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annotate barcode_sort.pl @ 3:2d1df0e14208 draft

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planemo upload commit 48a9cb9e296a9afe54276d75b464da0513e95ddc
author tiagoantao
date Mon, 28 Mar 2016 19:39:35 -0400
parents d838c6a2ec0f
children 5cd3cc89bf59
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1 #!/usr/bin/perl -w
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2 use strict; use warnings;
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4 my $b = 8; # set barcode length (Hohenlohe lab 2015 bestRAD barcodes are 8bp)
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5 my $n = 2; # set number of nucleotides to expect before the barcode (bestRAD libraries run at U. Oregon have 2bp here)
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6 my $c = $b + $n; # set position for start of enzyme cutsite, which occurs after the initial nucleotides plus the barcode
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7 my $e = 6; # set length of remaining enzyme cutsite sequence (e.g. 6 for SbfI) -- for other than SbfI, need to change the actual sequence below!!!
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8 # note this is the length of what's left after enzyme digestion, NOT the full length of the enzyme recognition site
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9 # the program expects all correct forward reads to follow the pattern: $n initial nucleotides, then $b nucleotides of barcode, then $e nucleotides of the cutsite
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11 open (IN, $ARGV[0]); # read file with barcodes
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12 my %counts = (); # make a hash of barcodes that will be searched
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13 while(<IN>) { # counts of each barcode can be tracked with this hash, with a few more lines of code below
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14 chomp($_);
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15 $counts{$_} = 0;
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16 }
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17 close IN;
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19 open (IN1, $ARGV[1]); # read fastq file of raw forward reads
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20 open (IN2, $ARGV[2]); # read fastq file of raw reverse reads -- these must have pairs in identical order
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21 open (OUT1, ">$ARGV[3]"); # create fastq outfile for flipped forward reads (cutsite end)
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22 open (OUT2, ">$ARGV[4]"); # create fastq outfile for flipped reverse reads (randomly sheared end)
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23 my $forward; my $reverse; my $barcode; # establish string variables for all parts of fastq files
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24 my $name1; my $name2; my $third1; my $third2; my $qual1; my $qual2;
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25 while($name1 = <IN1>) { # start reading through the paired fastq input files
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26 $name2 = <IN2>; # read all parts of a single read pair (4 lines in each of the 2 fastq files)
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27 $forward = <IN1>;
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28 $reverse = <IN2>;
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29 $third1 = <IN1>; $third2 = <IN2>; $qual1 = <IN1>; $qual2 = <IN2>;
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30 my $which = 0; my $for; my $rev; # establish variables used below
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31 if(substr($forward, $c, $e) eq "TGCAGG") { # check for SbfI cutsite in the correct place in forward read
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32 if(substr($reverse, $c, $e) eq "TGCAGG") { # check for SbfI cutsite in the correct place in reverse read
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33 $for = substr($forward, $n, $b); # this is where a barcode should be if it's in the forward read
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34 $rev = substr($reverse, $n, $b); # this is where a barcode should be if it's in the reverse read
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35 if(exists $counts{$for} && (exists $counts{$rev}) == 0) { # if a correct barcode and cutsite are in forward but not reverse read...
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36 $which = 1; $barcode = $for; $counts{$for}++; # which = 1 means the pair is correctly oriented
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37 }
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38 elsif((exists $counts{$for}) == 0 && exists $counts{$rev}) { # if a correct barcode and cutsite are only in the reverse read...
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39 $which = 2; $barcode = $rev; $counts{$rev}++; # which = 2 means the pair needs to be flipped
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40 }
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41 }
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42 else { # the cutsite is only found in the forward read
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43 $barcode = substr($forward, $n, $b);
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44 if(exists $counts{$barcode}) {$which = 1; $counts{$barcode}++; } # if a correct barcode is in the forward read, the pair is correctly oriented
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45 }
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46 }
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47 elsif(substr($reverse, $c, $e) eq "TGCAGG") { # cutsite not in forward read but is in reverse read
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48 $barcode = substr($reverse, $n, $b);
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49 if(exists $counts{$barcode}) {$which = 2; $counts{$barcode}++; } # pair needs to be flipped
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50 } # if a cutsite and correct barcode has not been found in either read, which = 0 at this point
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51 if($which == 1) { # if the pair is correctly oriented, print out fastq format for the pair
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52 my $temp1 = substr($forward, $n); # trim initial nucleotides off read and quality scores...
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53 my $temp2 = substr($qual1, $n); # so that output keeps barcode and cutsite but not other nucleotides...
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54 print OUT1 "$name1$temp1$third1$temp2"; # and is ready to go into process_radtags
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55 print OUT2 "$name2$reverse$third2$qual2";
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56 }
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57 elsif($which == 2) { # if the pair needs to be flipped, print out fastq format for the flipped pair
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58 my $temp1 = substr($reverse, $n);
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59 my $temp2 = substr($qual2, $n);
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60 print OUT1 "$name2$temp1$third2$temp2";
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61 print OUT2 "$name1$forward$third1$qual1";
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62 } # if which == 0, nothing is printed out for the pair
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63 }
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64 close IN1;
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65 close IN2;
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66 close OUT1;
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67 close OUT2;
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68
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69 foreach my $key (sort keys %counts) {
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70 print "$key" . "\t" . "$counts{$key}\n";
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71 }
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