Micro Array Oligo design

Computer exercise by

Henrik Bjørn Nielsen

 

The exercises objective is to make you familiar with the parameters that are important for designing oligos for spotted arrays. This we hope you will achieve by this hands-on exercise.

 

To make things easy for yourself, login to genome.cbs.dtu.dk. Make an exercise directory and go there. Then type:

alias oligowiz /usr/opt/www/pub/CBS/services/DNAarray/OligoWiz/oligowiz.pl

ln -s ~mic00/microarray/yeast_ESTs.fsa yeast_ESTs.fsa

 

In this exercise you should design oligos for the 100 yeast EST’s in the file: yeast_ESTs.fsa now in your exercise folder. Alternatively you could find some other sequences to play with on the web but remember they must be in FASTA format. And if it is to make any sense you must restrict yourself to sequences originating from species that our program OligoWiz has databases for. OligoWiz has currently databases for the following species:

 

A. thaliana

B. subtilis

C. elegans

D. melanogaster

E. coli (K12)

Human (unigene)

M. musculus (unigene)

S. cerevisiae

 

First thing to do is to calculate the parameters for the EST sequences. This can be done by the program oligowiz.pl

To calculate the oligo parameters with default settings type:

oligowiz -in yeast_ESTs.fsa -species S.cerevisiae > yeast_ESTs.owz

This may take some time (1-5 min).

 

You can also play with the settings. To get help on oligowiz type:

oligowiz -h

 

If you want to design oligos for E. coli, which two parameters (besides the input file name) would you probably want to change then?

 

Now you have an owz file in your course directory it’s time to look at the parameters.

Download the oligowiz.jar program as well as your .owz and .fsa file.

Then run oligowiz.jar on your local machine.

 

In oligowiz.jar open the owz file (Alt-O). Select an entry from the bottom frame of oligowiz.jar and adjust the parameter weights in the middle frame. Note that you can hide some parameters to get a better overview.

Try to consider what parameters you find most relevant for an expression study, and try to consider the individual parameter distributions.

If you think you have found some nice weights try to press the “Apply to all” button and look trough some more entries to see if the parameters also looks good for these entries.

 

Finally try to export the oligos (Alt-E).

 

If you have extra time try to see how the parameters change if you design 30’mers instead of 50’mers

 

For supplementary reading on oligo design see

Kane_et_al.00.pdf

 

 

Backup.owz file!