BLAST

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20 Tips to Improve Your BLAST Searches

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for nearly identical sequences) isn’t expected to answer the question; too many

sequences have changed in the 500 million years that separate worms and humans.

8.3 Perform Controls, Especially in

the Twilight Zone

Controls are crucial to any scientific experiment. The random model underlying

BLAST statistics provides one kind of control, but performing an explicit control can

give you greater confidence in your results. This is especially true when looking for

weak similarities, commonly called the twilight zone. One of the simplest and most

effective ways to determine if an alignment is believable is to shuffle your query

sequence and repeat the search. If the shuffled sequence returns similar results, the

alignment is based on compositional biases or the search parameters aren’t specific

enough. The following Perl script shuffles a FASTA file:

#!/usr/bin/perl -w

use strict;

my ($def, @seq) = <>;

print $def;

chomp @seq;

@seq = split(//, join("", @seq));

my $count = 0;

while (@seq) {

my $index = rand(@seq);

my $base = splice(@seq, $index, 1);

print $base;

print "\n" if ++$count % 60 == 0;

}

Now let’s put this script into action. Let’s make the dubious hypothesis that ALU

repeats aren’t specific to primates but are present in all genomes. They haven’t ...