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1 #!/usr/bin/perl -w
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2 use strict;
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3
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4 #########################################################################
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5 # codingSnps.pl
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6 # This takes a bed file with the names being / separated nts
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7 # and a gene bed file with cds start and stop.
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8 # It then checks for changes in coding regions, reporting
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9 # those that cause a frameshift or substitution in the amino acid.
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10 #########################################################################
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11
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12 my $seqFlag = "2bit"; #flag to set sequence type 2bit|nib
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13 if (!@ARGV or scalar @ARGV < 3) {
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14 print "Usage: codingSnps.pl snps.bed genes.bed (/dir/*$seqFlag|Galaxy build= loc=) [chr=# start=# end=# snp=# keepColumns=1] > codingSnps.txt\n";
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15 exit;
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16 }
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17 my $uniq = 0; #flag for whether want uniq positions
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18 my $syn = 0; #flag for if want synonomous changes rather than non-syn
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19 my $keep = 0; #keep old columns and append new ones
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20 my $snpFile = shift @ARGV;
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21 my $geneFile = shift @ARGV;
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22 my $nibDir = shift @ARGV; #2bit or nib, depending on flag above
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23 if ($nibDir eq 'Galaxy') { getGalaxyInfo(); }
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24 my $col0 = 0; #bed like columns in default positions
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25 my $col1 = 1;
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26 my $col2 = 2;
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27 my $col3 = 3;
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28 #column positions 1 based coming in (for Galaxy)
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29 foreach (@ARGV) {
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30 if (/chr=(\d+)/) { $col0 = $1 -1; }
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31 elsif (/start=(\d+)/) { $col1 = $1 -1; }
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32 elsif (/end=(\d+)/) { $col2 = $1 -1; }
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33 elsif (/snp=(\d+)/) { $col3 = $1 -1; }
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34 elsif (/keepColumns=1/) { $keep = 1; }
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35 }
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36 if ($col0 < 0 || $col1 < 0 || $col2 < 0 || $col3 < 0) {
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37 print STDERR "ERROR column numbers are given with origin 1\n";
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38 exit 1;
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39 }
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40 my @genes; #bed lines for genes, sorted by chrom and start
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41 my %chrSt; #index in array where each chrom starts
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42 my %codon; #hash of codon amino acid conversions
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43 my $ends = 0; #ends vs sizes in bed 11 position, starts relative to chrom
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44 my $ignoreN = 1; #skip N
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45
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46 my %amb = (
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47 "R" => "A/G",
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48 "Y" => "C/T",
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49 "S" => "C/G",
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50 "W" => "A/T",
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51 "K" => "G/T",
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52 "M" => "A/C",
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53 "B" => "C/G/T",
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54 "D" => "A/G/T",
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55 "H" => "A/C/T",
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56 "V" => "A/C/G",
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57 "N" => "A/C/G/T"
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58 );
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59 fill_codon();
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60 open(FH, "cat $geneFile | sort -k1,1 -k2,2n |")
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61 or die "Couldn't open and sort $geneFile, $!\n";
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62 my $i = 0;
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63 while(<FH>) {
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64 chomp;
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65 if (/refGene.cdsEnd|ccdsGene.exonEnds/) { $ends = 1; next; }
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66 push(@genes, "$_");
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67 my @f = split(/\t/);
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68 if (!exists $chrSt{$f[0]}) { $chrSt{$f[0]} = $i; }
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69 $i++;
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70 }
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71 close FH or die "Couldn't close $geneFile, $!\n";
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72
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73 if ($ends) { print STDERR "WARNING using block ends rather than sizes\n"; }
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74
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75 #open snps sorted as well
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76 my $s1 = $col0 + 1; #sort order is origin 1
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77 my $s2 = $col1 + 1;
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78 open(FH, "cat $snpFile | sort -k$s1,$s1 -k$s2,${s2}n |")
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79 or die "Couldn't open and sort $snpFile, $!\n";
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80 $i = 0;
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81 my @g; #one genes fields, should be used repeatedly
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82 my %done;
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83 while(<FH>) {
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84 chomp;
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85 if (/^\s*#/) { next; } #comment
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86 my @s = split(/\t/); #SNP fields
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87 if (!@s or !$s[$col0]) { die "ERROR missing SNP data, $_\n"; }
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88 my $size = $#s;
