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#!/usr/bin/perl
use strict;
use warnings;
...
        print if not $tested{$_};  
    }
}

C:\Old_Data\perlp>type o33.txt
>chr9:133738100-133738472_0 chr20:62159728-62161126_840
...
>chr9:133747466-133747650_0 chr5:108523084-108532592_960
>chr9:133747466-133747650_60    chr20:62159728-62161126_900
>chr9:133747466-133747650_65