BLAST Exercise: Detecting and Interpreting Genetic Homology

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dramatically decrease the computational time needed for BLAST searches. For this tutorial, however, we will use the generic databases. Figure 5. The FlyBase web site (http://www.flybase.indiana.edu) Figure 6. A blastx search against the Swissprot database Open the file dmel_seq2.fasta and copy the sequence onto the clipboard. Then open a new browser window and navigate to the BLAST web server at NCBI (www.ncbi.nlm.nih.gov/BLAST/) and select blastx. Paste the contents of the clipboard into the search box. Under the “Choose database” drop-down menu, select “swissprot”. Click “BLAST!” (Figure 6). 6
At the initial BLAST response page, click “Format!” and wait for the results (this may take a while). If you don’t want to go through these steps, the blastx output is also available in the tutorial package (the file blastxresults.html within the BLASTresults directory). Question 4: How many blastx hits to distinct sequences were returned What are the best and worst E-values reported Are the matches with poor E-values consistent with those with better E-values When searching a large database, it is good practice to ignore matches with poor E-values. In principle, a match with an E-value less than 1 is a highly significant hit (since only 1 match is expected to be found by chance alone when searching the database). In practice, you should allow a large margin of safety in interpreting E-values. This is because the probabilistic model on which E-values are calculated relies on strong independence assumptions that may not accurately reflect the behavior of real biological sequences. As a rule of thumb, you should be suspicious of DNA sequence matches with E-values higher than about 1e-10 and extremely skeptical of such matches with E-values above 1e-5. Figure 7. Changing the E-value for the blastx search By default, BLAST reports matches with E-values that are less than or equal to 10. You can change this behavior by changing the Expect field (for example, to “1e-10”) under the “Options for advanced blasting” section (Figure 7). Question 5: Considering only the most reliable matches, what does BLAST say about the content of this sequence What caveats might you consider in interpreting these results 7
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BLAST Exercise: Detecting and Interpreting Genetic Homology

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