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89 if ($col0 > $size || $col1 > $size || $col2 > $size || $col3 > $size) {
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90 print STDERR "ERROR file has fewer columns than requested, requested columns (0 based) $col0 $col1 $col2 $col3, file has $size\n";
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91 exit 1;
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92 }
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93 if ($s[$col1] =~ /\D/) {
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94 print STDERR "ERROR the start point must be an integer not $s[$col1]\n";
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95 exit 1;
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96 }
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97 if ($s[$col2] =~ /\D/) {
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98 print STDERR "ERROR the start point must be an integer not $s[$col2]\n";
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99 exit 1;
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100 }
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101 if ($s[$col3] eq 'N' && $ignoreN) { next; }
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102 if (exists $amb{$s[$col3]}) { $s[$col3] = $amb{$s[$col3]}; }
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103 if (!@g && exists $chrSt{$s[$col0]}) { #need to fetch first gene row
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104 $i = $chrSt{$s[$col0]};
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105 @g = split(/\t/, $genes[$i]);
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106 if (scalar @g < 12) {
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107 print STDERR "ERROR the gene file must be the whole genes in BED format\n";
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108 exit 1;
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109 }
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110 }elsif (!@g) {
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111 next; #no gene for this chrom
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112 }elsif ($s[$col0] ne $g[0] && exists $chrSt{$s[$col0]}) { #new chrom
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113 $i = $chrSt{$s[$col0]};
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114 @g = split(/\t/, $genes[$i]);
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115 }elsif ($s[$col0] ne $g[0]) {
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116 next; #no gene for this chrom
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117 }elsif ($s[$col1] < $g[1] && $i == $chrSt{$s[$col0]}) {
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118 next; #before any genes
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119 }elsif ($s[$col1] > $g[2] && ($i == $#genes or $genes[$i+1] !~ $s[$col0])) {
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120 next; #after all genes on chr
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121 }else {
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122 while ($s[$col1] > $g[2] && $i < $#genes) {
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123 $i++;
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124 @g = split(/\t/, $genes[$i]);
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125 if ($s[$col0] ne $g[0]) { last; } #end of gene
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126 }
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127 if ($s[$col0] ne $g[0] or $s[$col1] < $g[1] or $s[$col1] > $g[2]) {
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128 next; #no overlap with genes
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129 }
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130 }
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131
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132 processSnp(\@s, \@g);
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133 if ($uniq && exists $done{"$s[$col0] $s[$col1] $s[$col2]"}) { next; }
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134
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135 my $k = $i + 1; #check for more genes without losing data of first
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136 if ($k <= $#genes) {
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137 my @g2 = split(/\t/, $genes[$k]);
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138 while (@g2 && $k <= $#genes) {
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139 @g2 = split(/\t/, $genes[$k]);
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140 if ($s[$col0] ne $g2[0]) {
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141 undef @g2;
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142 last; #not same chrom
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143 }else {
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144 while ($s[$col1] > $g2[2] && $k <= $#genes) {
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145 $k++;
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146 @g2 = split(/\t/, $genes[$k]);
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147 if ($s[$col0] ne $g2[0]) { last; } #end of chrom
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148 }
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149 if ($s[$col0] ne $g2[0] or $s[$col1] < $g2[1] or $s[$col1] > $g2[2]) {
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150 undef @g2;
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151 last; #no overlap with more genes
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152 }
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153 processSnp(\@s, \@g2);
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154 if ($uniq && exists $done{"$s[$col0] $s[$col1] $s[$col2]"}) { last; }
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155 }
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156 $k++;
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157 }
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158 }
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159 }
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160 close FH or die "Couldn't close $snpFile, $!\n";
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161
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162 exit;
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163
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164 ########################################################################
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165 sub processSnp {
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166 my $sref = shift;
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167 my $gref = shift;
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168 #overlaps gene, but maybe not coding seq
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169 #inside cds
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170 if ($sref->[$col1] + 1 < $gref->[6] or $sref->[$col2] > $gref->[7]) {
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171 return; #outside of coding
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172 }
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173 #now check exon
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174 my $i = 0;
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175 my @st = split(/,/, $gref->[11]);
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176 my @size = split(/,/, $gref->[10]);
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177 if (scalar @st ne $gref->[9]) { return; } #cant do this gene #die "bad gene $gref->[3]\n"; }
