SEQUENCE ALIGNMENT

Alignment is the result of a comparison of two or more gene or protein sequences in order to determine their degree of base or amino acid similarity. Sequence alignments are used to determine the similarity, homology, function or other degree of relatedness between two or more genes or gene products. The likelihood that the two sequences are related is represented in an alignment score. This score is calculated by totaling the scores for each matched pair of residues at each position in the alignment.

The general approach for similarity searching involves the use of a set of algorithms such as the BLAST programs to compare a query sequence to all the sequences in a specified database. Comparisons are made in a pairwise fashion. Each comparison is given a score reflecting the degree of similarity between the query and the sequence being compared. The higher the score, the greater the degree of similarity. The similarity is measured and shown by aligning two sequences. Alignments can be global or local. A global alignment is an optimal alignment that includes all characters from each sequence, whereas a local alignment is an optimal alignment that includes only the most similar local region or regions. Discriminating between real and artifactual matches is done using an estimate of probability that the match might occur by chance. The similarity itself, is not a sufficient indicator of function.

Blast

The BLAST programs (Basic Local Alignment Search Tools) are a set of sequence comparison algorithms introduced in 1990 that are used to search sequence databases for optimal local alignments to a query. The BLAST programs improved the overall speed of searches while retaining good sensitivity (important as databases continue to grow) by breaking the query and database sequences into fragments ("words"), and initially seeking matches between fragments. The initial search is done for a word of length "W" that scores at least "T" when compared to the query using a given substitution matrix. Word hits are then extended in either direction in an attempt to generate an alignment with a score exceeding the threshold of "S". The "T" parameter dictates the speed and sensitivity of the search.

The quality of each pair-wise alignment is represented as a score and the scores are ranked. Scoring matrices are used to calculate the score of the alignment base by base (DNA) or amino acid by amino acid (protein). A unitary matrix is used for DNA pairs because each position can be given a score of +1 if it matches and a score of zero if it does not. Substitution matrices are used for amino acid alignments. These are matrices in which each possible residue substitution is given a score reflecting the probability that it is related to the corresponding residue in the query. The alignment score will be the sum of the scores for each position. Various scoring systems (e.g. PAM, BLOSUM and PSSM) for quantifying the relationships between residues have been used.