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1 #!/usr/bin/perl
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2
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3 if(@ARGV<8) { print "usage: $0 [options] -L=<read_length> -D=<maxdist> -1=<input_file1(mate1)> -2=<input_file2(mate2)> -U=<Utable_prefix(fullpath, before .gU.table or .iU.table)> --g2m=<gene2NM_file> --g2s=<gene2symbol_file> -b=<bowtie_dir(eg.bin/bowtie-0.12.7)> --bi=<bowtieindex> -o=<outputdir> -s=<sample_name>\n\nOrder of arguments can be interchangeable.\n\n"; exit; }
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4
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5
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6 ## options ##
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7 ($fastoption)= grep /^-f=/, @ARGV;
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8 if(!defined($fastoption)){$fastoption='q'; }
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9 else { $fastoption =~s/^-f=//; if($fastoption !~/^[fq]$/) { die "ERROR: wrong file type (-f).\n"; } }
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10
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11 ($coding_option)= grep /^-c=/, @ARGV;
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12 if(!defined($coding_option)){$coding_option='n'; }
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13 else { $coding_option =~s/^-c=//; if($coding_option !~/^[nc]$/) { die "ERROR: wrong coding option (-c).\n"; } }
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14
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15 ($datatype)= grep /^-d=/, @ARGV;
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16 if(!defined($datatype)){$datatype='R'; }
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17 else { $datatype =~s/^-d=//; if($datatype !~/^[RE]$/) { die "ERROR: wrong data type (-d).\n"; } }
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18
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19 ($parallel)= grep /^-p=/, @ARGV;
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20 if(!defined($parallel)){$parallel=1; }
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21 else { $parallel =~s/^-p=//; if($parallel !~/^[\d]+$/) { die "ERROR: wrong parallel (multi-thread) option (-p).\n"; } }
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22
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23 ($EUMAcut)= grep /^-t=/, @ARGV;
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24 if(!defined($EUMAcut)){$EUMAcut=50; }
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25 else { $EUMAcut =~s/^-t=//; if($EUMAcut !~/^[\d\.]+$/) { die "ERROR: wrong EUMAcut (-t).\n"; } }
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26
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27 ($StrandSpecificity)= grep /^--str=/, @ARGV;
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28 if(!defined($StrandSpecificity)){ $StrandSpecificity='N'; }
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29 else { $StrandSpecificity =~s/^--str=//; if($StrandSpecificity !~/^[SN]+$/) { die "ERROR: wrong strand specificity (--str).\n"; } }
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30
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31 ($skip_init)= grep /^--skip_init/, @ARGV;
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32 if(!defined($skip_init)){ $skip_init=0; }
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33 else { $skip_init=1; }
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34
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35 ($only_init)= grep /^--only_init/, @ARGV;
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36 if(!defined($only_init)){ $only_init=0; }
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37 else { $only_init=1; }
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38
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39 if($only_init==1 && $skip_init==1) { die "ERROR: --only_init and --skip_init cannot be used simultaneously.\n"; }
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40
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41 ($noNIR)= grep /^--noNIR/, @ARGV;
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42 if(!defined($noNIR)){ $noNIR=0; }
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43 else { $noNIR=1; }
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44
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45 ($noNEUMA)= grep /^--noNEUMA/, @ARGV;
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46 if(!defined($noNEUMA)){ $noNEUMA=0; }
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47 else { $noNEUMA=1; }
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48
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49 if($noNIR==1 && $noNEUMA==0) { die "ERROR: --noNIR must be used together with --noNEUMA.\n"; }
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50
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51 ($get_gReadcount)= grep /^--gReadcount/, @ARGV;
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52 if(!defined($get_gReadcount)){ $get_gReadcount=0; }
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53 else { $get_gReadcount=1; }
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54
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55 ($mm)= grep /^--mm=/, @ARGV;
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56 if(!defined($mm)){ $mm=0; }
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57 else { $mm =~s/^--mm=//; if($mm !~/^[\d\.]+$/) { die "ERROR: wrong number of mismatches (--mm).\n"; } }
