Bioinformatics, Error: explicit package name

[mlsmit10]

I'm working on a script that will take a genome and cut it at two restriction sites. The original script gave me the numbers of size fragments flanked by these two cut sites that would be produced. Now, I am trying to add a third restriction enzyme and exclude from the final counts any fragments containing this cut site. I've tried using grep to exclude such fragments, but am running into one problem I can't figure out.

This is the portion of the code I have altered: (for the rest of the code, see the end of the post)

        my @third_fragments = grep -v ($rsite3), $second_fragments[$i]
    
        my @final_fragment1 = $seqname."_".$i."_1";    
        my @final_fragment2 = $seqname."_".$i."_2";    
        $final_fragments{$final_fragment1} = $third_fragments[0];
        $final_fragments{$final_fragment2} = $third_fragments[scalar @
+third_fragments - 1];

I receive the following error message when I try to run the code:

syntax error at /Users/smithcabinets26/Desktop/RAD/Digester/Improving/
+MyTripleDigester-3.pl line 68, near "my "
Global symbol "@final_fragment1" requires explicit package name at /Us
+ers/smithcabinets26/Desktop/RAD/Digester/Improving/MyTripleDigester-3
+.pl line 68.
Global symbol "$final_fragment1" requires explicit package name at /Us
+ers/smithcabinets26/Desktop/RAD/Digester/Improving/MyTripleDigester-3
+.pl line 70.
Global symbol "$final_fragment2" requires explicit package name at /Us
+ers/smithcabinets26/Desktop/RAD/Digester/Improving/MyTripleDigester-3
+.pl line 71.
Execution of /Users/smithcabinets26/Desktop/RAD/Digester/Improving/MyT
+ripleDigester-3.pl aborted due to compilation errors.

Here is the complete code:

#! /usr/bin/perl -w
# a script to get fragments of a genome based on restriction enzyme
# retrieves the fragments greater than XX bp and less than XX bp
# whole genome in one gzip file (fasta format)!
# calculates the number of fragments you will get per genome for RADse
+q
# perl genomeFragmentor_doubleDigest.pl genome.gz restriction_site1 re
+striction_site2 organism
# perl genomeFragmentor_doubleDigest.pl Anolisgenome.gz CCTGCAGG GGATC
+C Anolis

use strict;

my %genome = fasta_read_gzip_alt($ARGV[0]);
#my %genome = fasta_read_alt($ARGV[0]);
my $rsite1 = $ARGV[1];
my $rsite2 = $ARGV[2];
my $rsite3 = $ARGV[3];

my $totalCount = 0;            # count of all fragments in the genome
my $totalBP = 0;            # the total number of base pairs in the se
+lected fragments

my $organism = $ARGV[4];

my $radtagfile = $organism."_Radseq_fragments_doubleDigest_".$rsite1."
+_".$rsite2."_".$rsite3.".fasta";
my $sizesfile = $organism."_Radseq_fragments_doubleDigest_".$rsite1."_
+".$rsite2."_".$rsite3."_sizes.txt";
open (OUT, ">$radtagfile");
open (SIZE, ">$sizesfile");
print SIZE "length    organism\n";

# prepare a summary file
my ($Second, $Minute, $Hour, $Day, $Month, $Year, $WeekDay, $DayOfYear
+, $IsDST) = localtime(time);
$Month++;
$Year += 1900;
my $date = $Month."_".$Day."_".$Year;
my $summaryfile = $organism."_summary_doubleDigest_".$rsite1."_".$rsit
+e2."_".$rsite3."_".$date.".txt";
open (RESULTS, ">$summaryfile");

my %final_fragments;

my @all_size_fragments;

# start creating the fragments
foreach my $seqname (keys %genome) {
    print "Working on sequence $seqname.\n";
    
    my @first_fragments = split("$rsite1", $genome{$seqname});        
+    # split the fragments up based on the enzyme motif
    
    # add the restriction site motif back onto the fragments
    $first_fragments[0] .= $rsite1;    
    $first_fragments[scalar @first_fragments - 1] = $rsite1.$first_fra
+gments[scalar @first_fragments - 1];     
    
    for (my $i = 0; $i < scalar @first_fragments; ++$i) {
        if ($i != 0 or $i != (scalar @first_fragments - 1)) {
            $first_fragments[$i] = $rsite1.$first_fragments[$i].$rsite
+1;
        }
        
        # now split the fragment using $rsite2
        # repair the first and last fragments to include $rsite2
        # these are the only fragments to contain both restriction sit
+es, so keep them in @final_fragments
        my @second_fragments = split($rsite2, $first_fragments[$i]);
        
        $second_fragments[0] .= $rsite2;
        $second_fragments[scalar @second_fragments - 1] = $rsite2.$sec
+ond_fragments[scalar @second_fragments - 1];
        foreach my $fragment (@second_fragments) {
            push(@all_size_fragments, length($fragment));
        }
        my @third_fragments = grep -v ($rsite3), $second_fragments[$i]
    
        my @final_fragment1 = $seqname."_".$i."_1";    
        my @final_fragment2 = $seqname."_".$i."_2";    
        $final_fragments{$final_fragment1} = $third_fragments[0];
        $final_fragments{$final_fragment2} = $third_fragments[scalar @
+third_fragments - 1];
    }
    
