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1 #######################################################################################
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2 # R-code: Protein Name and Tukey's Normalization
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3 # Author: Adam L Borne
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4 # Contributers: Paul A Stewart, Brent Kuenzi
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5 #######################################################################################
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6 # Assigns uniprot id from MaxQuant peptides file. Filters and normalizes the
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7 # intensities of each proteins. Resulting in a one to one list of intensities to
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8 # uniprot id.
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9 #######################################################################################
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10 # Copyright (C) Adam Borne.
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11 # Permission is granted to copy, distribute and/or modify this document
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12 # under the terms of the GNU Free Documentation License, Version 1.3
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13 # or any later version published by the Free Software Foundation;
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14 # with no Invariant Sections, no Front-Cover Texts, and no Back-Cover Texts.
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15 # A copy of the license is included in the section entitled "GNU
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16 # Free Documentation License".
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17 #######################################################################################
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18 ## REQUIRED INPUT ##
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19
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20 # 1) peptides_file: MaxQuant peptides file.
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21 #######################################################################################
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22
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23
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24 ins_check_run <- function() {
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25 if ("affy" %in% rownames(installed.packages())){}
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26 else {
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27 source("https://bioconductor.org/biocLite.R")
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28 biocLite(c('mygene','affy'))
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29 }
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30 if ('data.table' %in% rownames(installed.packages())){}
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31 else {
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32 install.packages('data.table', repos='http://cran.us.r-project.org')
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33 }
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34 if ('stringr' %in% rownames(installed.packages())){}
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35 else {
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36 install.packages('stringr', repos='http://cran.us.r-project.org')
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37 }
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38 if ('VennDiagram' %in% rownames(installed.packages())){}
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39 else {
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40 install.packages('VennDiagram', repos='http://cran.us.r-project.org')
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41 }
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42 }
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43
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44 ins_check_run()
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45 library(data.table)
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46 library(affy)
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47 library(stringr)
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48 library(mygene)
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49 library(VennDiagram)
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50
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51
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52 main <- function(peptides_file, db_path) {
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53 peptides_file = read.delim(peptides_file,header=TRUE,stringsAsFactors=FALSE,fill=TRUE)
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54 peptides_txt = peptides_file
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55 intensity_columns = names(peptides_txt[,str_detect(names(peptides_txt),"Intensity\\.*")])
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56 # Pulls out all lines with Intensity in them.
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57 intensity_columns = intensity_columns[2:length(intensity_columns)]
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58 # Removes the first column that does not have a bait.
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59 peptides_txt_mapped = as.data.frame(map_peptides_proteins(peptides_txt))
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60 # This function as below sets every line to a 1 to 1 intensity to each possible protein.
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61 peptides_txt_mapped$Uniprot = str_extract(peptides_txt_mapped$mapped_protein, "[OPQ][0-9][A-Z0-9]{3}[0-9]|[A-NR-Z][0-9]([A-Z][A-Z0-9]{2}[0-9]){1,2}") #Pulls out just Uniprot id from the script.
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62 peptides_txt_mapped = subset(peptides_txt_mapped,!is.na(Uniprot))
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63 # Removes reverse sequences and any that didn't match a uniprot accession.
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64 columns_comb = c("Uniprot", intensity_columns)
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65 peptides_mapped_intensity = subset(peptides_txt_mapped, select = columns_comb)
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66 # Subsets out only the needed cloumns for Tukeys (Uniprot IDS and baited intensities)/
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67 swissprot_fasta = scan(db_path, what="character")
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68 peptides_txt_mapped_log2 = peptides_mapped_intensity
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69 # Takes the log2 of the intensities.
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70 for (i in intensity_columns) {
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71 peptides_txt_mapped_log2[,i] = log2(subset(peptides_txt_mapped_log2, select = i))
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72 }
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73 # Get the minimum from each column while ignoring the -Inf; get the min of these mins for the
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74 # global min; breaks when there's only one intensity column.
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75 global_min = min(apply(peptides_txt_mapped_log2[,2:ncol(peptides_txt_mapped_log2)],2,function(x) {
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76 min(x[x != -Inf])
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77 }))
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78 peptides_txt_mapped_log2[peptides_txt_mapped_log2 == -Inf] <- NA
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79 #uniprot accessions WITHOUT isoforms; it looks like only contaminants contain isoforms anyways.
