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open my $fh, '<', $file or die "Can't read $file: $!";

        my $start = $1; $start--; #increment down for substr
        my $end = $2; $end--; #increment down for substr

sub html_out {
    my ( $total_muts, $mutant_genes_ref ) = @_;
...
    my %mutant_genes = %{$mutant_genes_ref};
    # ...etc...
}