TBLASTn and Protein Sequence Analysis

When Nucleotides Don't Work

Occasionally BLASTing genes with just a nucleotide sequence doesn't work. This can be due to a number of reasons:

• Lack of conservation between species
• Incomplete genome database
• Rearrangement of introns/exons

But assuming that you're working with a gene that should be highly conserved and you have allowed for the exon/intron issue, it may be time to go through some extra steps to make sure everything is working as it should:

First -- Check to make sure that your database is made correctly.

• You should have three additional files made from the original fasta file:

Second -- Check that your commands are entered correct.

• Ensure that you are in the correct directory (i.e. Desktop) and BLASTing with the correct file name
• Sequences being blasted should be in TextEdit and saved without any extension (such as .txt)

Third -- Ensure that the databases/commands are working.

• Take a known sequence from the database being blasted

• Typically copy a small portion of the genome into a separate text file

• Run a BLAST with that sequence against the database you took it from

• If it doesn't show a hit in the scaffold that you took it from, something is wrong in your programming

If you've gone through these steps and still can't find the issue, it might be time to move away from nucleotide sequences and try some amino acid sequences.