Oh no, sorry! That was a "duh" moment from me! :)

How should the script be modified to use the original IDs?

I did add a few lines to your script, to reflect the possibility of Ns in the input, and those lines seem to work. I also just discovered that there appear to be some OTHER characters apart from A/C/G/T/N. The OTHERS could be one of R/Y/S/W/K/M/B/D/H/V. What is your advice for how to account for these? I hard coded those changes in a modified version of your script, as shown below, does it look alright?

Importantly, what is the part of your script that generates randomness in the sequence? Is it the map(letter(), 1 .. $count)?

I ask because of 2 reasons:

1. The goal is to randomly shuffle DNA, which is the obvious reason.

2. So that I can be sure that any two outputs with the same input are going to be different, due to this randomness

Many thanks!

#!/usr/bin/perl
#tybalt89_DNAfreq_Random_Generator.pl

# https://perlmonks.org/?node_id=1228191

use strict;
use warnings;

my $window = 1e6;
my $A = my $C = my $G = my $T = my $N = my $all = 0;
my $R=0; my $Y=0; my $S=0; my $W=0; my $K=0; my $M=0; my $B=0; my $D=0
+; my $H=0; my $V=0;

my (@sizes, $tmp, $start);
my $in = shift @ARGV;
my $out = shift @ARGV;
#    print $in, "\n";
#    print $out, "\n";

open IN, '<', $in or die "$! opening $in";
open OUT, '>', $out or die "$! opening $out";

sub letter
  {
  my $n = int rand $all--;
  $n < $A ? ($A--, return 'A') :
    $n < $A + $C ? ($C--, return 'C') :
    $n < $A + $C + $G ? ($G--, return 'G') :
    $n < $A + $C + $G + $T ? ($T--, return 'T') :

    $n < $A + $C + $G + $T + $R ? ($R--, return 'R') :
    $n < $A + $C + $G + $T + $R + $Y ? ($Y--, return 'Y') :
    $n < $A + $C + $G + $T + $R + $Y + $S ? ($S--, return 'S') :
    $n < $A + $C + $G + $T + $R + $Y + $S + $W ? ($W--, return 'W') :
    $n < $A + $C + $G + $T + $R + $Y + $S + $W + $K ? ($K--, return 'K
+') :
    $n < $A + $C + $G + $T + $R + $Y + $S + $W + $K + $M ? ($M--, retu
+rn 'M') :
    $n < $A + $C + $G + $T + $R + $Y + $S + $W + $K + $M + $B ? ($B--,
+ return 'B') :
    $n < $A + $C + $G + $T + $R + $Y + $S + $W + $K + $M + $B + $D ? (
+$D--, return 'D') :
    $n < $A + $C + $G + $T + $R + $Y + $S + $W + $K + $M + $B + $D + $
+H ? ($H--, return 'H') :
    $n < $A + $C + $G + $T + $R + $Y + $S + $W + $K + $M + $B + $D + $
+H + $V ? ($V--, return 'V') :
    return 'N';
  }

sub output
  {
  for my $count ( @sizes )
    {
#    print ">ID", $start++, "\n", map(letter(), 1 .. $count), "\n";
    print OUT ">ID", $start++, "\n", map(letter(), 1 .. $count), "\n";
    }
  @sizes = ();
  }

while( <IN> )
  {
  if( /^>/ )
    {
    $start //= s/\D+//gr;
    }
  elsif( /^[ACGTRYSWKMBDHVN]/ )
    {
    $A += tr/A//;
    $C += tr/C//;
    $G += tr/G//;
    $T += tr/T//;
    $R += tr/R//;
    $Y += tr/Y//;
    $S += tr/S//;
    $W += tr/W//;
    $K += tr/K//;
    $M += tr/M//;
    $B += tr/B//;
    $D += tr/D//;
    $H += tr/H//;
    $V += tr/V//;
    $N += tr/N//;
    $all += $tmp = tr/ACGTRYSWKMBDHVN//;
    push @sizes, $tmp;
    $all >= $window and output();
    }
  }
$all and output();
close IN;
close OUT;
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