When the search completes, all of the results will be downloaded from NCBI and placed in the newly created folder. By default, the search results should be ordered by their E Value which indicates the expected frequency of occurrence of each alignment by chance. If your results are not ordered by E value, click the E Value column header. Your hit table should look something like the table below, but the actual hits may vary slightly as new sequences are added to Genbank all the time.
For E values, the smaller the number the better. These are displayed using exponents. The top hit shown here as 1.18e-107 is the same as 1.18x10-107. This is a very small number and indicates that it is highly unlikely that this alignment would ever occur by chance. You may even have examples where the E Value reads 0.00e+00 and this is telling you that statistically there is no likelihood that this alignment has happened by chance. You should take these statistics as a guide as there can still be interesting alignments that appear far less significant.
In addition to the E Value, there is also a column labelled % Pairwise Identity. This is also useful as it will indicate how similar the sequence found in the database is to the one you used as a query. You can see that many of the hits in this example are 100% identical to the query over the length of the alignment, but have different Sequence Lengths. This is because the alignment produced is a local similarity alignment and it has aligned the maximum region it could find between the two sequences. The identity refers only to the aligned region so it is possible to have very short alignments which have high identity. This is why alignments tend to be ranked by their E Value rather than identity. Geneious also produces a Grade score, which combines query coverage, e-value and identity values for each hit with weights 0.5, 0.25 and 0.25 respectively, allowing you to determine the longest, highest identity hits.
Now that you have a set of search results, you should look at some alignments. Click on the hit to NP_001014408 and you should see something like this:
You can see from the green identity graph above the alignment that the two sequences are identical. Like any other alignment in Geneious, you can zoom into display the bases, change the color settings, and highlight agreements or disagreements to the consensus in the General controls to the right of the viewer.
This alignment view only shows the region of alignment between the query and the hit sequence. The blast hit document returned is a summary document and does not contain the full Genbank record for that sequence. To get the full sequence and annotations for the blast hit, click Download Full Sequence(s). Once the full sequence is downloaded you'll see that a Sequence View tab is added to the viewer. This displays the full, annotated sequence of the BLAST hit, with a new "BLAST Hit" annotation showing which region of the sequence matches the query.
Query-centric view is useful for visualising all the hits against your query in one window, allowing you to see where conserved regions of your sequence are. Click on the Query Centric View tab at the top of the Hit table, then turn off the annotations in the Annotations and Tracks tab, and in the
Display tab choose to highlight Disagreements to Reference. Your display should look something like this:
The query sequence is presented as a reference sequence, with yellow shading, at the top of the alignment. You can see that many of the top hits are extremely similar to the query, indicating that this protein is highly conserved across most of its length. The first 20 residues of the query may be less well-conserved as many of the hits do not span this region. The sequences are aligned in order of E-value and if you scroll down you'll see that the sequences become more distantly related to the query as the E-value decreases.
Section 3: Batch searching
Section 4: Search options