Exercise 2: Search for homologues in sequence databases

Objective: retrieve homologues of human insulin in the genome of the nematode C. elegans.
 

Step 1: search for homologues of the human insulin protein (see exercise 1) with BLAST at NCBI ( protein blast), using default parameters:

• Database: Non-redundant protein sequences (nr)
• Algorithm: blastp (protein-protein BLAST)


Look at the results ( *** ) and the taxonomy report (link at the top of the result page) ( *** ). How many nematode insulin homologues did you find ? (NB: you can use the menu "Edit/Find" to search for nematode in the taxonomy report).
 

 
• Database: Non-redundant protein sequences (nr)
• Algorithm: blastp (protein-protein BLAST)
• Algorithm parameters:
• Max target sequences: 5000
• Expect threshold: 100
How many nematode insulin homologues did you find ?

NB: to display only the results from the nematode (Caenorhabditis elegans) you can use the link "Reformat these Results" at the top of the result file, and then in the field "Organism" enter: Caenorhabditis elegans

Step 3: Now repeat the BLAST search with the same parameters, except the "Organism" in the Choose Search Set section:

• Database: Non-redundant protein sequences (nr)
• Organism: Caenorhabditis elegans
• Algorithm: blastp (protein-protein BLAST)
• Algorithm parameters:
• Max target sequences: 5000
• Expect threshold: 100

Look at the results (  ***  ). How many nematode insulin homologues did you find ? How can you explain the difference with what you obtained in step 2 ?

Step 4: Now repeat the search but using the PSI-BLAST algorithm (NB: do not limit the search to Caenorhabditis elegans):

• Database: Non-redundant protein sequences (nr)
• Algorithm: PSI-BLAST (Position-Specific Iterated BLAST)
• Algorithm parameters:
• Max target sequences: 5000
• Expect threshold: 100

Run 3 PSI-BLAST iterations, with the same  parameters, and then look at the result and the taxonomy report . How many  insulin homologues did you find ? Look at the Score and E-value of the nematode hits.

Run 2 other PSI-BLAST iterations. Compare the Score and E-value of the nematode hits obtained after 3 and 5 iterations (***).