BLAST Exercise: Detecting and Interpreting Genetic Homology

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Question 7: What is the orientation of the Swallow gene relative to our query sequence Question 8: Look at all the matches to SWA_DROME in your blastx output. Is the entire protein matched (Hint: create a drawing of the blastx hits relative to the Swallow sequence. Check the individual alignments.) If not, which residues are missing Are there any regions of the protein that are aligned to multiple places in our query sequence [Hint: check the amino acid identities of the aligned fragments in the Graphical Representation of the blastx output (Figure 13).] Figure 12. Graphical Representation of alignments to our unknown sequence There is considerable confusion (missing residues and multiple hits to the same residues) in the BLAST alignments of the SWA_DROME protein with our putative coding sequence (Figure 12). As an annotator, your job is to produce order from this chaos. Let’s start with the missing residues. Go back to the Swissprot entry for SWA_DROME and find the part of the protein that is not represented by any of the BLAST alignments of SWA_DROME with our sequence, as shown by your analysis for Question 8. Question 9: Which amino acids predominate in the missing region Given that blastx likes to mask low-complexity sequence in the query before a search, do you have a reasonable explanation for why this part of the protein is missing How can you prove your hypothesis (Hint: repeat your blastx search with the lowcomplexity filter turned off; compare maps showing HSP’s relative to your query sequence.) 10
Figure 13. blastx with repeat-masked query sequence Around amino acid 190 of the protein (subject) sequence, you will see a string of X’s representing masked residues (Figure 13) in the query. BLAST apparently decided that the protein in the region, rich in serine/asparagines, should be marked as low-complexity. Aligning a residue to an X yields a negative score. Question 10: Given blastx seems happy enough to include masked residues in its alignments, why didn’t it include residues 78-90 of the protein [Hint: look at the frames (specified in the heading) of the matches ending at 77 and beginning at 91. What happens if you add negative-scoring residue pairs to the end of an alignment] Now we need to deal with the duplicated matches. The best way to make sense of the output is to sketch out the relative positions of all the matches to SWA_DROME in the query on a piece of paper (see Figure 14). Note the residues of the SWA_DROME protein that match with each part of the query. Question 11: How many distinct features (i.e. genes or pseudogenes) seem to be present at this locus Which one seems most likely to be the true Swallow gene What might the other matches be, and what biological mechanisms might have produced them Further Exploration Using blastn: To make further progress in determining the correct annotation for our sequence, we will try to obtain additional evidence at the nucleotide level, specifically looking at Drosophila mRNAs. In order to detect homology at the nucleotide level, we will use blastn instead of blastx. As was the case for the blastx search, there are some decisions we must make before we perform the search. For example, our choices of a database against which to search our query include the GenBank nonredundant nucleotide database (also known as nr), or one of the EST databases. 11
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BLAST Exercise: Detecting and Interpreting Genetic Homology

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