The following exercises will introduce you to the principle of database searches using the blast tools.
These tools allow the biologist to find sequences similar to his query sequence. They are routinely used to identify homologuesof proteins, distantly related sequences, functional domains, etc.

Many web-based blast-services are available to the biologist. They differ in the structure of the web-interface, the amountof parameters that can be modified and the number of different databases. The frequency with which the databases are updated to reflectthe latest changes, may also vary between servers.

• Some well known blast servers:
  

  Swiss EMBnet basic BLAST	This server gives access to a simplified blast web page. It is easy to use and understand, but does not allowto change all parameters. Use this link as entry point for the following practicals !
  Swiss EMBnet advanced BLAST	Allows to modify all the parameters of the blast tools. It is more powerfull when handling difficult queries.The course and practicals should allow you to understand these parameters and use them if necessary. Use this link as entry point for the following practicals !
  NCBI BLAST Server	Very well maintained blast server located at the NCBI; where the blast tools are developped.

During this practical please use either EMBnet's basic blastor advanced blast pages.

Reminder: some links for the recovery of sequences

[ Easily fetch sequences ][ Search SWISS-PROT ][ NCBI's Entrez ]

Use the protein sequence CC16_SCHPO as query to search for similar sequences in theSwiss-Prot database, using the program blastp. Use the default parameters andtry to answer the following questions:

• How is the output organized ? What information can be found in the different sections ?
• Find the section describing the database searched. How many sequences does the database contain ? How many amino-acids ?
• What do the score and the E-value stand for ?
• Do we find homologous sequences in the database ? Don't hesitate to take a look at the Swiss-Prot entries to get moreinformation about the sequences.
• Try using different substitution matrices like blosum90 or blosum45.What are the differences ? Can you see an influence on sensitivity or selectivity ?
• On the advanced blast form try changingthe default gap opening and extension penalties. The parameter help page showsvalid combinations of gap parameters according to the matrix used.
• Try to disactivate the low complexity filter. What are the differences ?

The EMBL W99073 sequence is a mouse EST. Compare this nucleotide sequence to the Swiss-Protdatabase using the blastx program on the basic blast page.
Try to answer the following questions:

• What is the purpose of the blastx program ? What are the differences with blastp and blastn ?
• Does this EST correspond to a coding sequence ?
• Does the EST cover the complete sequence of this mouse protein ?
• Look at the similar regions. Where are they located in the EST ? and in the protein ?What is known about the conserved regions ? (Don't hesitate to use Dotlet)
• Try to disactivate the low complexity filter. What are the differences ?

Take a look at the database choice available on the advanced blast page. More information about the databases is available on thedatabase help page.