BLAST Exercise: Detecting and Interpreting Genetic Homology

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ESTs are pretty noisy and do not come with easily accessible annotations, so we will use the nucleotide nr database. Figure 14. blastn search againt the nr database To do the blastn search, go back to the BLAST page at (http://www.ncbi.nlm.nih.gov/BLAST) and select Nucleotide-nucleotide BLAST (blastn). In the “choose database” menu, make sure that “nr” is selected. Open the file containing our sequence, copy it onto the clipboard, and paste it into the search box (Figure 14). Click “BLAST!” then click “Format!” Alternatively, the blastn output is also available in the tutorial package (see the file blastnResults.html within the BLASTresults directory). Question 12: Had your query contained a repetitive element such as a transposon, what would have happened had you forgotten to repeat-mask the query before running it Figure 15. Refseq matches have accession numbers that begins with “ref” The sequences in the GenBank nr database come from numerous sources, including genomic contigs from genome sequencing projects and mRNAs/cDNAs. A particular useful class of 12
mRNA entries is the NCBI Refseqs, which come from a curated database of full-length mRNAs for various genes. You can find out more information about the Refseq database at http://www.ncbi.nlm.nih.gov/RefSeq/. Refseq matches can be easily recognized in the BLAST output because their accession numbers always start with “ref” (Figure 15). Question 13: What is the best Refseq match to the query How good is the match to what you think is the true Swallow gene (Hint: Create a map showing blastn hits to the query sequence as you did for Question 9.) Based on this alignment, how many exons does the gene have, and roughly where do the introns occur Question 14: How well does the Refseq match the other part of the query Can you see matches that were not visible at the protein level Why might this be The main question at this point is what is the other set of matches outside the region that we believe to be the D. melanogaster Swallow gene Do these matches reveal a real gene or pseudogene Pseudogenes are rare in Drosophila compared to mammals, but they are not unknown. There are at least two types of mutation that strongly suggest that a putative match to a gene might be a pseudogene. One is a stop codon that truncates the protein prematurely in the middle of a coding exon. You can see such internal stop codons in a blastx alignment as star (“*”) characters. Another diagnostic mutation is a gap in the middle of an exon that would cause a frameshift. Typically, this type of gap is visible only at the DNA level, since a frameshift will quickly terminate a blastx alignment. Question 15: Keeping in mind the exon boundaries you inferred above, can you find evidence of premature stop codons and/or frameshift-inducing gaps that would cause you to diagnose a pseudogene adjacent to the Swallow gene Describe any evidence you find. Summary: Question 16: Based on all the evidence gathered in this lab, how would you annotate the query sequence What uncertainties remain Compose a short (a few sentences) paragraph that you could add to an annotation database summarizing your findings. Last Update: 06/15/2006 13
Page 1 and 2: BLAST Exercise: Detecting and Inter
Page 3 and 4: Figure 1. RepeatMasker Web Server a
Page 5 and 6: In the next section, we will be wor
Page 7 and 8: At the initial BLAST response page,
Page 9 and 10: Figure 10. Results when searching f
Page 11: Figure 13. blastx with repeat-maske

BLAST Exercise: Detecting and Interpreting Genetic Homology

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