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Pairwise Sequence Alignment using BLAST


BLAST Procedure 


This is the common procedure for any BLAST program.


Step 1: Select the BLAST program.

Step 2: Enter a query sequence or upload a file containing sequence.

Step 3: Select the database to search.

Step 4: Select the algorithm and the parameters of the algorithm for the search.

Step 5: Run the BLAST program.


Step 1: Select the BLAST program


User have to specify the type of BLAST programs from the database like BLASTp, BLASTn, BLASTx, tBLASTn, tBLASTx.


Step 2: Enter a query sequence or upload a file containing sequence


Enter a query sequence by pasting the sequence in the query box or uploading a FASTA file which is having the sequence for similarity search. This step is similar for all BLAST programs. The user can give the accession number or gi number or even a raw FASTA sequence. Go to simulator tab to know more about how to retrieve query sequence.



Figure 1: Enter a query sequence or upload a file containing sequence


Step 3: Select database to search


User first has to know what all databases are available and what type of sequences are present in those databases. Sequence similarity search involves searching of similar sequences of the query sequence from the selected databases (Figure 2).



Figure 2: Select database to search 


Step 4: Select the algorithm and the parameters of the algorithm for the search


There are different algorithms for some of the BLAST program. User has to specify the algorithm for the BLAST program. Nucleotide BLAST uses algorithms like MegaBLAST which searches for highly similar sequences, discontiguous MegaBLAST which searches for more dissimilar sequences and BLASTn which searches for somewhat similar sequences. Meanwhile for protein BLAST algorithms like BLASTp, searches for similarity between protein query and protein database, PSI-BLAST performs position specific search iteratively, PHI-BLAST searches for a particular pattern (user has to enter the pattern to search in the PHI pattern box provided) that is present in the sequence against the sequences in the database, DELTA-BLAST is Domain Enhanced Lookup Time Accelerated BLAST. It searches multiple sequence and aligns them to find protein homology. The different algorithmic parameters are, Target sequences, Short queries, E-value, Word size, Query range, scoring parameters (Match/Mismatch scores, and Gap penalties) and filters (Filter and Mask) which are required to run BLAST programs. Default values are provided but the user can adjust the values accordingly which is shown in figure 3.




Figure 3: Algorithm and the parameters


Step 5: Run the BLAST program


Submission of the BLAST program can be done by clicking the BLAST button at the end of the page. Screen shot of result can be shown in figure 4.



Figure 4: Run the BLAST program


BLAST Result:


After submitting the query sequence for sequence similarity search, the result page will appear along with the information like Query id, Description, Molecule type, Length of sequence, Database name and BLAST program. It shows the putative conserved domains that have been detected while undergoing sequence similarity search.

The query sequence represented as a numbered red bar below the color key. Database hits are shown below the query (red) bar according to the alignment score. Among the aligned sequences, the most related sequences are kept near to the query sequence. User can find more description about these alignments, by dragging the mouse to the each colored bar which is shown below in figure 5.



Figure 5: BLAST result


The alignment is preceded by the sequence identities, along with the definition line, length of the matched sequence, followed by the score and E-value. The line also contains the information about the identical residues in alignment (identities), number of positivity’s, number of gaps used in the alignment. Finally it shows the actual alignment, along with the query sequence on the top and database sequence below the query. The number on either sides of the alignment indicates the position of amino acids/nucleotides in sequence which can be represented in figure 6.



Figure 6: BLAST result







 This experiment uses : Basic Local Allignment Search Tool (BLAST) , blast.ncbi.nlm.nih.gov




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