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178 my @pos;
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179 my $in = 0;
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180 for($i = 0; $i < $gref->[9]; $i++) {
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181 my $sta = $gref->[1] + $st[$i] + 1; #1 based position
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182 my $end = $sta + $size[$i] - 1; #
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183 if ($ends) { $end = $size[$i]; $sta = $st[$i] + 1; } #ends instead of sizes
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184 if ($end < $gref->[6]) { next; } #utr only
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185 if ($sta > $gref->[7]) { next; } #utr only
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186 #shorten to coding only
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187 if ($sta < $gref->[6]) { $sta = $gref->[6] + 1; }
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188 if ($end > $gref->[7]) { $end = $gref->[7]; }
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189 if ($sref->[$col1] + 1 >= $sta && $sref->[$col2] <= $end) { $in = 1; }
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190 elsif ($sref->[$col1] == $sref->[$col2] && $sref->[$col2] <= $end && $sref->[$col2] >= $sta) { $in = 1; }
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191 push(@pos, ($sta .. $end)); #add exon worth of positions
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192 }
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193 #@pos has coding positions for whole gene (chr coors),
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194 #and $in has whether we need to continue
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195 if (!$in) { return; } #not in coding exon
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196 if ((scalar @pos) % 3 != 0) { return; } #partial gene? not even codons
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197 if ($sref->[$col3] =~ /^-+\/[ACTG]+$/ or $sref->[$col3] =~ /^[ACTG]+\/-+$/ or
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198 $sref->[$col3] =~ /^-+$/) { #indel or del
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199 my $copy = $sref->[$col3];
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200 my $c = ($copy =~ tr/-//);
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201 if ($c % 3 == 0) { return; } #not frameshift
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202 #handle bed4 or any interval file
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203 if (!$keep) {
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204 print "$sref->[$col0]\t$sref->[$col1]\t$sref->[$col2]\t$sref->[$col3]";
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205 print "\t$gref->[3]\tframeshift\n";
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206 }else {
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207 my @s = @{$sref};
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208 print join("\t", @s), "\t$gref->[3]\tframeshift\n";
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209 }
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210 $done{"$sref->[$col0] $sref->[$col1] $sref->[$col2]"}++;
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211 return;
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212 }elsif ($sref->[$col1] == $sref->[$col2]) { #insertion
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213 my $copy = $sref->[$col3];
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214 my $c = ($copy =~ tr/\[ACTG]+//);
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215 if ($c % 3 == 0) { return; } #not frameshift
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216 #handle bed4 or any interval file
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217 if (!$keep) {
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218 print "$sref->[$col0]\t$sref->[$col1]\t$sref->[$col2]\t$sref->[$col3]";
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219 print "\t$gref->[3]\tframeshift\n";
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220 }else {
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221 my @s = @{$sref};
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222 print join("\t", @s), "\t$gref->[3]\tframeshift\n";
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223 }
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224 $done{"$sref->[$col0] $sref->[$col1] $sref->[$col2]"}++;
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225 return;
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226 }elsif ($sref->[$col3] =~ /-/) { #indel and sub?
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227 return; #skip
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228 }
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229 #check for amino acid substitutions
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230 my $s = $sref->[$col1] + 1;
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231 my $e = $sref->[$col2];
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232 my $len = $sref->[$col2] - $sref->[$col1];
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233 if ($gref->[5] eq '-') {
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234 @pos = reverse(@pos);
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235 my $t = $s;
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236 $s = $e;
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237 $e = $t;
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238 }
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239 $i = 0;
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240 my $found = 0;
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241 foreach (@pos) {
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242 if ($s == $_) {
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243 $found = 1;
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244 last;
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245 }
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246 $i++;
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247 }
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248 if ($found) {
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249 my $fs = $i; #keep original start index
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250 #have index where substitution starts
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251 my $cp = $i % 3;
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252 $i -= $cp; #i is now first position in codon
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253 my $cdNum = int($i / 3) + 1;
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254 my $ls = $i;
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255 if (!defined $ls) { die "ERROR not defined ls for $fs $sref->[$col2]\n"; }
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256 if (!@pos) { die "ERROR not defined array pos\n"; }
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257 if (!defined $pos[$ls]) { die "ERROR not defined pos at $ls\n"; }
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258 if (!defined $e) { die "ERROR not defined e for $pos[0] $pos[1] $pos[2]\n"; }
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259 while ($ls <= $#pos && $pos[$ls] ne $e) {
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260 $ls++;
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261 }
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262 my $i2 = $ls + (2 - ($ls % 3));
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263 if ($i2 > $#pos) { return; } #not a full codon, partial gene?
