annotate barcode_sort.pl @ 0:5dc02b8a2a57 draft default tip

planemo upload commit 70654da77972b29181d3b388035836b6fa84d59d-dirty

#!/usr/bin/perl -w
use strict; use warnings;

my $cut_site = $ARGV[7]; #let the user specify the cut site keyword (e.g. "TGCA" for the SbfI 6 bp cutter)

my $b = $ARGV[6]; #let the user specify barcode length
my $n = 2;        # set number of nucleotides to expect before the barcode (bestRAD libraries run at U. Oregon have 2bp here)
my $c = $b + $n;  # set position for start of enzyme cutsite, which occurs after the initial nucleotides plus the barcode
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!!!
my $e = length($cut_site); #calculate the length of the cut site
# note this is the length of what's left after enzyme digestion, NOT the full length of the enzyme recognition site
# 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

my $is_resilient;

open (IN, $ARGV[0]);    # read file with barcodes
my %counts = ();        # make a hash of barcodes that will be searched
while(<IN>) {           # counts of each barcode can be tracked with this hash, with a few more lines of code below
    chomp($_);
    $counts{$_} = 0;
}
close IN;

open (IN1, $ARGV[1]);        # read fastq file of raw forward reads
open (IN2, $ARGV[2]);        # read fastq file of raw reverse reads  -- these must have pairs in identical order
open (OUT1, ">$ARGV[3]");    # create fastq outfile for flipped forward reads (cutsite end)
open (OUT2, ">$ARGV[4]");    # create fastq outfile for flipped reverse reads (randomly sheared end)
my $forward; my $reverse; my $barcode;        # establish string variables for all parts of fastq files
my $name1; my $name2; my $third1; my $third2; my $qual1; my $qual2;

$is_resilient = $ARGV[5];  # true if is resilient to errors - Brian's additions


sub strDiffMaxDelta {

    my ( $s1, $s2, $maxDelta ) = @_;
    my $diffCount = () = ( $s1 ^ $s2 ) =~ /[^\x00]/g;
    return $diffCount <= $maxDelta;

}

if ($is_resilient eq "true") {

    my $max_errors = $ARGV[8];

    while($name1 = <IN1>) {        # start reading through the paired fastq input files
        $name2 = <IN2>;            # read all parts of a single read pair (4 lines in each of the 2 fastq files)
        $forward = <IN1>;
        $reverse = <IN2>;
        $third1 = <IN1>; $third2 = <IN2>; $qual1 = <IN1>; $qual2 = <IN2>;
        my $which = 0; my $for; my $rev;        # establish variables used below

        if(strDiffMaxDelta(substr($forward, $c, $e), $cut_site, $max_errors)) {            # check for SbfI cutsite in the correct place in forward read
            if(strDiffMaxDelta(substr($reverse, $c, $e), $cut_site, $max_errors)) {        # check for SbfI cutsite in the correct place in reverse read
                $for = substr($forward, $n, $b);            # this is where a barcode should be if it's in the forward read
                $rev = substr($reverse, $n, $b);            # this is where a barcode should be if it's in the reverse read
                if(exists $counts{$for} && (exists $counts{$rev}) == 0) {        # if a correct barcode and cutsite are in forward but not reverse read...
                    $which = 1; $barcode = $for; $counts{$for}++;                # which = 1 means the pair is correctly oriented
                }
                elsif((exists $counts{$for}) == 0 && exists $counts{$rev}) {    # if a correct barcode and cutsite are only in the reverse read...
                    $which = 2; $barcode = $rev; $counts{$rev}++;                # which = 2 means the pair needs to be flipped
                }
            }
            else {                                            # the cutsite is only found in the forward read
                #$barcode = substr($forward, $n, $b);
                #if(exists $counts{$barcode}) {$which = 1; $counts{$barcode}++; }    # if a correct barcode is in the forward read, the pair is correctly oriented
                $which = 1;