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58
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59
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60
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61 ## required arguments ##
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62
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63 ($READ_LENGTH)= grep /^-L=/, @ARGV;
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64 if(!defined($READ_LENGTH)){ die "ERROR: READ_LENGTH must be specified (-L).\n"; }
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65 else { $READ_LENGTH =~s/^-L=//; if($READ_LENGTH !~/^[\d]+$/) { die "ERROR: wrong READ_LENGTH (-L).\n"; } }
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66
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67 ($MAXDIST)= grep /^-D=/, @ARGV;
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68 if(!defined($MAXDIST)){ die "ERROR: MAXDIST must be specified (-D).\n"; }
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69 else { $MAXDIST =~s/^-D=//; if($MAXDIST !~/^[\d]+$/) { die "ERROR: wrong MAXDIST (-D).\n"; } }
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70
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71 ($input_file1)= grep /^-1=/, @ARGV;
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72 if(!defined($input_file1)){ die "ERROR: input_file1 must be specified (-1).\n"; }
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73 else { $input_file1 =~s/^-1=//; if(!-e $input_file1) { die "ERROR: wrong input_file1 (-1).\n"; } }
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74
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75 ($input_file2)= grep /^-2=/, @ARGV;
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76 if(!defined($input_file2)){ die "ERROR: input_file2 must be specified (-2).\n"; }
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77 else { $input_file2 =~s/^-2=//; if(!-e $input_file2) { die "ERROR: wrong input_file2 (-2).\n"; } }
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78
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79 ($Utable_prefix)= grep /^-U=/, @ARGV;
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80 if($only_init==0 && !defined($Utable_prefix)){ die "ERROR: Utable_prefix must be specified (-U).\n"; }
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81 else { $Utable_prefix =~s/^-U=//; if(!-e $Utable_prefix.".gU.table" || !-e $Utable_prefix.".iU.table") { die "ERROR: wrong Utable_prefix (-U).\n"; } }
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82
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83 ($gene2NM_file)= grep /^--g2m=/, @ARGV;
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84 if($only_init==0 && !defined($gene2NM_file)){ die "ERROR: gene2NM_file must be specified (--g2m).\n"; }
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85 else { $gene2NM_file =~s/^--g2m=//; if(!-e $gene2NM_file) { die "ERROR: wrong gene2NM_file (--g2m).\n"; } }
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86
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87 ($gene2symbol_file)= grep /^--g2s=/, @ARGV;
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88 if($only_init==0 && !defined($gene2symbol_file)){ die "ERROR: gene2symbol_file must be specified (--g2s).\n"; }
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89 else { $gene2symbol_file =~s/^--g2s=//; if(!-e $gene2symbol_file) { die "ERROR: wrong gene2symbol_file (--g2s).\n"; } }
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90
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91 ($bowtie_dir)= grep /^-b=/, @ARGV;
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92 if(!defined($bowtie_dir)){ die "ERROR: bowtie_dir must be specified (-b).\n"; }
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93 else { $bowtie_dir =~s/^-b=//; if(!-d $bowtie_dir || !-e "$bowtie_dir/bowtie") { die "ERROR: wrong bowtie_dir(please avoid using '~') (-b).\n"; } }
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94
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95 ($bowtieindex)= grep /^--bi=/, @ARGV;
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96 if(!defined($bowtieindex)){ die "ERROR: bowtieindex must be specified (--bi).\n"; }
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97 else { $bowtieindex =~s/^--bi=//; if(!-e "$bowtieindex.1.ebwt") { die "ERROR: wrong bowtieindex (--bi).\n"; } }
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98
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99 ($basedir)= grep /^-o=/, @ARGV;
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100 if(!defined($basedir)){ die "ERROR: output dir must be specified (-o).\n"; }
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101 else { $basedir =~s/^-o=//; if($basedir=~/~/) { die "ERROR: wrong base_dir(please avoid using '~') (-o).\n"; } }
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102
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103 ($sample)= grep /^-s=/, @ARGV;
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104 if(!defined($sample)){ die "ERROR: sample name must be specified (-s).\n"; }
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105 else { $sample =~s/^-s=//; }
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106
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107
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108 ##############
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109
