Before employing Oligowiz for design of oligonucleotides you need to prepare a FASTA file with target sequences. Each target corresponds to the sequence of an mRNA transcript. For each sequence a separate entry must be given. Your FASTA file must be in ASCII format, i.e.
a text file,
not MSWord.doc or the like. (an example, with 30 Bacillus subtilis coding regions is placed here
First thing you must do is to download the client program from
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
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
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
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
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
-"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"
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
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.
Description of interface
Below is listed a number of legends reffering to arrows pointing at different items in the OligoWiz interface depicted hereunder.
1) Graphs represent scores (y-axis) along the input sequence (x-axis). The different graphs represent different score types indicated by their color (arrow 5). For all scores it counts that 1 is optimal and 0 is worst.
2) Color bar representing the Total (weighted) score. The Total score is also represented as a graph (thick red line). On the color bar red represent total scores higher than 0.8
3) The oligonucleotide selected/predicted. The red line under the square represent a selected oligo, placed along the length of an input sequence. The Predicted/ Custom bottom (arrow 9) indicates the status of the oligo. The oligo can be moved to a desired position by dragging the red square. Note that the score graphs indicated at the 5'end of the oligo represent the score for the oligo along its entire length. Resulting in an extension of the orange input sequence representation to the right of the graph arrear.
4) The sequence of the oligonucleotide selected (arrow 3)
5) Score function manipulation interface. Here the score weight and score graph can be manipulated. Hint: deselecting some scores using the "show" option may help overview the remaining scores.
6) Sequence info field. Describing the input sequence selected in the table pointed to by arrow 7.
7) The input sequence table. Here the entries in the input FASTA file are represented. Selection of an entry in this table has effect on all other fields on the page.
8) The Total score function manipulation interface.
9) The "Apply to all" bottom applies score weights of the selected entry to all the entries, influencing the predicted optimal oligonucleotide for all the entries.
10) The predicted/custom bottom. Indicating if the oligonucleotide in question (arrow 3) is predicted by the program or selected by the user (by dragging the red box). Clicking the "Predicted" bottom moves the oligonucleotide to the highest total score position.
11) The "Oligo info" field describes the oligonucleotide selected (arrow 3). The W-score is the total weighted score for the selected oligonucleotide.
12) "oligos" per entry field. Here additional oligos for the same entry can be added or removed and will be displayed.