}

# keep a score of how many fragments fall within a particular size ran
+ge

my $size_651_750 = 0;
my $size_551_650 = 0;
my $size_501_550 = 0;
my $size_401_500 = 0;
my $size_301_400 = 0;
my $size_small = 0;
my $size_large = 0;    
    
foreach my $fragment (keys %final_fragments) {        
    # add on $rsite1 to both sides of the fragment
    my $fragmentLength = length($final_fragments{$fragment});
    print OUT ">$fragment", "_", "1\n";
    print OUT substr($final_fragments{$fragment}, 0, 96), "\n";
    print OUT ">$fragment", "_", "2rc\n";
    print OUT revcom(substr($final_fragments{$fragment}, $fragmentLeng
+th - 96, 96)), "\n";
    $totalBP += $fragmentLength;
    
    if ($fragmentLength >= 300 and $fragmentLength <= 400) {
        ++$size_301_400;
    } elsif ($fragmentLength > 400 and $fragmentLength <= 500) {
        ++$size_401_500;
    } elsif ($fragmentLength > 500 and $fragmentLength <= 550) {
        ++$size_501_550;
    } elsif ($fragmentLength > 550 and $fragmentLength <= 650) {
        ++$size_551_650;
    } elsif ($fragmentLength > 650 and $fragmentLength <= 750) {
        ++$size_651_750;
    } elsif ($fragmentLength < 300) {
        ++$size_small;
    } elsif ($fragmentLength > 750) {
        ++$size_large;
    }
}
    
$totalCount = scalar keys %final_fragments;            # count of all 
+fragments in the genome
print RESULTS "The restriction sites used were:\n";
print RESULTS $ARGV[1], "\n";
print RESULTS $ARGV[2], "\n\n";
print RESULTS $ARGV[3], "\n\n\n";

print RESULTS "There were ", $totalCount, " fragments from the whole g
+enome.\n\n";

print RESULTS "For MiSeq v3 there are ", $size_651_750, " fragments\n"
+;
print RESULTS "For MiSeq v2 there are ", $size_551_650, " fragments\n"
+;
print RESULTS "For HiSeq 2X150 there are ", $size_401_500, " fragments
+\n";
print RESULTS "For HiSeq 2X100 there are ", $size_301_400, " fragments
+\n\n";

print RESULTS "There were ", $size_small, " fragments smaller than 100
+ bp.\n";
print RESULTS "There were ", $size_large, " fragments larger than 800 
+bp.\n\n";

print RESULTS "There are ", $totalBP, " base pairs in the fragments.\n
+";

print RESULTS "\nJust some notes:\n";
print RESULTS "3RAD adapters are ~140bp, select below +/- 50bp\n"; 
print RESULTS "MiSeq v3 2X300 you will want 700bp fragments.\n"; 
print RESULTS "MiSeq v2 2X250 you will want 600bp fragments.\n"; 
print RESULTS "HiSeq Fast 2X150 you will want 450bp fragments.\n"; 
print RESULTS "HiSeq 2X100 you will want 350bp fragments.\n"; 

close OUT;
close RESULTS;

foreach my $length (@all_size_fragments) {
    print SIZE $length, "\t", $organism, "\n";
}
exit;

sub fasta_read_gzip_alt {
    
    # reads in a gzip fasta file and pases it into a hash
    # version 1.0
    
    (my $filename) = @_;        # be sure to include the path
    my %fasta;
    open(FASTA, "gunzip -c $filename |") || die "can't open pipe to $f
+ilename";
    my $fastaData;
    my $sequence = '';
    my $name = '';
    while(<FASTA>) {
        $fastaData = $_;
        $fastaData =~ s/\n//gms;
        if ($fastaData =~ />/) {
            if ($sequence) {                # if there is a sequence, 
+then the sequence belongs to the last name
                $fasta{$name} = $sequence;
            }
            
            # reinitialize everything
            $sequence = '';                # start over!
            $name = $fastaData;
            $name =~ s/>//gms;
        } elsif (eof FASTA) {
            $fasta{$name} = $sequence;
        } else {
            $sequence .= $fastaData;
        }
    }
    close FASTA;
    
    return %fasta;

}

sub revcom {
    (my $sequence) = @_;
    $sequence = reverse($sequence);
    $sequence =~ tr/AGCTRYMKSWHBVDNagctrymkswhbvdn/TCGAYRKMSWDVBHNtcga
+yrkmswdvbhn/;
    return $sequence;
}

sub fasta_read_alt {
    
    # reads in a fasta file and pases it into a hash
    # version 1.0
    
    (my $filename) = @_;        # be sure to include the path
    my %fasta;
    open(FASTA, $filename);
    my $fastaData;
    my $sequence = '';
    my $name = '';
    while(<FASTA>) {
        $fastaData = $_;
        $fastaData =~ s/\n//gms;
        if ($fastaData =~ />/) {
            if ($sequence) {                # if there is a sequence, 
+then the sequence belongs to the last name
                $fasta{$name} = $sequence;
            }
            
            # reinitialize everything
            $sequence = '';                # start over!
            $name = $fastaData;
            $name =~ s/>//gms;
        } elsif (eof FASTA) {
            $fasta{$name} = $sequence;
        } else {
            $sequence .= $fastaData;
        }
    }
    close FASTA;
    return %fasta;

}

My apologies if the answer is obvious or the question is posed poorly. I'm new to coding, and I haven't been able to solve this by looking at previous posts. Thanks for the help.

Problem solved! Thanks for all the help, everyone. After I fixed the semicolon issue, I played around with the grep function a bit (which I now see some of you suggested), and was able to get the script up and running. In case anyone else has a similar situation where they want to use grep in this way, the modifications I made to the grep line are included below (I've only included the one line, and it should just replace the grep line in the above code). The script now does exactly what I wanted it to do. Again, thanks for the help!

my @third_fragments = grep !/$rsite3/, $second_fragments[$i];