
#
# Significant modifications to the original methodology include:
# * taking a linear regression of log data rather than trial-and-error curve fitting of non-log data
# * using a linear regression for the final intercept value rather than using the average intercept value for all conditions
# -- both of these should increase the accuracy of the final FDR value.

version <- "1.0.2"
cat(paste("polii.gene.call v",version,"\n", sep=""))

library(tools)

### Read CLI options
input.args <- commandArgs(trailingOnly = TRUE)

in.files <- vector()
read.ops <- function (x) {
  for (op in x) {
	if (any(grepl("^--",op))) {
		op <- gsub("^--","",op)
		y <- unlist(strsplit(op,"="))
		
		if (y[1] == "help") {
		  cat(paste("Usage: Rscript polii.gene.call.r --genes.file=[some_genes_file.gff] [list of .gatc.bedgraph or .gatc.gff ratio files to process]\n\n", sep=""))
		  
		  cat("Options:\n")
		  for (n in names(op.args)) {
			cat(paste("  ",n,"=",op.args[[n]],"\n",sep=""))
		  }
		  cat("\n")
		  quit("no",1)
		}
		
		if (!is.null(op.args[[ y[1] ]])) {
		  op.args[[ y[1] ]] <<- y[2]
		} else {
		  cat("Error: Option",y[1],"not recognised ...\n")
		  quit("no",1)
		}
	} else {
		in.files <<- c(in.files,op)
	}
  }
}

write.ops <- function () {
  out.df <- data.frame()
  for (n in names(op.args)) {
	v <<- as.character(op.args[[n]])
	df.line <- data.frame(
	  option=n,
	  value=v
	)
	out.df <- rbind(out.df, df.line)
  }
  write.table(out.df,"input.args.single.txt",row.names=F)
}

op.args <- list(
  "genes.file" = "/mnt/data/Genomes/dmel_release/DmR6/DmR6.genes.gff",
  "iter" = 50000,
  "fdr" = 0.01
)

read.ops(input.args)

if (length(in.files) == 0) {
	cat("Usage: Rscript polii.gene.call.r [list of .gatc.gff ratio files to process]\n\n")
	quit("no",1)
}

write.ops()

### save random seed for future reproducibility
dump.random <- runif(1)
my.seed  <- .Random.seed
write.table(my.seed,".randomseed")

### read genes file
cat("Reading genes data file ...\n")
genes.file=op.args[["genes.file"]]
genes <- read.table(genes.file, comment.char="#", sep="\t", quote="", fill=T)
names(genes) <-  c('chr','source','type','start','end','score','strand','c','details')

# only subset if there is a type termed "gene"
if (any(genes$type == 'gene')) {
  genes <- subset(genes, type=='gene')
}

genes$name <- sapply(genes$details, FUN = function (x) {regmatches(x,gregexpr("(?<=Name=).*?(?=;)", x, perl=T))} )
genes <- genes[,c('chr','start','end','strand','name')]
genes$chr <- gsub("^chr","",genes$chr,perl=T)

if (nrow(genes) == 0) {
	cat("Error: unable to extract gene information from genes file\n\n")
	quit("no",1)
}

### functions
read.gff <- function (x,name="score") {
  fn.ext <- file_ext(x)                                                                                                 ##identifies the extension of the file as alphanumeric string
  
  if (grepl("gff",ignore.case=T,fn.ext)) {                                                                              ##'ignore.case=T' indicates to treat small & capital letters similarly
	temp.data <- read.table(x,row.names=NULL)
	if (ncol(temp.data) > 5) {                                                                                            ##checking only for 'ncol()>5' implies that only 'gff'- or 'bedgraph'-files would be parsed 
	  # GFF
	  trim.data <- temp.data[,c(1,4,5,6)]
	} else {
		cat("Error: file does not appear to be in GFF format\n\n")
		quit("no",1)
	}
  } else if (grepl("bed",ignore.case=T,fn.ext)) {
	temp.data <- read.table(x,row.names=NULL,skip=1)                                                                     ##skip indicates to ignore the header of the bedgraph-file
	if (ncol(temp.data) == 4) {
		# bedgraph
		trim.data <- temp.data
	} else {
		cat("Error: file does not appear to be in bedGraph format\n\n")
		quit("no",1)
	}
  } else {
	cat("Error: input file does not appear to be in bedGraph or GFF format ...\n\n")
	quit("no",1)
  }
  
  names(trim.data) <- c("chr","start","end",name)
  
  trim.data$chr <- gsub("^chr","",trim.data$chr,perl=T)
  
  return(trim.data)
}

gene.exp <- function (input.df, buffer=0, iter=50000, debug=F) {
  
  avg.exp <- data.frame(input.df[1,c(4:(length(names(input.df))))])
  avg <- vector(length=(length(names(input.df)) - 4))
  avg.exp <- avg.exp[0,]                                                                                              ##reset the entire dataframe
  
