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