NCBI Blast, Sequence Analysis & Result Interpretation: Lecture 2 part 2 by Dr. Muhammad Naveed

NCBI Blast, Sequence Analysis, Result Interpretation & Result Submission.
Background
BLAST, which The New York Times called the Google of biological research,[2] is one of the most widely used bioinformatics programs for sequence searching.[3] It addresses a fundamental problem in bioinformatics research. The heuristic algorithm it uses is much faster than other approaches, such as calculating an optimal alignment. This emphasis on speed is vital to making the algorithm practical on the huge genome databases currently available, although subsequent algorithms can be even faster.

Input
Input sequences (in FASTA or Genbank format) and weight matrix.

Output
BLAST output can be delivered in a variety of formats. These formats include HTML, plain text, and XML formatting. For NCBI's web-page, the default format for output is HTML. When performing a BLAST on NCBI, the results are given in a graphical format showing the hits found, a table showing sequence identifiers for the hits with scoring related data, as well as alignments for the sequence of interest and the hits received with corresponding BLAST scores for these. The easiest to read and most informative of these is probably the table.

If one is attempting to search for a proprietary sequence or simply one that is unavailable in databases available to the general public through sources such as NCBI, there is a BLAST program available for download to any computer, at no cost. This can be found at BLAST+ executables. There are also commercial programs available for purchase. Databases can be found from the NCBI site, as well as from Index of BLAST databases (FTP).

Process
Using a heuristic method, BLAST finds similar sequences, by locating short matches between the two sequences. This process of finding similar sequences is called seeding. It is after this first match that BLAST begins to make local alignments. While attempting to find similarity in sequences, sets of common letters, known as words, are very important. For example, suppose that the sequence contains the following stretch of letters, GLKFA. If a BLAST was being conducted under normal conditions, the word size would be 3 letters. In this case, using the given stretch of letters, the searched words would be GLK, LKF, KFA. The heuristic algorithm of BLAST locates all common three-letter words between the sequence of interest and the hit sequence or sequences from the database. This result will then be used to build an alignment. After making words for the sequence of interest, the rest of the words are also assembled. These words must satisfy a requirement of having a score of at least the threshold T, when compared by using a scoring matrix.


Algorithm
To run the software, BLAST requires a query sequence to search for, and a sequence to search against (also called the target sequence) or a sequence database containing multiple such sequences. BLAST will find sub-sequences in the database which are similar to sub sequences in the query. In typical usage, the query sequence is much smaller than the database, e.g., the query may be one thousand nucleotides while the database is several billion nucleotides.
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