Once the search finishes, Geneious will download the alignments into a results folder, which will be a subfolder of the folder containing your query. The results can be sorted in a variety of ways. Sort them by "E Value" - this should be the first column. The sorting should be set so that the least likely hit is at the top (ie. the small triangular arrow will point up).
Question 1: What is the E-value for the top match? > |
Look at what happens to the Bit-Score, % Pairwise Identity and so on as you scroll down the list (you may need to scroll across to see these values).
Above all, you should always look at the alignments. Click through some of the hits to view the alignments. The Alignment View shows the query sequence aligned to the database (result) sequence (for more information on pairwise alignments, see the Pairwise Alignments tutorial). You should notice here that the length of the alignment differs from the full length of the database sequence for many of the hits. To see the base numbers for the hit and the query, open the Advanced settings tab (on the right hand side of the alignment view window). Tick the box next to Numbering and in the dropdown box next to this select All Sequences. The alignment length differs because BLAST implements a local alignment which means it does not try to align whole sequences, just the parts that match best. For BLAST hits to be truly signficant, the alignment should be a substantial subset of the original aligned pairs and there should be good levels of identity.
The Alignment View shows a graph displaying the identity between the query sequence and the hit. To view the identity graph, open the graphs tab (on the right hand side of the alignment view window) and tick the boxes next to Show graphs and Identity.
Question 2: In what ways does the query differ from the hit? > |
If you wish to display all of the BLAST hits against the query sequence in one view, click the Query-centric view tab at the top of the Document table. In this view the query sequence is displayed as a reference sequence with all the hits mapped to it.
Switch back to the Hit Table view, then select the hit to Medicago polymorpha, Accession number DQ311981 (you can filter the hit table using the Search function in the top right hand corner of Geneious). Click the Download full sequences button in the sequence viewer. Geneious will download the full NCBI GenBank record for this sequence including available annotations and add a Sequence View tab which shows the hit sequence annotated with "BLAST hit" where the query matches. If the annotations are not currently visible, open the annotations tab and check that the boxes next to Show annotations and next to each of the annotation types are ticked.
Question 3: What domains are indicated by the annotations for this sequence? > |
Click on the Text View tab to see the NCBI record (you may have to scroll down past the alignment). This text record provides the information for Geneious to display its graphical view, in particular the annotations shown against the sequence in the "Sequence View" tab.
Copy this sequence to your plant DNA folder by simply dragging the sequence entry in the Document Table to your folder.
Congratulations! You have completed a BLAST search.