The following procedure guides you through utilizing BLAST against NCBI’s sequence databases.

1. Launch DNASIS MAX and import the Tutorial1.fsa file which is located in the
C:\HSK_DB\TutorialData directory.

Fig1. After loading the tutorial data.

2. Click the DNA – Compare tab and select the Internet – Blast Search option.

Fig2. DNA – Compare tag.

3. The Analysis dialog will appear.

Fig3. Analysis Dialog – In the Analysis Dialog, you will find the Parameter and Execute buttons.
The Parameter button is used for changing the parameters of an analysis.

4. Press the Parameter button and a dialog box will appear.
NCBI BLAST is set as the default URL.

Fig4. Internet Blast Search Parameterset dialog box.

5. Press the Setting… button to edit the parameters of NCBI BLAST.

Fig5.　NCBI Advanced BLAST Search dialog.

6. Once the configurations have been set, press the OK button.
To discard any changes, press the Cancel button.

7. Press the Execute button to send the query.
The time it takes to retrieve your results will depend on the size of the sequence, the database, and your internet service provider.  Once your results come back, it will be displayed like in Fig6.

Fig6. A sample result of the BLAST search in DNASIS MAX.

1. Launch DNASIS MAX and import the Tutorial1.fsa file which is located in the
C:\HSK_DB\TutorialData directory.

Fig1. After loading the tutorial data.

2. Click the DNA - Compare tab and select the Blast Search option.

Fig2. DNA – Compare tag.

3. The Analysis dialog will appear.

Fig3. Analysis Dialog – In the Analysis Dialog, you will find the Parameter and Execute buttons.
The Parameter button is for changing the parameters of an analysis.

4. Press the Parameter button and a dialog box will appear.

Fig4. BlastParameters dialog box.

5. Select the Blast program type from the Program name box.

• blastp – compares an amino acid query sequence against a protein sequence database
• blastn – compares a nucleotide query sequence against a nucleotide sequence database
• blastx – compares a nucleotide query sequence translated in all reading frames against a protein sequence database
• tblastn – compares a protein query sequence against a nucleotide sequence database dynamically translated in all reading frames
• tblastx – compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.

6. Set the E-value or Expect value in the Expectation value field. This value represents the number of times you would expect to see such a match (or better) merely by chance. The closer the value is to zero, the less likely the event is. To re-iterate, the smaller the E-value, the more significant the match is and the lower number of hits you’ll see.  The default value is 10.

7. Select the intended database from the Nucleotide Database.
Select the In-house-NA for this tutorial.

8. Once the configurations have been set, press the OK button.
To discard any changes, press the Cancel button.

9. Press the Execute button to start the blast analysis.

Fig5. A sample result of the Blast search in DNASIS MAX.

For more information on DNASIS MAX, please visit the DNASIS MAX product page or download the FREE trial.

Focusing on local alignments, BLAST uses a heuristic algorithm to detect relationships between sequences that may only share isolated regions of similarity. BLAST results take sequence length and the nucleotide/peptide compositions of the query into account when assigning alignment scores. For sequences shorter than 200 residues, an effective length is used to compensate for “edge effects”. Sequence alignment scores are reported by BLAST programs as E-values that reflect the strength of alignment between a given sequence in the database and a query. E-values are reported instead of the traditional P-value, to improve resolution between low scoring alignments, but for closely related sequences (P < 0.01), these values are nearly equal.

For more detailed information on how BLAST scores are calculated, visit:

http://www.ncbi.nlm.nih.gov/BLAST/tutorial/Altschul-1.html

For most first-time users of BLAST, choosing the right sub-program may be difficult. BLAST offers a variety of search tools for different types of queries. In general, the best choice of program depends upon the sequence length, the database being searched, and the information requested in the search.

Nucleotide BLAST is a collection of programs allowing users to compare a query sequence against other nucleotides in the database. BLAST accepts sequences in a variety of formats, including FASTA, GenBank, and Accession/GI numbers, and compares these with the NCBI databases. MEGABLAST is a concatenating algorithm for quickly aligning sequences longer than 28 residues. For shorter sequences, such as primers, standard nucleotide-nucleotide BLAST offers automatic parameter settings suited to these queries.

Protein BLAST is a collection of programs used to find protein sequences similar to a query. These programs accept sequences in the same file formats as Nucleotide BLAST. PSI-BLAST is a position specific, iterating algorithm that searches sequences from each round as the basis for scoring sequences searched in the next round. It distinguishes between highly and weakly conserved positions in the sequence, resulting in increased sensitivity with each iteration. PSI BLAST also offers the option of including regular expression patterns in the search, allowing users to identify sequences that include a pattern and are homologous to the query protein sequence. As with Nucleotide BLAST, Protein BLAST includes automatic parameter settings for shorter sequences.

Translating BLAST operates in a similar fashion to both the nucleotide and protein search routines. BLASTX translates nucleotide sequences into protein sequences in each of the 6 reading frames, prior to comparing the query to the protein databases. TBLASTN compares a protein sequence query against a database of nucleotide sequences previously translated in each of the 6 reading frames.

Users can refer to the NCBI BLAST program selection guide for more information:

http://www.ncbi.nlm.nih.gov/blast/producttable.shtml.

Users can access BLAST tools directly through the web, or through a variety of software applications, such as MiraiBio’s DNASIS SmartNote, which helps users find and organize sequences, and automatically submit them to the BLAST programs. DNASIS SmartNote has the additional ability to BLAST multiple sequences “in batch” without tediously copying/pasting each sequence and waiting for each result to come back.

To learn more about DNASIS SmartNote, visit http://smartnote.miraibio.com.