Re: [DIYbio] BLAST query analysis

I think you need to take a different approach to this for a couple of reasons.

1. the frequency of particular mutations in the database reflects what is deposited - so common mistakes, common biases etc. not what is necessarily the most frequent.  

2. BLAST is a local rather than global aligner and is not the best tool for what is essentially a phylogenetics task - as Dakota mentions

3. having your sequences in Word and Excel makes them very difficult to handle and can result in creating and propagating errors (for example http://www.biomedcentral.com/1471-2105/5/80)

My approach would be to get as many of the sequences into an Arb database http://www.arb-silva.de/ (free) align them using a global aligner (clustal, MUSCLE etc) and then manually check the quality of the alignment.  Use Arb's PT server to generate a quickly searchable database of sequences and then get it to select potential primers for you that discriminate between subsets of the sequences (can be grouped via phylogenetic tree) or mutations.  The nice thing with this approach is that you have a database that you can add to when new sequences become available and check whether you need to update your primers or not.  

How common the particular mutations are and therefore how useful to you each primer set is can be overlaid afterwards, if you have the frequency information as part of the primer search then database errors are propagated.  Better to define what different types there are first and then to look at frequency second (or as an experiment).  

Where you may come across difficulties with the above is that I'm more used to doing this with single genes or operons rather than whole genomes which are likely to be harder to align with higher variability (though viral genomes aren't that big).  Is there a typing scheme for hepB?  Can you align representatives within the subtypes first?  How many whole genome sequences are available and has anyone done any comparative genomics?  The above approach will definitely work if you're only interested in designing primers for one of the genes.  

Cheers

Mike