First thing you must do is to download the client program from the OligoWiz web page and possible Java 1.3 or newer.

From a Windows PC or Macintosh just click on the program icon and wait a few seconds.

In a unix or linux operative system you must write:
java -jar OligoWiz.jar

Set up your run

Click on the image for full size      From the "Query" page you can launch calculations on the OligoWiz Server program running on the CBS web server. To launch a calculation you must: In the "Main" field under Parameters: - Specify an input file - Specify an output file (OligoWiz will suggest one) - Select a database for the complexity and homology calculations in the "Species" field. The databases currently available are described here Now you may start your run, using default settings, or customizing your run Customizing your run The OligoWiz client allows you to customize a series of parameters for the calculations of the scores. These parameters can be set in the "Scores" field to the right of "Main" field. Here you will find five "page selectors" named: General, Tm, Homology and Position, described hereunder.

- Specify the length of the oligonucleotides.
If the "Optimize oligo length to fit Tm" is selected, the aim length is used to calculate the optimal Tm for the delta-Tm score, i.e. the mean Tm for all oligos of this length is the aim Tm.

- Specify the Min and Max oligo length to define the length interval within which OligoWiz selects oligos.

Here you may specify an optimal Tm, but we recommend you leave that to OligoWiz.

Here you can specify the criteria's for BLAST hits to be included in the Homology score calculation.

- "Minimum homology" (%) specifies the lowest degree of similarity to be taken into account for the homology calculation.

- "Minimum length of homology stretch" (bp) specifies the shortest overlap a BLAST hit can have with an oligo without being ignored from the score calculation.

Kane et al. (2000) show that for 50mers more than 75-80% homology or stretches over 15bp can course cross-hybridization.

- "Maximum homology" and "(total) Max length cutoff" is used to filter BLAST hits coursed by homology to the database version of the transcript in question, or homology to very closely related paralogs.
The "Max length cutoff" sets a maximum fraction of the entire length of the query sequence, which can be covered by an accepted BLAST hit exceeding "Maximum homology". Both "Maximum homology" and "(total) Max length cutoff" should be violated for a BLAST hit to be rejected.

Position refers to the position within the query sequence.  The score models the reverse transcription process that produces cDNA.
There are two exclusive options.

- "Poly-T primer" specifies a score based on a 3' poly-T primer. The further a probe is placed from the 3' end the lower score.

- "Random primer" specifies a score based on a random primer that anneals various places along the transcript. The chance of having a primer anneal downstream is larger for transcripts toward the 5'end, still taking into account that the reverse transcriptase can drop off the transcript.

-"RT. drop off probability" is the probability that the reverse transcriptase will drop off at any given base along the transcript.

Starting your run

Click on the image for full size      Having specified the parameters you press the Submit button and wait. The server will return an owz file and the client will tell you when the calculations and the file transfer are "Completed" (in the "Query list" winodw). The time required for the server to finish the calculations depending on the server load, the query length and the database selected (Species). The sample data set, with 30 B. subtilis sequences will typically take 30 seconds to complete. When "Completed" double click on the completed query in the Query list. Now a new page appears.

Processing results: placing oligos

Click on the image for full size      On the new page: Select an entry from the bottom frame and adjust the parameter weights in the upper frame. Note that you can hide some parameters to get a better overview. The orange bar under the curves indicate the query sequence, and the curves above describe the values of the different parameters for the oligonucleotide with its 5'end in that position. Note! The orange bar extend the curve's in the 3'end. 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 good 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. The red line indicates the selected oligo. By default the selected oligo is the one with the best total score, but you have the option to interact directly by dragging the little read box on the orange bar. To undo that press the Predicted button. When satisfied with the selected oligonucleotides, you can export them (Alt-E). OligoWiz exports the oligos in FASTA format.