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264
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265 if ($i2 - $i < 2) { die "not a full codon positions $i to $i2 for $sref->[3]\n"; }
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266 my $oldnts = getnts($sref->[$col0], @pos[$i..$i2]);
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267 if (!$oldnts) { die "Failed to get sequence for $sref->[$col0] $pos[$i] .. $pos[$i2]\n"; }
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268 my @vars = split(/\//, $sref->[$col3]);
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269 if ($gref->[5] eq '-') { #complement oldnts and revcomp vars
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270 $oldnts = compl($oldnts);
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271 if (!$oldnts) { return; } #skip this one
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272 $oldnts = join('', (reverse(split(/ */, $oldnts))));
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273 foreach (@vars) {
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274 $_ = reverse(split(/ */)); #needed for indels
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275 $_ = compl($_);
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276 }
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277 }
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278 my $r = $fs - $i; #difference in old indexes gives new index
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279 my @newnts;
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280 my $changed = '';
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281 foreach my $v (@vars) {
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282 if (!$v or length($v) != 1) { return; } #only simple changes
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283 my @new = split(/ */, $oldnts);
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284 $changed = splice(@new, $r, $len, split(/ */, $v));
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285 #should only change single nt
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286 push(@newnts, join("", @new));
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287 }
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288 #now compute amino acids
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289 my $oldaa = getaa($oldnts);
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290 my @newaa;
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291 my $change = 0; #flag for if there is a change
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292 foreach my $v (@newnts) {
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293 my $t = getaa($v);
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294 if ($t ne $oldaa) { $change = 1; }
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295 push(@newaa, $t);
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296 }
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297 if (!$change && $syn) {
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298 if (!$keep) {
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299 print "$sref->[$col0]\t$sref->[$col1]\t$sref->[$col2]\t$sref->[$col3]";
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300 print "\t$gref->[3]\t$oldaa:", join("/", @newaa), "\n";
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301 }else {
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302 my @s = @{$sref};
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303 print join("\t", @s),
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304 "\t$gref->[3]\t$oldaa:", join("/", @newaa), "\n";
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305 }
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306 return;
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307 }elsif ($syn) { return; } #only want synonymous changes
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308 if (!$change) { return; } #no change in amino acids
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309 if (!$keep) {
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310 print "$sref->[$col0]\t$sref->[$col1]\t$sref->[$col2]\t$sref->[$col3]";
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311 if ($gref->[5] eq '-') { $changed = compl($changed); } #use plus for ref
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312 if (!$changed) { return; } #skip this one
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313 print "\t$gref->[3]\t$oldaa:", join("/", @newaa), "\t$cdNum\t$changed\n";
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314 }else {
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315 my @s = @{$sref};
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316 print join("\t", @s);
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317 if ($gref->[5] eq '-') { $changed = compl($changed); } #use plus for ref
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318 if (!$changed) { return; } #skip this one
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319 print "\t$gref->[3]\t$oldaa:", join("/", @newaa), "\t$cdNum\t$changed\n";
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320 }
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321 $done{"$sref->[$col0] $sref->[$col1] $sref->[$col2]"}++;
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322 }
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323 }
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324
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325 sub getnts {
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326 my $chr = shift;
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327 my @pos = @_; #list of positions not necessarily in order
