I think your problem is

• you have a large file
• you have created a new file with line endings removed
• now you want to read in characters between two positions in the new file

use Fcntl qw(SEEK_SET);

my $input = '...';

my $start  = 1_490_117;
my $end    = 1_492_312;
my $length = $end - $start +1;

open my $fh, '<', $input or die "Can't open $input: $!";
sysseek($fh, $start, SEEK_SET) or die "Can't seek to $start in $input:
+ $!";
my $sequence;
my $read = sysread $fh, $sequence, $length;
die "Failed to read $length bytes from $input, got $read"
  if $length != $read;
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Note: sysread and sysseek use unbuffered IO, don't mix calls to them on a filehandle using other functions such as read, <> or eof.