            }
        }
        elsif(strDiffMaxDelta(substr($reverse, $c, $e), $cut_site, $max_errors)){    # cutsite not in forward read but is in reverse read
            #$barcode = substr($reverse, $n, $b);
            #if(exists $counts{$barcode}) {$which = 2; $counts{$barcode}++; }        # pair needs to be flipped
            $which = 2;
        }                                                    # if a cutsite and correct barcode has not been found in either read, which = 0 at this point
        if($which == 1) {                                    # if the pair is correctly oriented, print out fastq format for the pair
            my $temp1 = substr($forward, $n);                # trim initial nucleotides off read and quality scores...
            my $temp2 = substr($qual1, $n);                    # so that output keeps barcode and cutsite but not other nucleotides...
            print OUT1 "$name1$temp1$third1$temp2";            # and is ready to go into process_radtags
            print OUT2 "$name2$reverse$third2$qual2";
        }
        elsif($which == 2) {                                # if the pair needs to be flipped, print out fastq format for the flipped pair
            my $temp1 = substr($reverse, $n);
            my $temp2 = substr($qual2, $n);
            print OUT1 "$name2$temp1$third2$temp2";
            print OUT2 "$name1$forward$third1$qual1";
        }                                                    # if which == 0, nothing is printed out for the pair
    }
    close IN1;
    close IN2;
    close OUT1;
    close OUT2;

    foreach my $key (sort keys %counts) {
        print "$key" . "\t" . "$counts{$key}\n";
    }

}


else{ #Paul's code

    while($name1 = <IN1>) {        # start reading through the paired fastq input files
        $name2 = <IN2>;            # read all parts of a single read pair (4 lines in each of the 2 fastq files)
        $forward = <IN1>;
        $reverse = <IN2>;
        $third1 = <IN1>; $third2 = <IN2>; $qual1 = <IN1>; $qual2 = <IN2>;

        my $which = 0; my $for; my $rev;        # establish variables used below
        if(substr($forward, $c, $e) eq "TGCAGG") {            # check for SbfI cutsite in the correct place in forward read
            if(substr($reverse, $c, $e) eq "TGCAGG") {        # check for SbfI cutsite in the correct place in reverse read
                $for = substr($forward, $n, $b);            # this is where a barcode should be if it's in the forward read
                $rev = substr($reverse, $n, $b);            # this is where a barcode should be if it's in the reverse read
                if(exists $counts{$for} && (exists $counts{$rev}) == 0) {        # if a correct barcode and cutsite are in forward but not reverse read...
                    $which = 1; $barcode = $for; $counts{$for}++;                # which = 1 means the pair is correctly oriented
                }
                elsif((exists $counts{$for}) == 0 && exists $counts{$rev}) {    # if a correct barcode and cutsite are only in the reverse read...
                    $which = 2; $barcode = $rev; $counts{$rev}++;                # which = 2 means the pair needs to be flipped
                }
            }
            else {                                            # the cutsite is only found in the forward read
                $barcode = substr($forward, $n, $b);
                if(exists $counts{$barcode}) {$which = 1; $counts{$barcode}++; }    # if a correct barcode is in the forward read, the pair is correctly oriented
            }
        }
        elsif(substr($reverse, $c, $e) eq "TGCAGG") {        # cutsite not in forward read but is in reverse read
            $barcode = substr($reverse, $n, $b);
            if(exists $counts{$barcode}) {$which = 2; $counts{$barcode}++; }        # pair needs to be flipped
        }                                                    # if a cutsite and correct barcode has not been found in either read, which = 0 at this point
        if($which == 1) {                                    # if the pair is correctly oriented, print out fastq format for the pair
            my $temp1 = substr($forward, $n);                # trim initial nucleotides off read and quality scores...
            my $temp2 = substr($qual1, $n);                    # so that output keeps barcode and cutsite but not other nucleotides...
            print OUT1 "$name1$temp1$third1$temp2";            # and is ready to go into process_radtags
            print OUT2 "$name2$reverse$third2$qual2";
        }
        elsif($which == 2) {                                # if the pair needs to be flipped, print out fastq format for the flipped pair
            my $temp1 = substr($reverse, $n);
            my $temp2 = substr($qual2, $n);
            print OUT1 "$name2$temp1$third2$temp2";
            print OUT2 "$name1$forward$third1$qual1";
        }                                                    # if which == 0, nothing is printed out for the pair


    }
    close IN1;
    close IN2;
    close OUT1;
    close OUT2;

    foreach my $key (sort keys %counts) {
        print "$key" . "\t" . "$counts{$key}\n";
    }
}