Your code is full of mistakes/"sins". If you want to work with fasta files please take a look at fasta on CPAN. It might be a better idea to use an existing module to work with fasta files than to roll your own.

Some general comments:

• check for the success of open, don't assume everything is fine.
• use the the 3-arg form of open, see open.
• put use strict; and use warnings; in your code. It would catch most of the errors in your script.

Where is the question? You have just posted some Perl code, no description of the problem, your input or your output.

The question title is just your user name, and not descriptive of your problem, and your closing code tag should be </code> and not <code>.

Please read Writeup Formatting Tips and How do I post a question effectively?.

Martin

  $count_G = ( $seq =~ tr/G//);
  $ratio_G = ($count_G/$len);
# print "\n$ratio_G\n";
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  $ratio_G = Calculate( 'G' );
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• get rid of all of the temporary $count_* variables
• remove duplicated code
• make future changes (e.g. you want percentage rather than ratio) more centralized
• make the intentions of your code much clearer

sub Passref {
    my ( $define, $seq, $count, $outfh ) = @_;
    chomp($define);
    my $len   = length($$seq);
    my %count = ( Miss => 0+( $$seq =~ m/^YCWLPHQIMTNKSRVADEGF/ig ) );
    # YCWLPHQIMTNKSRVADEGF doesn't appear in test file
    my %ratio = ( Miss => 0+( $count{Miss} / $len ) );
    for my $letter (qw[ E D A V R S K N T M I Q H P L W C Y F ]) {
        $count{$letter} = ( $$seq =~ m/$letter/g );
        $ratio{$letter} = $count{$letter} / $len;
    }
    print {$outfh} join "\t", $define, $len,
      @ratio{qw[ E D A V R S K N T M I Q H P L W C Y F Miss  ]}, "\n";
}
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For count you need

$count{$letter} = ()= $$seq =~ m/$letter/g;
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