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110
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111 $gUtable_file = $Utable_prefix.".gU.table";
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112 $iUtable_file = $Utable_prefix.".iU.table";
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113
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114 my $coding='';
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115 if($coding_option eq 'n') {$coding='';}
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116 elsif($coding_option eq 'c') {$coding='-C';}
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117 else { die "ERROR: Error: invalid coding option.\n"; }
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118
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119 if($datatype eq 'R') { $mapping_stat_column = 4; }
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120 elsif($datatype eq 'E') { $mapping_stat_column = 6; }
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121 else { die "ERROR: Error: wrong datatype.\n"; }
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122
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123 if($StrandSpecificity eq 'S') { $bowtie_strand_option = "--norc"; }
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124 else { $bowtie_strand_option = ""; }
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125
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126 ($scriptdir) = $0 =~ /(.+)\/[^\/]+$/;
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127
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128 $bowtieoutdir = "$basedir/bowtieout";
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129 $readcount_dir = "$basedir/readcount";
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130 $lendis_dir = "$basedir/insertlendis";
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131 $EUMAdir = "$basedir/EUMA";
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132 $FVKM_dir = "$basedir/FVKM";
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133 $LVKM_dir = "$basedir/LVKM";
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134
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135 $mapping_stat_file = "$basedir/mapping_stat.$sample";
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136
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137 ($midrefix) = $bowtieindex=~/([^\/]+)$/;
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138
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139
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140 if(!-d $bowtieoutdir) { `mkdir -p $bowtieoutdir`; }
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141 if(!-d $readcount_dir) { `mkdir -p $readcount_dir`; }
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142 if(!-d $lendis_dir) { `mkdir -p $lendis_dir`; }
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143 if(!-d $EUMAdir) { `mkdir -p $EUMAdir`; }
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144 if(!-d $FVKM_dir) { `mkdir -p $FVKM_dir`; }
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145 if(!-d $LVKM_dir) { `mkdir -p $LVKM_dir`; }
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146
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147
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148 #`dos2unix $gene2NM_file`;
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149 #`dos2unix $gene2symbol_file`;
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150
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151
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152
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153 #$perlcommand = "perl -I $scriptdir";
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154 $perlcommand = "perl";
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155 $bestbowtieout_script = "$scriptdir/filter.best.from.bowtieout.3.pl";
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156 $bowtie2insertlendis_script = "$scriptdir/bowtieout2insertlendis.PE.pl";
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157 $bowtieout2mappingstat_script = "$scriptdir/bowtieout2mappingstat.3.pl";
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158 $bowtieout2readcount_script = "$scriptdir/bowtie2genecount.11.pl";
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159
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160 $calc_gEUMA_script = "$scriptdir/calculate_gEUMA.2.pl";
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161 $calc_iEUMA_script = "$scriptdir/calculate_iEUMA.2.pl";
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162 $NIR2LVKM_script = "$scriptdir/NIR2LVKM.pl";
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163
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164
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165 system("date");
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166
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167
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168 if($skip_init==0){
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169 print STDERR "Mapping reads using bowtie...\n";
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170 system("$bowtie_dir/bowtie -$fastoption $coding --minins 0 --maxins $MAXDIST -v $mm -a --suppress 5,6,7 -p $parallel $bowtie_strand_option $bowtieindex -1 $input_file1 -2 $input_file2 > $bowtieoutdir/$sample.$midrefix.maxins$MAXDIST.mm$mm.bowtieout");
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171
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172 print STDERR "Filtering best-matching alignments...\n";
