Pairwise and Multiple Sequence Alignments, and Similarity Searches

>RPE_MYCPN
     MLNLVVNREI AFSLLPLLHQ FDRKLLEQFF ADGLRLIHYD VMDHFVDNTV FQGEHLDELQ
     QIGFQVNVHL MVQALEQILP VYLHHQAVKR ISFHVEPFDI PTIKHFIAQI KQAGKQVGLA
     FKFTTPLVNY ERLVQQLDFV TLMSVPPGKG GQAFNSAVFN NLKQAHKYHC SIEIDGGIKL
     DNIHQIQDDV NFIVMGSGFI KLERWQRQQL LKTNQ

Please make a global alignment (choosing the option "needle") and also a local alignment (choosing the option "water"). What are the differences between the two outputs? Do you think that these two sequences are related?

Now try to align the two sequnces using different substitution matrices and changing the gap opening and gap extension penalties. Use, for example, BLOSUM62 and BLOSUM40. Do you see any difference? (you can check the pre-computed results here).

How could we decide which of the alignments obtained is better?

Similarity Search with BLAST

Use the sequence RPE_YEAST, to make a BLAST search on a protein database.

For the moment, please use the EMBL or EBI BLAST servers, since it is easier to retrieve the sequences identified by the BLAST search, and we will use them later, in another exercise.

If you are using the EMBL BLAST server, use the following parameters:

database=Swiss-Prot (nrdb95 provides more coverage, but we would obtain too many related sequences, making the analysis more complicated).
filter=none
descriptions=250
alignments=250

Once the results of the BLAST search have been returned, you can retrieve the sequences that have been identified as similar, by clicking on "Get selected sequences".

Here you will find pre-computed results for the EMBL BLAST search.

By default, those sequences with the best p-values appear checked, but you could select more or less sequences.

Those selected by default have been saved in this file.

Now you can try to use the NCBI BLAST server and compare (the EMBL BLAST server uses WU-BLAST, which is different to the original BLAST developed at the NCBI).

Here you will find pre-computed results for the NCBI BLAST search.

Now look for RPE_MYCPN in the output of the two BLAST searches using RPE_YEAST as a query. Check the associated e-value (or p-value) Is the similarity between the to sequences significant?

Note: to retrieve NCBI sequences you need to to choose the sequences you want by hand (!) and then click on "get sequences". Then on the next page choose the two options "FASTA" and display as "text" in the drop down menus".

Multiple Sequence Alignment of Sequences Identified After a BLAST Search.

In this exercise you will make a multiple sequence alignment of the sequences that have been identified in a similarity search with BLAST.

CREATING A MULTIPLE SEQUENCE ALIGNMENT WITH ClustalW.

Use a ClustalW web server.

Leave the parameters as appear by default, but change the output format:

output format= GCG (or GCG-msf).

Visualisation of Multiple Sequence Alignments (MSAs)

Multiple sequence alignments can be more easily interpreted if the columns in the alignment are coloured following some criteria (for example, the degree of conservation).

If you are running ClustalW at the web server of the EBI, you will have the option of getting a coloured alignment. You will have also the options of visualizing the alignment with JalView or constructing a tree.

The previously obtained MSA would look like this with Belvu, a popular visualisation program .
With Belvu, it is possible to manipulate or perform several kinds of analyses with the alignment, as for example, sorting the sequences according to several criteria, removing redundant sequences, or generating a tree from the MSA.
Other MSA visualization tools, that you could have installed, are Pfaat and JalView, which are available for Windows. In addition, you can use BoxShade, which is available as a web server.

Michael Tress
Protein Design Group