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80 mapped_protein_uniprotonly = str_extract(peptides_txt_mapped_log2$Uniprot,"[OPQ][0-9][A-Z0-9]{3}[0-9]|[A-NR-Z][0-9]([A-Z][A-Z0-9]{2}[0-9]){1,2}")
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81 mapped_protein_uniprot_accession = str_extract(peptides_txt_mapped_log2$Uniprot,"[OPQ][0-9][A-Z0-9]{3}[0-9](-[0-9]+)?|[A-NR-Z][0-9]([A-Z][A-Z0-9]{2}[0-9]){1,2}(-[0-9]+)?|[OPQ][0-9][A-Z0-9]{3}[0-9]|[A-NR-Z][0-9]([A-Z][A-Z0-9]{2}[0-9]){1,2}")
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82 peptides_txt_mapped_log2$mapped_protein = mapped_protein_uniprotonly
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32
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83 names_db = str_extract(swissprot_fasta,"[OPQ][0-9][A-Z0-9]{3}[0-9]|[A-NR-Z][0-9]([A-Z][A-Z0-9]{2}[0-9]){1,2}")
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84 names_db = names_db[!is.na(names_db)]
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28
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85 # Runs the Tukey function returning completed table.
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32
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86 peptides_txt_mapped_log2 = subset(peptides_txt_mapped_log2,mapped_protein %in% names_db)
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28
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87 if (nrow(peptides_txt_mapped_log2) == 0) {
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88 print("Uniprot Database does not have any of the proteins in the peptides file")
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89 quit()
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90 }
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91 protein_intensities_tukeys = get_protein_values(peptides_txt_mapped_log2,intensity_columns)
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92 protein_intensities_tukeys[protein_intensities_tukeys == 1] <- 0
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30
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93 # Eliminates all pure NA rows.
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94 protein_intensities_tukeys = protein_intensities_tukeys[rowSums(is.na(protein_intensities_tukeys)) != (ncol(protein_intensities_tukeys)-1),]
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95 write.table(protein_intensities_tukeys, "./tukeys_output.txt", row.names = FALSE, col.names = TRUE, quote = FALSE, sep = "\t")
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28
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96
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97 }
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98
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99 map_peptides_proteins = function(peptides_in) {
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100 peptides_in = subset(peptides_in,peptides_in$Proteins != "")
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101 results_list = list()
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102 k = 1
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103 for (i in 1:nrow(peptides_in)) {
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104 protein_names = peptides_in[i,"Proteins"]
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105 protein_names_split = unlist(str_split(protein_names,";"))
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106 for (j in 1:length(protein_names_split)) {
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107 peptides_mapped_proteins = data.frame(peptides_in[i,],mapped_protein=protein_names_split[j],stringsAsFactors=FALSE)
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108 results_list[[k]] = peptides_mapped_proteins
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109 k = k+1
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110
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111 }
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112 }
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113 return(rbindlist(results_list))
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114 }
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115
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116 get_protein_values = function(mapped_peptides_in,intensity_columns_list) {
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117 unique_mapped_proteins_list = unique(mapped_peptides_in$mapped_protein)
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118 # Gets list of all peptides listed.
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119 # Generates a blank data frame with clomns of Intensities and rows of Uniprots.
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120 Tukeys_df = data.frame(mapped_protein = unique_mapped_proteins_list, stringsAsFactors = FALSE )
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121 for (q in intensity_columns_list) {Tukeys_df[,q] = NA}
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122 for (i in 1:length(unique_mapped_proteins_list)) {
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123 mapped_peptides_unique_subset = subset(mapped_peptides_in, mapped_protein == unique_mapped_proteins_list[i])
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124 # Calculate Tukey's Biweight from library(affy); returns a single numeric.
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125 # Results_list[[i]] = data.frame(Protein=unique_mapped_proteins_list[i],Peptides_per_protein=nrow(mapped_peptides_unique_subset)).
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126 for (j in intensity_columns_list) {
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127 # Populates with new Tukeys values.
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128 Tukeys_df[i,j] = 2^(tukey.biweight(na.omit(mapped_peptides_unique_subset[,j])))
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129 }
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130 }
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131 return(Tukeys_df)
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132 }
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133
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134
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135 args <- commandArgs(trailingOnly = TRUE)
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136 main(args[1], args[2])
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