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328 #list may be reversed or have gaps(introns), at least 3 bps
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329 my $seq = '';
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330 if (scalar @pos < 3) { die "too small region for $chr $pos[0]\n"; }
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331 if ($pos[0] < $pos[1]) { #not reversed
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332 my $s = $pos[0];
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333 for(my $i = 1; $i <= $#pos; $i++) {
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334 if ($pos[$i] == $pos[$i-1] + 1) { next; }
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335 if ($seqFlag eq '2bit') {
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336 $seq .= fetchSeq2bit($chr, $s, $pos[$i-1]);
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337 }else {
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338 $seq .= fetchSeqNib($chr, $s, $pos[$i-1]);
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339 }
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340 $s = $pos[$i];
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341 }
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342 if (length $seq != scalar @pos) { #still need to fetch seq
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343 if ($seqFlag eq '2bit') {
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344 $seq .= fetchSeq2bit($chr, $s, $pos[$#pos]);
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345 }else {
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346 $seq .= fetchSeqNib($chr, $s, $pos[$#pos]);
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347 }
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348 }
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349 }else { #reversed
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350 my $s = $pos[$#pos];
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351 for(my $i = $#pos -1; $i >= 0; $i--) {
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352 if ($pos[$i] == $pos[$i+1] + 1) { next; }
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353 if ($seqFlag eq '2bit') {
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354 $seq .= fetchSeq2bit($chr, $s, $pos[$i+1]);
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355 }else {
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356 $seq .= fetchSeqNib($chr, $s, $pos[$i+1]);
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357 }
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358 $s = $pos[$i];
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359 }
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360 if (length $seq != scalar @pos) { #still need to fetch seq
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361 if ($seqFlag eq '2bit') {
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362 $seq .= fetchSeq2bit($chr, $s, $pos[0]);
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363 }else {
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364 $seq .= fetchSeqNib($chr, $s, $pos[0]);
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365 }
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366 }
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367 }
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368 }
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369
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370 sub fetchSeq2bit {
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371 my $chr = shift;
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372 my $st = shift;
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373 my $end = shift;
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374 my $strand = '+';
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375 $st--; #change to UCSC numbering
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376 open (BIT, "twoBitToFa -seq=$chr -start=$st -end=$end $nibDir stdout |") or
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377 die "Couldn't run twoBitToFa, $!\n";
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378 my $seq = '';
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379 while (<BIT>) {
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380 chomp;
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381 if (/^>/) { next; } #header
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382 $seq .= uc($_);
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383 }
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384 close BIT or die "Couldn't finish twoBitToFa on $chr $st $end, $!\n";
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385 return $seq;
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386 }
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387
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388 sub fetchSeqNib {
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389 my $chr = shift;
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390 my $st = shift;
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391 my $end = shift;
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392 my $strand = '+';
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393 $st--; #change to UCSC numbering
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394 open (NIB, "nibFrag -upper $nibDir/${chr}.nib $st $end $strand stdout |") or die "Couldn't run nibFrag, $!\n";
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395 my $seq = '';
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396 while (<NIB>) {
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397 chomp;