  ### FDR calcs ###
  # Method based off perl scripts by Tony Southall (TDS) as published in
  # Southall et al. (2013). Dev Cell, 26(1), 101–12. doi:10.1016/j.devcel.2013.05.020
  #
  # Significant modifications to the original methodology include:
  # * taking a linear regression of log data rather than trial-and-error curve fitting of non-log data
  # * using a linear regression for the final intercept value rather than using the average intercept value for all conditions
  # -- both of these should increase the accuracy of the final FDR value.
  
  input.len <- length(input.df[,1])
  frag.samp <- c(1,2,3,4,6,8,10,12,15)
  thres.samp <- c(0.1,0.2,0.3,0.4,0.5,0.65,0.8,1.0,1.5,2.0)
  rand <- list()
  
  for (thres in thres.samp) {
    
    cat(paste("  Calculating FDR for threshold",thres,"\n",sep=" "))
    
    # init vars
    for (f in frag.samp) {                                                                                           ##create list of different threshold-set-ups
      # List names are, e.g. frag 1, thres 0.2: rand[[thres.1.0.2]]
      rand[[paste("thres.",f,".",thres,sep="")]] <- 0;
    }
    
    for (i in 1:iter) {
      if (i %% 200 == 0) {cat(paste("  iter",i,"\r"))}                                                               ##TRUE, whenever i is multiple of 200
      # get random sample for different fragment lengths
      
      rand.samp <- list()
      
      for (f in frag.samp) {
        # Using the fourth column as we're only calculating FDR for one sample ...
        rand.samp[[paste("rand.",f,sep="")]] <- mean(input.df[runif(f,1,input.len),4])                               ##pick f-number of random values from the entire column 4 (i.e., "val") in the *.bedgraph-file, calculate the mean &
      }                                                                                                              ##store in the 'rand.sample'-list --> for every iteration, a new set of 9 values (=frag.sample) generated
      
      # count number of times exp > thres
      for (f in frag.samp) {                                                                                         ##register the number of times the mean of the randomized sampling exceeds the specified threshold
        if (rand.samp[[paste("rand.",f,sep="")]] > thres) {rand[[paste("thres.",f,".",thres,sep="")]] <- rand[[paste("thres.",f,".",thres,sep="")]] + 1}
      }
    }
  }
  
  rand.fdr <- list()
  
  for (thres in thres.samp) {
    for (f in frag.samp) {                                                                                          ##calculate the normalized counts by dividing them with the amount of iterations used
      rand.fdr[[paste("thres.",f,".",thres,sep="")]] <- rand[[paste("thres.",f,".",thres,sep="")]]/iter
    }
  }
  
  cat("Fitting curves ...\n")
  
  # curve fit: fdr vs thresholds
  var.thres <- list()
  
  for (thres in thres.samp) {
    for (f in frag.samp) {                                                                                         ##enhances the newly generated var.thres by the 'f'-related corresponding entries for rand.fdr, i.e., concatenation
      var.thres[[paste("frags.",f,sep="")]] <- append(var.thres[[paste("frags.",f,sep="")]], rand.fdr[[paste("thres.",f,".",thres,sep="")]])
    }
  }
  
  inf.log.lm <- function (v) {
    non.inf <- log(v) != -Inf
    ret <- lm(log(v)[non.inf] ~ thres.samp[non.inf])
    return(ret)
  }
  
  # The relationship is exponential, so we need log data for a linear regression
  # (in R, linear regression is: y = lm$coefficients[[2]]x + lm$coefficients[[1]] ... )
  var.lm <- list()
  for (f in frag.samp) {                                                                                          ##parses all values from rand.fdr/var.thres to the inf.log.lm-function, where these values are correlated with the thres.sample-values
    var.lm[[paste("frags.",f,sep="")]] <- inf.log.lm(var.thres[[paste("frags.",f,sep="")]])
  }
  
  # ... and now we do a linear regression on the slopes and intercepts of our previous regressions
  # (This is the clever bit, and it actually seems to work.  The correlation of slope to fragment size is linear ...
  # By doing this on the slope and intercept, we can now predict the FDR for any number of fragments with any expression.)
  slope <- vector()
  for (f in frag.samp) {
    slope <- append(slope, var.lm[[paste("frags.",f,sep="")]]$coefficients[[2]])
  }
  
  # slope regression predicts the average slope
  slope.lm <- lm(slope ~ frag.samp)
  
  # TDS used an average intercept value for the intercept, however ...
  inter <- vector()
  for (f in frag.samp) {
    inter <- append(inter, var.lm[[paste("frags.",f,sep="")]]$coefficients[[1]])
  }
  
  # ... there's actually quite a bit of variation of the intercept with real data,
  # so we're going to do a lin regression on the intercept instead.
  #
  # (I'm not convinced it's a true linear relationship.  But it's close to linear,
  # and will certainly perform better than taking the mean intercept ...)
  #
  # If you're interested, set the debug flag to TRUE and take a look at the plots generated below ...
  inter.lm <- lm(inter ~ frag.samp)
  
  if (debug == T) {
    # plots for debugging/checking
    plot.debug <- function (y,x,l,name="Debug plot") {
      plot(y ~ x)
      abline(l)
      
      lsum <- summary(l)
      r2 <- lsum$r.squared
      
      legend("topleft",legend=r2,bty='n')
      title(name)
      
      dev.copy(png,paste(name,".png",sep=""));dev.off()
    }
    
    plot.debug(slope,frag.samp,slope.lm,name="correlation of slope")
    plot.debug(inter,frag.samp,inter.lm,name="correlation of intercepts")
  }
  
