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1 ###############################################################################
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2 # In summary this script normalizes each plate of input data to each of its negative
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3 # controls.
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4 #
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5 # Args:
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6 # input.data.frame contains one or more columns of plate data. The first column
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7 # must contain the well annotation ie A01, A02 etc. Each subsequent column represents
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8 # a plate of data where the column name is the plate name.
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9 #
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10 # plate.conf.data.frame is a file of the following format:
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11 # well, type (syntax must be either "poscontr" or "negcontr" or "empty"), name (name of control)
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12 #
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13 # Returns:
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14 # plate values normalized to each negative control type per plate.
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15 #
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16 # Author: kate gould
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17 ###############################################################################
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18
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19 use strict;
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20 use warnings;
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21 use IO::Handle;
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22 use File::Temp qw/ tempfile tempdir /;
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23 my $tdir = tempdir( CLEANUP => 0 );
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24
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25 # check to make sure having correct input and output files
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26 my $usage = "usage: normalizeMedianNegCntrl.pl [TABULAR.in] [TABULAR.in] [TABULAR.out] \n";
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27 die $usage unless @ARGV == 3;
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28
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29 #get the input arguments
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30 my $plateData = $ARGV[0];
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31 my $plateConfig = $ARGV[1];
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32 my $normMedNeg = $ARGV[2];
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33
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34 #open the input files
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35 open (INPUT1, "<", $plateData) || die("Could not open file $plateData \n");
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36 open (INPUT2, "<", $plateConfig) || die("Could not open file $plateConfig \n");
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37 open (OUTPUT1, ">", $normMedNeg) || die("Could not open file $normMedNeg \n");
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38
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39 #variable to store the name of the R script file
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40 my $r_script;
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41
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42 # R script to implement the calcualtion of q-values based on multiple simultaneous tests p-values
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43 # construct an R script file and save it in a temp directory
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44 chdir $tdir;
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45 $r_script = "normalizeMedianNegCntrl.r";
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46
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47 open(Rcmd,">", $r_script) or die "Cannot open $r_script \n\n";
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48 print Rcmd "
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49 #options(show.error.messages = FALSE);
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50 header <- read.table(\"$plateData\", head=F, sep=\"\\t\", comment=\"\", nrows=1);
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51 input.data.frame <- read.table(\"$plateData\", head=F, sep=\"\\t\", skip=1);
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52 plate.conf.data.frame <- read.table(\"$plateConfig\", head=T, sep=\"\\t\", comment=\"\");
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53
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54 normalise <- function(x, y){if (!is.na(x)) return (x/y) else (return (NA))}
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55
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56 # assumed second column is type ie negcontr and third column is name of control
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57 negative.controls.list <- unique(plate.conf.data.frame[which (plate.conf.data.frame[,2] == \"negcontr\"),][,3]);
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58
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59 normalized.data.frame <- data.frame(well=input.data.frame[,1], stringsAsFactors=FALSE);
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60 column.name.list <- c();
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61 for (negative.control in negative.controls.list){
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62 negative.control.wells <- plate.conf.data.frame[which (plate.conf.data.frame[3] == negative.control),][,1];
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63
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64 for (i in 2:length(colnames(input.data.frame))){
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65 column.name <- paste(colnames(input.data.frame)[i], \".normalised.to.\", negative.control, sep=\"\");
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66 column.name.list <- append(column.name.list, column.name);
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67
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68 negative.control.values <- input.data.frame[((input.data.frame[,1] %in% negative.control.wells)&(!(is.na(input.data.frame[,i])))),][,i];
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69 median.negative.value <- median(negative.control.values);
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70
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71 normalised.to.median.negative.control.value <- round(sapply(input.data.frame[,i], FUN=normalise, median.negative.value), 2);
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72 normalized.data.frame <- cbind(normalized.data.frame, normalised.to.median.negative.control.value);
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73 }
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74 }
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75 header <- c(header[1], column.name.list)
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76 write.table(header, file=\"$normMedNeg\", quote=F, sep=\"\\t\", row.names=F, col.names=F);
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77 write.table(normalized.data.frame, file=\"$normMedNeg\", quote=F, sep=\"\\t\", row.names=F, col.names=F, append=T);
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78 #eof\n";
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79
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80 close Rcmd;
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81
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82 system("R --no-restore --no-save --no-readline < $r_script > $r_script.out");
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83
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84 #close the input and output files
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85 close(OUTPUT1);
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86 close(INPUT1);
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87 close(INPUT2);
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