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398 if (/^>/) { next; } #header
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399 $seq .= $_;
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400 }
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401 close NIB or die "Couldn't finish nibFrag on $chr $st $end, $!\n";
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402 return $seq;
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403 }
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404
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405 sub compl {
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406 my $nts = shift;
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407 my $comp = '';
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408 if (!$nts) { die "ERROR called compl with nts undefined"; }
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409 foreach my $n (split(/ */, $nts)) {
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410 if ($n eq 'A') { $comp .= 'T'; }
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411 elsif ($n eq 'T') { $comp .= 'A'; }
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412 elsif ($n eq 'C') { $comp .= 'G'; }
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413 elsif ($n eq 'G') { $comp .= 'C'; }
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414 elsif ($n eq 'N') { $comp .= 'N'; }
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415 elsif ($n eq '-') { $comp .= '-'; } #deletion
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416 else { $comp = undef; }
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417 }
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418 return $comp;
|
|
419 }
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420
|
|
421 sub getaa {
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422 my $nts = shift; #in multiples of 3
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423 my $aa = '';
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424 my @n = split(/ */, $nts);
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425 while (@n) {
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426 my @t = splice(@n, 0, 3);
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427 my $n = uc(join("", @t));
|
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428 if (!exists $codon{$n}) { $aa .= 'N'; next; }
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|
429 $aa .= $codon{$n};
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|
430 }
|
|
431 return $aa;
|
|
432 }
|
|
433
|
|
434 sub fill_codon {
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|
435 $codon{GCA} = 'Ala';
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436 $codon{GCC} = 'Ala';
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437 $codon{GCG} = 'Ala';
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|
438 $codon{GCT} = 'Ala';
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|
439 $codon{CGG} = 'Arg';
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440 $codon{CGT} = 'Arg';
|
|
441 $codon{CGC} = 'Arg';
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|
442 $codon{AGA} = 'Arg';
|
|
443 $codon{AGG} = 'Arg';
|
|
444 $codon{CGA} = 'Arg';
|
|
445 $codon{AAC} = 'Asn';
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|
446 $codon{AAT} = 'Asn';
|
|
447 $codon{GAC} = 'Asp';
|
|
448 $codon{GAT} = 'Asp';
|
|
449 $codon{TGC} = 'Cys';
|
|
450 $codon{TGT} = 'Cys';
|
|
451 $codon{CAG} = 'Gln';
|
|
452 $codon{CAA} = 'Gln';
|
|
453 $codon{GAA} = 'Glu';
|
|
454 $codon{GAG} = 'Glu';
|
|
455 $codon{GGG} = 'Gly';
|
|
456 $codon{GGA} = 'Gly';
|
|
457 $codon{GGC} = 'Gly';
|
|
458 $codon{GGT} = 'Gly';
|
|
459 $codon{CAC} = 'His';
|
|
460 $codon{CAT} = 'His';
|
|
461 $codon{ATA} = 'Ile';
|
|
462 $codon{ATT} = 'Ile';
|
|
463 $codon{ATC} = 'Ile';
|
|
464 $codon{CTA} = 'Leu';
|
|
465 $codon{CTC} = 'Leu';
|
|
466 $codon{CTG} = 'Leu';
|
|
467 $codon{CTT} = 'Leu';
|
|
468 $codon{TTG} = 'Leu';
|
|
469 $codon{TTA} = 'Leu';
|
|
470 $codon{AAA} = 'Lys';
|
|
471 $codon{AAG} = 'Lys';
|
|
472 $codon{ATG} = 'Met';
|
|
473 $codon{TTC} = 'Phe';
|
|
474 $codon{TTT} = 'Phe';
|
|
475 $codon{CCT} = 'Pro';
|
|
476 $codon{CCA} = 'Pro';
|
|
477 $codon{CCC} = 'Pro';
|
|
478 $codon{CCG} = 'Pro';
|
|
479 $codon{TCA} = 'Ser';
|
|
480 $codon{AGC} = 'Ser';
|
|
481 $codon{AGT} = 'Ser';
|
|
482 $codon{TCC} = 'Ser';
|
|
483 $codon{TCT} = 'Ser';
|
|
484 $codon{TCG} = 'Ser';
|
|
485 $codon{TGA} = 'Stop';
|
|
486 $codon{TAG} = 'Stop';
|
|
487 $codon{TAA} = 'Stop';
|
|
488 $codon{ACT} = 'Thr';
|
|
489 $codon{ACA} = 'Thr';
|
|
490 $codon{ACC} = 'Thr';
|
|
491 $codon{ACG} = 'Thr';
|
|
492 $codon{TGG} = 'Trp';
|
|
493 $codon{TAT} = 'Tyr';
|
|
494 $codon{TAC} = 'Tyr';
|
|
495 $codon{GTC} = 'Val';
|
|
496 $codon{GTA} = 'Val';
|
|
497 $codon{GTG} = 'Val';
|
|
498 $codon{GTT} = 'Val';
|
|
499 }
|
|
500
|
|
501 sub getGalaxyInfo {
|
|
502 my $build;
|
|
503 my $locFile;
|
|
504 foreach (@ARGV) {
|
|
505 if (/build=(.*)/) { $build = $1; }
|
|
506 elsif (/loc=(.*)/) { $locFile = $1; }
|
|
507 }
|
|
508 if (!$build or !$locFile) {
|
|
509 print STDERR "ERROR missing build or locfile for Galaxy input\n";
|
|
510 exit 1;
|
|
511 }
|
|
512 # read $locFile to get $nibDir (ignoring commets)
|
|
513 open(LF, "< $locFile") || die "open($locFile): $!\n";
|
|
514 while(<LF>) {
|
|
515 s/#.*$//;
|
|
516 s/(?:^\s+|\s+$)//g;
|
|
517 next if (/^$/);
|
|
518
|
|
519 my @t = split(/\t/);
|
|
520 if ($t[0] eq $build) { $nibDir = $t[1]; }
|
|
521 }
|
|
522 close(LF);
|
|
523 if ($nibDir eq 'Galaxy') {
|
|
524 print STDERR "Failed to find sequence directory in locfile $locFile\n";
|
|
525 }
|
|
526 $nibDir .= "/$build.2bit"; #we want full path and filename
|
|
527 }
|
|
528
|