• don't read STDIN, use @ARGV
• use tr/// to count characters, see perlop, perlfaq4#How can I count the number of occurrences of a substring within a string?
• forget about Tie::File
• don't read the whole file into memory, read the file line by line ( see readline )
• [^ATGC] doesn't do what you're hoping it does, perlretut
• write programs to learn how while loops work, how readline works... the basics, perlintro, http://learn.perl.org/books/beginning-perl/, Tutorials
• http://serverfault.com/questions/133122/ulimit-for-windows
• Super Search reveals some interesting ATCG related threads :)
  • ?node_id=3989;BIT=ATGC
  • millions of records in a Hash
  • A memory efficient hash, trading off speed - does it already exist?
  • Hash lookups, Database lookups, and Scalability
  • Comparing strings (exact matches) in LARGE numbers FAST
  • Limitation of extra large hash
  • Random sampling a variable record-length file.
  • Reducing memory footprint when doing a lookup of millions of coordinates
  • remove part of string (DNA)
  • Combining 3 files
  • Count similar characters in a row - only once
  • Increasing the efficiency of a viral clonal expansion model
  • concatenating identical sequences
  • Efficient way to handle huge number of records?
  • Why does this sub grab the whole hash
  • mismatching characters in dna sequence
  • Splitting a multi-sequence fasta file into individual sequences in individual arrays
  • problems with converting a string character by character
  • Fast Identification Of String Difference
  • Refomating a large fasta file...

update: oh, I see you've gotten some of this advice before in Request to detect the mistake in a perl script for finding inter-substring distance from a large text file, I would definnitely start with http://learn.perl.org/books/beginning-perl/ and take it slow, write lots of little programs to learn how readline et all work

Good luck

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