Just some of my thoughts on this, maybe no solution to your question, but these just came up to my mind:

• I would reformat the script; especially the indentation
• I think the line numbers of the warnings don't fit to the posted code and its line numbers
• make the code for reading file1 shorter:

  while ( <$fh> ) {
      chomp;
      my ( $key, $pos ) = split /\s+/;
  
      # fill HoA automagically
      push @{ $positions{$key} }, $pos;
  }
  [download]

• Make clear, that $value contains the position; rename $value into $position
• In line 99 of your code, you compare the length of $sequence{$key} with the array reference in $positions{$key}; shouldn't you compare with $value (resp. the suggested $position)?