Difference between revisions of "TBLASTn and Protein Sequence Analysis"
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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: | 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: | :::*You should have three additional files made from the original fasta file: | ||
| − | ::==='''''Second''''' - Check that your commands are entered correct.=== | + | ::==='''''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 | :::*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) | :::*Sequences being blasted should be in TextEdit and saved without any extension (such as .txt) | ||
| − | ::==='''''Third''''' - Ensure that the databases/commands are working.=== | + | ::==='''''Third''''' -- Ensure that the databases/commands are working.=== |
:::*Take a known sequence from the database being blasted | :::*Take a known sequence from the database being blasted | ||
::::*Typically copy a small portion of the genome into a separate text file | ::::*Typically copy a small portion of the genome into a separate text file | ||
Revision as of 19:11, 22 February 2011
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:
- First -- Check to make sure that your database is made correctly.
- ===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)
- ===Second -- Check that your commands are entered correct.===
- ===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
- ===Third -- Ensure that the databases/commands are working.===
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.
