The main issue really was that you provided an end position to substr while it expects a length. This has already been corrected by choroba above. I would like to propose to directly construct the intervals for the Ns under the assumption that your input intervals are sorted and non-overlapping. So instead of looking at 1 => 3, 5 => 6, etc I propose to add a zero at the beginning and the length of the string at the end to get 0 => 1, 3 => 5, 6 => ... which give you directly the intervals to put Ns into.

use warnings;
use strict;

my $mask_input = '0000000000';
my %intervals = ( 0, ( do { local $/; <DATA> } =~ /(\d+)/g ), 1+length
+ $mask_input );

while( my( $start, $end ) = each %intervals ) {
        substr( $mask_input, $start, $end-$start-1 ) =~ tr/N/N/c;
}

print "$mask_input\n";

__DATA__
chrX      1      3
chrX      5      6
chrX      8      9
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