How can I get a tfasta output aligned with Pileup?

The tfasta output (or the tblastn output) are not suitable for automatic parsing, so it is difficult to get a list of sequence names from the output that you can pop into Pileup.

However, a better way to do this would be to run a fasta search against the GenPept database, instead of Genbank. GenPept is maintained on the helix systems and is updated bimonthly. It contains all the putative translations from the last Genbank release. You can search GenPept using 'GenPept:*' in Fasta. In Pileup, you can then use the Begin= and End= parameters to define the ends of the region for alignment. Each sequence can have its own range limitation.

Another option is to use framesearch instead of fasta. You could then create a list file using the begin, end and range information from the framesearch output, and use that as input into Pileup. However, framesearch is much slower than Fasta.

How do I design a primer?

• melting temperatures of primer and product
• GC content
• ambiguous bases
• self-complementarity -- how to avoid primer dimer formation and primer secondary structure
• size of primer
• PCR product length
• repeats, e.g. similarity to c