BLAST searches

MacVector can directly perform BLAST searches of the sequence databases available on the NCBI server over the Internet.

To perform a BLAST search, a nucleic acid or a protein sequence window must be the active window.

The query sequence cannot exceed 10,000 residues. If your sequence is longer than this you will be required to specify a region of the sequence to be used as the query.

PERFORMING A standard BLAST search

The MacVector interface to the BLAST server has been designed to give the same results as the NCBI web page interface. To accomplish this, a standard search is provided on a dialog box with few options. The version number of the selected BLAST program is displayed on the right of the Program selector.

1. Choose Database | Online search for Similar Sequences (BLAST).

   While the connection is being made to the NCBI server, an information box is displayed. The Online search for Similar Sequences (BLAST) dialog box is then displayed.NOTE: If any part of the connection process fails, an appropriate error message is displayed and the entire BLAST search is aborted.

2. Choose a search program from the Program drop-down menu.

   MacVector only displays program options relevant to the current query sequence type.

3. Choose a database to search from the Database drop-down menu.

   MacVector only displays databases relevant to the current search program you have requested.

4. Choose a threshold score for the BLAST search from the Expect drop-down menu.

   Lower Expect thresholds are more stringent, leading to fewer chance matches being reported.

5. If you do not want a gapped search, click in the Perform gapped alignment check box to deselect it.
6. You can limit the query sequence to a region of an entire sequence. One way to do this is to type, in the Region text boxes, the numbers of the first and last residues in the region. Another way is to select a region from the features table drop-down menu, which appears to the right of the text boxes.
7. Select More choices to expand the dialog box and modify the default settings.

   NOTE: If you have a Fewer Choices button, you already have the expanded form of the BLAST Search dialog box displayed. The extra settings are described in "Performing an advanced BLAST search", below.

8. Select OK to perform the BLAST search.

Alternatively, select Defaults to restore the default settings, or Cancel to close the dialog box without performing the analysis.

An information box indicates the progress of the BLAST search. The number of sequences searched and the number of matches found are updated appropriately. During the search phase, the number of characters read is displayed in the progress dialog box. It is impossible for MacVector to predict the volume of data that will be returned: this feature lets you know that your Internet connection is intact and that data retrieval is proceeding normally.

PERFORMING AN advanced BLAST search

MacVector enables you to change the standard BLAST search options, by expanding the Online search for Similar Sequences (BLAST) dialog box.

1. Choose Database | Online search for Similar Sequences (BLAST).

   While the connection is being made to the NCBI server, an information box is displayed. The Online search for Similar Sequences (BLAST) dialog box is then displayed.

   NOTE: If any part of the connection process fails, an appropriate error message is displayed and the entire BLAST search is aborted.

2. Set the Program, Database and Expect values as described in the procedure above.
3. Select More choices to expand the dialog box.

   NOTE: If you have a Fewer choices button, you already have the expanded form of the Online search for Similar Sequences (BLAST) dialog box displayed.

   The dialog box expands to show additional options.

4. The default setting is to perform a gapped alignment. If you do not want this, click in the Perform gapped alignment check box to deselect it.
5. Set the gapped alignment controls as required (these will be disabled if you deselected Perform gapped alignment in the previous step):
   • Use the Open cost drop-down menu to choose a penalty value for inserting a gap

     A high value will favor alignments with as few gaps as possible (range 3 - 19, default 11)

   • Use the Extend cost drop-down menu to choose a penalty value for extending a gap

     A high value will favor alignments with many short gaps over ones with fewer longer gaps range 1 - 3, default 1)

6. Choose a scoring matrix for the BLAST programs from the Matrix drop-down menu.

   NOTE: This item is disabled if the BLASTN program is selected in the program drop-down menu.

7. Select the Low Complexity Filter check box to enable filtering.

   This feature uses sequence similarity to mask regions that are non-specific for protein identification using sequence similarity. It can eliminate spuriously high scores that reflect compositional bias rather than specific pairwise alignment (e.g., hits against proline-rich regions or poly-A tails). Queries searched with the blastn program are filtered with DUST. Other programs use SEG.

8. Choose a genetic code from the genetic code drop-down menu.

   This is the code used to translate the query sequence, and defaults to the standard universal genetic code.

   NOTE: This item is only enabled if the BLASTX program is selected under the program drop-down menu.

9. Chose a value from the Output descriptions drop-down menu to restrict the number of short descriptions of matching sequences reported.
10. You can limit the query sequence to a region of the entire sequence. To do this, type in the numbers that bracket the region in the Region text boxes, or select a region from the features table drop-down menu that appears to the right of the text boxes.
11. Select OK to perform the BLAST search.

    Alternatively, select Defaults to restore the default settings, or Cancel to close the dialog box without performing the analysis.

    An information box indicates the progress of the BLAST search. The number of sequences searched and the number of matches found are updated appropriately. During the search phase, the number of characters read is displayed in the progress dialog box. It is impossible for MacVector to predict the volume of data that will be returned: this feature lets you know that your Internet connection is intact and that data retrieval is proceeding normally.

    NOTE: At the conclusion of a BLAST search, any matching sequences are not automatically retrieved from the remote NCBI server. If you want to perform additional analyses on matching sequences, you must first retrieve them. For details, see the help topic "Retrieving matching sequences".

When the search is complete, the Search Results dialog box is displayed.

Related Topics.

Online search for Similar Sequences (BLAST)

Displaying BLAST search results

Retrieving matching sequences