BLAST

BLAST (Basic Local Alignment Search Tool) is a popular program for searching biosequences against databases. BLAST was developed and is maintained by a group at the National Center for Biotechnology Information (NCBI). Salient characteristics of BLAST are:

Local alignments

BLAST tries to find patches of regional similarity, rather than trying to find the best alignment between your entire query and an entire database sequence.

Ungapped alignments

Alignments generated with BLAST do not contain gaps. BLAST's speed and statistical model depend on this, but in theory it reduces sensitivity. However, BLAST will report multiple local alignments between your query and a database sequence.

Explicit statistical theory

BLAST is based on an explicit statistical theory developed by Samuel Karlin and Steven Altschul (PNAS 87:2284-2268. 1990) The original theory was later extended to cover multiple weak matches between query and database entry PNAS 90:5873. 1993).

CAUTION: the repetitive nature of many biological sequences (particularly naive translations of DNA/RNA) violates assumptions made in the Karlin & Altschul theory. While the P values provided by BLAST are a good rule-of-thumb for initial identification of promising matches, care should be taken to ensure that matches are not due simply to biased amino acid composition.

CAUTION: The databases are contaminated with numerous artifacts. The intelligent use of filters can reduce problems from these sources. Remember that the statistical theory only covers the likelihood of finding a match by chance under particular assumptions; it does not guarantee biological importance.

Rapid

BLAST is extremely fast. You can either run the program locally or send queries to an E-mail server maintained by NCBI

blast@ncbi.nlm.nih.gov

Heuristic

BLAST is not guaranteed to find the best alignment between your query and the database; it may miss matches. This is because it uses a strategy which is expected to find most matches, but sacrifices complete sensitivity in order to gain speed. However, in practice few biologically significant matches are missed by BLAST which can be found with other sequence search programs. BLAST searches the database in two phases. First it looks for short subsequences which are likely to produce significant matches, and then it tries to extend these subsequences.

A substitution matrix is used during all phases of protein searches (BLASTP, BLASTX, TBLASTN)

Both phases of the alignment process (scanning & extension) use a substitution matrix to score matches. This is in contrast to FASTA, which uses a substitution matrix only for the extension phase. Substitution matrices greatly improve sensitivity.

BLAST comes in 4 flavors:

BLASTP: search a Protein Sequence against a Protein Database.
BLASTN: search a Nucleotide Sequence against a Nucleotide Database.
TBLASTN: search a Protein Sequence against a Nucleotide Database, by translating each database Nucleotide sequence in all 6 reading frames.
BLASTX: search a Nucleotide Sequence against a Protein Database, by first translating the query Nucleotide sequence in all 6 reading frames.

BLASTN, BLASTP, TBLASTN were described in the original BLAST paper (JMB 215:403-410); a later paper describes BLASTX (Nature Genetics 3:266-272). Analysis of BLAST reports can be aided by a number of tools such as the filters XNU, SEG, and XBLAST.

This document is intended to serve as a guide to using certain bioinformatics programs. It cannot be guaranteed to be free of errors or completely up-to-date. If you know of errors or other shortcomings of this document, please mail them to Keith Robison (Church Lab, HMS Genetics)

KRobison@nucleus.harvard.edu