Sequence database searching

The most obvious first stage in the analysis of any new sequence is to perform comparisons with sequence databases to find homologues. These searches can now be performed just about anywhere and on just about any computer. In addition, there are numerous web servers for doing searches, where one can post or paste a sequence into the server and receive the results interactively:

There are many methods for sequence searching. By far the most well known are the BLAST suite of programs. One can easily obtain versions to run locally (either at NCBI or Washington University), and there are many web pages that permit one to compare a protein or DNA sequence against a multitude of gene and protein sequence databases. To name just a few:

One of the most important advances in sequence comparison recently has been the development of both gapped BLAST and PSI-BLAST (position specific interated BLAST). Both of these have made BLAST much more sensitive, and the latter is able to detect very remote homologues by taking the results of one search, constructing a profile and then using this to search the database again to find other homologues (the process can be repeated until no new sequences are found). It is essential that one compares any new protein sequence to the database with PSI-BLAST to see if known structures can be found prior to doing any of the other methods discussed in the next sections.

Other methods for comparing a single sequence to a database include:

It is also possible to use multiple sequence information to perform more sensitive searches. Essentially this involves building a profile from some kind of multiple sequence alignment. A profile essentially gives a score for each type of amino acid at each position in the sequence, and generally makes searches more sentive. Tools for doing this include:

A different approach for incorporating multiple sequence information into a database search is to use a MOTIF. Instead of giving every amino acid some kind of score at every position in an alignment, a motif ignores all but the most invariant positions in an alignment, and just describes the key residues that are conserved and define the family. Sometimes this is called a "signature". For example, "H-[FW]-x-[LIVM]-x-G-x(5)-[LV]-H-x(3)-[DE]" describes a family of DNA binding proteins. It can be translated as "histidine, followed by either a phenylalanine or tryptophan, followed by an amino acid (x), followed by leucine, isoleucine, valine or methionine, followed by any amino acid (x), followed by glycine,... [etc.]".

PROSITE (ExPASy Geneva) contains a huge number of such patterns, and several sites allow you to search these data:

It is best to search a few different databases in order to find as many homologues as possible. A very important thing to do, and one which is sometimes overlooked, is to compare any new sequence to a database of sequences for which 3D structure information is available. Whether or not your sequence is homologous to a protein of known 3D structure is not obvious in the output from many searches of large sequence databases. Moreover, if the homology is weak, the similarity may not be apparent at all during the search through a larger database.

One last thing to remember is that one can save a lot of time by making use of pre-prepared protein alignments. Many of these alignments are hand edited by experts on the particular protein families, and thus represent probably the best alignment one can get given the data they contain (i.e. they are not always as up to date as the most recent sequence databases). These databases include:

Generally one can compare a protein sequence to these databases via a variety of techniques. These can also be very useful for the domain assignment.

Next Homologue in PDB? or Secondary structure prediction or Domain assignment

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