Introduction to sequence alignment

Proteins that have a significant biological relationship to one another often share only isolated regions of sequence similarity. For identifying relationships of this nature, the ability to find local regions of optimal similarity is advantageous over global alignments that optimize the overall alignment of two entire sequences. BLAST , or Basic Local Alignment Search Tool (1), is an alignment tool thatuses a measure of local similarity to score sequence alignments in such a way as to identify regions of good local alignment.

The basic BLAST algorithm can be implemented in DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. There are 5 BLAST options on the NCBI web site. BLASTP compares any amino acid query sequence against a protein sequence database. Similarly, BLASTN compares a nucleotide sequence against a nucleotide sequence database. BLASTX takes a nucleotide query sequence and translates it in all reading frames for comparison against a protein sequence database. TBLASTN compares a protein sequence against a nucleotide sequence database, translating the nucleotide database sequences in all possible reading frames. TBLASTX compares translations of a nucleotide query sequence against translations of a nucleotide sequence database. Sequences must be input in one of three formats; FASTA sequence format, NCBI Accession numbers, or GIs (GenBank Identifiers). The FASTA format is the only format where you can input a sequence, instead of a number or code identifying a gene that has already been deposited in GenBank, so this is the format that must be used for nonpublished sequences. Take a moment to view the FASTA format description .

Consider the following nucleotide sequence, which is in FASTA format, but lacks the single-line description that identifies it source:

Access the NCBI BLAST home page , and click on the link for nucleotide BLAST (BLASTN), the standard BLAST for searching a nucleotide database with anucleotide query sequence. Highlight and copy the FASTA sequence above, and paste it into the first box on the BLAST query page.BLAST allows the option of querying over only a subset of the query sequence, identified by numerical order in the "query subrange" boxes next to the query. If it were desirable to query with only the subsequence beginningwith the 50th nucleotide of the example sequence and ending with the 250th nucleotide, then the number 50 would be entered into the "From" box and 250would be entered into the "To" box. Here in this example the entire sequence should be used as the query, so these boxes should be left blank.The database can be selected under "Choose Search Set". There are many available database choices you can view from the pull down menu. The "nr/nt" database is the largest nucleotide database available through NCBI BLAST; select the "nr/nt" database for this exercise. It includes all GenBank, RefSeq Nucleotides, EMBL (European nucleotide database), DDBJ (Japanese nucleotide database) and PDB (Protein Data Bank) sequences, but no EST, STS, GSS, or phase 0, 1 or 2 htgs (unfinished high throughput genomic) sequences. The NCBI nr database originally got its name from the phrase "nonredundant" nucleotide database, but there is no longer any claim to nonredundancy in the sequence set. Choosing the largest database is not always best; in fact, often thegoal is to find a match from a specific organism, for example, a lab that works with flies ( "Drosophila melanogaster" ) as the model organism might be only interested in their organism's sequences. In that case, there is a box entitled Organism, where a user interested can type in "Drosophila melanogaster" .