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173 if($mm > 0) {
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174 system("$perlcommand $bestbowtieout_script -d $datatype --rm $bowtieoutdir/$sample.$midrefix.maxins$MAXDIST.mm$mm.bowtieout 1");
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175 }
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176 else {
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177 system("mv $bowtieoutdir/$sample.$midrefix.maxins$MAXDIST.mm$mm.bowtieout $bowtieoutdir/$sample.$midrefix.maxins$MAXDIST.mm$mm.best.bowtieout");
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178 }
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179
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180 print STDERR "Computing fragment length distribution...\n";
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181 system("$perlcommand $bowtie2insertlendis_script $bowtieoutdir/$sample.$midrefix.maxins$MAXDIST.mm$mm.best.bowtieout $READ_LENGTH > $lendis_dir/$sample.$midrefix.maxins$MAXDIST.mm0.i.insertlendis");
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182 }
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183
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184 if($only_init==0) {
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185
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186 print STDERR "Computing the mapping stat...\n";
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187 system("$perlcommand $bowtieout2mappingstat_script -d $datatype -m $MAXDIST -l $READ_LENGTH $bowtieoutdir/$sample.$midrefix.maxins$MAXDIST.mm$mm.best.bowtieout $sample 1 > $mapping_stat_file");
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188 print STDERR "Mapping stat :\n";
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189 system("cat $mapping_stat_file");
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190
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191 if($noNIR==0){
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192 print STDERR "Computing NIRs...\n";
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193 system("$perlcommand $bowtieout2readcount_script -d $datatype -m $MAXDIST -l $READ_LENGTH --gNIR -f -s $sample -v $gene2NM_file $bowtieoutdir/$sample.$midrefix.maxins$MAXDIST.mm$mm.best.bowtieout 1 > $readcount_dir/$sample.$midrefix.maxins$MAXDIST.mm$mm.gNIR");
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194 system("$perlcommand $bowtieout2readcount_script -d $datatype -m $MAXDIST -l $READ_LENGTH --iNIR -f -s $sample -v $gene2NM_file $bowtieoutdir/$sample.$midrefix.maxins$MAXDIST.mm$mm.best.bowtieout 1 > $readcount_dir/$sample.$midrefix.maxins$MAXDIST.mm$mm.iNIR");
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195 }
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196
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197 if($get_gReadcount==1) {
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198 print STDERR "Computing total gene read counts (gReadcount)...\n";
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199 print STDERR "$perlcommand $bowtieout2readcount_script -d $datatype -m $MAXDIST -l $READ_LENGTH -f -s $sample -v $gene2NM_file $bowtieoutdir/$sample.$midrefix.maxins$MAXDIST.mm$mm.best.bowtieout 1 > $readcount_dir/$sample.$midrefix.maxins$MAXDIST.mm$mm.gReadcount\n";
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200 system("$perlcommand $bowtieout2readcount_script -d $datatype -m $MAXDIST -l $READ_LENGTH -f -s $sample -v $gene2NM_file $bowtieoutdir/$sample.$midrefix.maxins$MAXDIST.mm$mm.best.bowtieout 1 > $readcount_dir/$sample.$midrefix.maxins$MAXDIST.mm$mm.gReadcount");
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201 }
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202
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203 if($noNEUMA==0){
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204 print STDERR "Computing gEUMA and iEUMA...\n";
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205 system("$perlcommand $calc_gEUMA_script $gUtable_file $lendis_dir/$sample.$midrefix.maxins$MAXDIST.mm0.i.insertlendis $READ_LENGTH > $EUMAdir/$sample.$midrefix.maxins$MAXDIST.mm$mm.gEUMA");
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206 system("$perlcommand $calc_iEUMA_script $iUtable_file $lendis_dir/$sample.$midrefix.maxins$MAXDIST.mm0.i.insertlendis $READ_LENGTH > $EUMAdir/$sample.$midrefix.maxins$MAXDIST.mm$mm.iEUMA");
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207
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208 print STDERR "Computing FVKM and LVKM...\n";
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209 system("$perlcommand $NIR2LVKM_script $sample $readcount_dir $midrefix.maxins$MAXDIST.mm$mm $EUMAdir $FVKM_dir $LVKM_dir $EUMAcut $mapping_stat_column $mapping_stat_file 1 2 $gene2NM_file $gene2symbol_file $datatype $scriptdir");
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210
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211 }
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212 }
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213
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214 print STDERR "Done.\n";
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215
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216 system("date");
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217
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218
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