  # ok, so putting that all together ...
  fdr <- function (frags, expr) {
    inter.test <- inter.lm$coefficients[[2]] * frags + inter.lm$coefficients[[1]]
    slope.test <- slope.lm$coefficients[[2]] * frags + slope.lm$coefficients[[1]]
    
    fdr.out <- exp(slope.test * expr + inter.test)
    return(fdr.out)
  }
  
  ### Gene expression values ###  
  cat("Calculating gene values ...\n")
  
  count <- 0                                                                                                      ##resets the counter
  
  # unroll chromosomes for speed:
  for (chromo in unique(genes$chr)) {                                                                             ##unfolds only the chromosomes that encode for the genes of interest, loops through the individual chromosomes
    input.chr <- subset(input.df, chr==chromo)                                                                    ##subset the current bedgraph-file to the current chromosome
    genes.chr <- subset(genes, chr==chromo)                                                                       ##subset the gene-list to the current chromosome
    for (i in 1:length(genes.chr$name)) {                                                                         ##loop through the genes encoded on the current chromosome
      # Roll through each gene
      
      # Note: the original script calculated expression values for a table of proteins,
      # here we just limit ourselves to the FDR for the first column past "chr", "start" and "end"
      # so if you're thinking of looking at chromatin, for example, put PolII as column 4 in your table!
      
      gene.start <- genes.chr[i,"start"] - buffer                                                                 ##extend the start & end position of the current gene by a predefined margin
      gene.end <- genes.chr[i,"end"] + buffer
      
      gene.start <- ifelse(gene.start < 1, 1, gene.start)                                                         ##if the gene-start-side is less than 1 due to the extension by the predefined margin, set the start-position back to '1'
      
      # Create data frames for all gatc fragments covering current gene
      exp <- data.frame(input.chr[(input.chr$start <= gene.end) & (input.chr$end >= gene.start),] )               ##subsets only the GATC-fragments falling fully into the gene body (inbetween the start- & end-coordinates)
      
      gatc.num <- length(exp[,1])
      
      # skip if no gatc fragments cover gene :(
      if (gatc.num == 0) {next}
      
      # trim to gene boundaries ...
      exp$start[1] <- gene.start                                                                                  ##extend the first and last GATC-fragment within the gene body to the start and end positions of the actual gene
      exp$end[length(exp[,1])] <- gene.end
      
      # gene length covered by gatc fragments
      len <- sum(exp$end-exp$start)                                                                               ##similar to 'exp$end[nrow(exp)]-exp$start[1]'
      
      # calculate weighted score for each column (representing different proteins)
      for (j in 4:length(names(input.chr))) {
        avg[j] <- (sum((exp$end-exp$start)*exp[j]))/len                                                           ##weigh the intensity associated to the individual GATC-fragments according to their length, sum these weighted fragments & divide by the entire gene length
      }
     
      # make data.frame of averages (to be appended to avg.exp)                                                   ##concatenate the weighted averages for the gene (gene_isoforms) in a dataframe by cbind
      df <- cbind(avg[1])
      for (k in 2:length(avg)) {
        df <- cbind(df,avg[k])
      }
      df <- cbind(df,gatc.num)                                                                                    ##add the maximum number of GATC-fragments associated with this particular gene (and its isoforms)
      
      # only fdr for first column for now ...
      gene.fdr <- fdr(gatc.num,avg[4])                                                                            ##invoke the fdr-function (see above) for this particular gene & the amount of GATC-fragments associated with it
      df <- cbind(df, gene.fdr)                                                                                   ##include it into the dataframe via cbind
      
      # append current gene to list
      avg.exp <- rbind(avg.exp,data.frame(name=as.character(genes.chr[i,"name"]), df))                            ##concatenate the information for the current gene to the avg.exp-dataframe
      count <- count+1
      if (count %% 50 == 0) {cat(paste(count,"genes averaged ...\r"))}
    }
  }
  
  avg.exp <- avg.exp[,c(1,5:(length(names(avg.exp))))]
  names(avg.exp) <- c("name",names(input.df)[c(4:(length(names(input.df))))],"gatc.num","FDR")
  
  return(avg.exp)
}

for (name in in.files) {
	cat(paste("\nNow working on",name,"...\n"))
	bname <- basename(name)
	fname <- sub("\\..*","",bname,perl=T)
	
	polii <- read.gff(name,"polii")
	
	polii.exp <- gene.exp(polii, iter=op.args[["iter"]])
	
	out <- subset(polii.exp,FDR < op.args[["fdr"]])
	
	write.table(polii.exp,paste(fname,"genes.details.csv",sep="."),row.names=F,col.names=T,quote=T,sep="\t")
	write.table(out$name, paste(fname,"genes",sep="."),row.names=F,col.names=F,quote=F)
}

cat("\nAll done.\n\n")