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DNA Software > Support
> Visual OMP > Tips and Techniques
Visual OMP Tips and Techniques
BASICS
1. System Requirements |
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“Captain! We are running out of power!”
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Please check out our system suggestions
at: http://www.dnasoftware.com/Products/VisualOMP/SystemRequirements/index.htm
It may also help to increase your virtual memory to 4096
MB, if it is available.
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SIMULATION
2. Moving Structures within DNA Display |
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Goto AddPivotPoints (Second to last icon
on the toolbar). Click once to add the points in your structure.
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Goto MovePivotPoints (Last icon on the
toolbar). With mouse arrow over a pivot point, drag to desired
location.
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Press Refresh too see the nucleotides
again.
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3. Freezing panes in results summary table |
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Goto the left side most vertical line
until your icon turns into a lock icon.
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Click on that line, hold the mouse down,
move the line to the column that you want to freeze.
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Now you can move the columns along with
frozen name columns.
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4. Copying results from one experiment file
to another experiment file |
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From your Design Results Table. Select
the row you want to copy
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Right click and add to experiment.
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Goto your Sequences Tab of the same experiment.
Your copied design result should be at the bottom of the
table.
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Select Cut Row(s).
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Goto your new experiment Sequence Tab.
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Select bottom empty row.
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Select Paste Row(s).
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Please do not cut and paste from your design
results summary page directly into your new experiment.
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DESIGN
5. Advanced Parameters for Designs |
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For a description of all parameters
press F1 on the probe/primer design tab and go to the
help-page about the advanced parameters.
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Choose a strategy (last column
of design-row) which best matches your design. “Balanced”
is recommended for normal probe or PCR primer design.
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Review the parameters and change
them to your needs if necessary. The most adjusted parameters
in general are:
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Oligo size, the length of
the primer/probe you are going to design. If you do
not care too much about its length, maintain minimum
and maximum values and change the values of the weighting
factors, if you like. For example, a primer of length
20 may have min=18 and max=22 with weight factors
as 0, 0 meaning that any primer of length between
18 to 22 nucleotides will be weighted the same.
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The same applies to amplicon
size in case of primer pair designs. For example,
if the intended amplicon is between 100-200 nucleotides
long, then you can adjust the minimum and maximum
values as such and change the weight factors to 0,
0 as well.
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Duplex Tm is the melting temperature
of the intended heterodimer. This needs to be set
a couple of degrees (5 to 10) higher than the assay
annealing temperature, so that heterodimer-concentrations
at the assay-temperature will be at a maximum.
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Solution distance, one of
the parameters on the right side of the screen. This
value indicates the minimum number of nucleotides
which are apart from each other in a set of design
solutions. For example, setting it to 3 means that
if a primer#1 starts from nucleotide #1 on your target,
then the next primer solution will have a minimum
starting point at target-nucleotide #4. For designs
which are not very restricted, the default of 3 is
good and will save computational time. But for difficult
designs where you are restricted to a certain region
for example, it’s recommended to decrease the value
of the solution distance to 1. This way more potential
probes/primers will be considered.
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6. Tackling "Infeasible Solutions" |
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If after running a design, VO
gives you “No feasible solutions were found”, it means
that none of the potential probes/primers have passed
the tests for all advanced parameters.
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In the “design information”
tab, the number of failed designs due to each parameter
setting will be displayed. The parameter which contains
the highest number of failed designs will appear in
pink shading.
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Relax this parameter in pink
shading first and then re-design. Keep repeating until
VO finds feasible solutions. With this method, the
constraints will be kept tighter and the designs which
pass will be more specific, as opposed to starting
out with very loose constraints.
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If after relaxing some of the
parameters, VO still gives “infeasible solutions” and
the main reason is the mishyb parameter, consider unchecking
one or more of the “eliminate extensible structure” settings
on the design settings tab. It is very dependent on your
design in which order you would do this. Taking PCR as
an example, the best order would most probably be:
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First uncheck the “eliminate
extensible homodimers” box, these will usually be
most innocent and at non-significant concentrations
because it involves bimolecular binding.
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Then uncheck the “eliminate
extensible monomers” box.
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The last option to also uncheck
the “eliminate extensible heterodimers” box is not
recommended. However if you are out of options, try
the design with this and assess your assay through
simulation which can show you if the unintended extensible
heterodimer(s) is (are) going to cause complications.
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It is usually recommended to assess
designed oligos by running a simulation, but especially
so in the case where “eliminate extensible structure”-boxes
have been unchecked. The simulation results will identify
extensible structures, their dG, concentration, and percent
bound. These resulting data may be used to check for potential
problems. For example: a monomer which is extensible by
one loosely bound basepair and present at a low concentration
is much less likely to cause artifacts in your assay than
an extensible homodimer with significant basepairing and
present at relatively high concentrations.
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In cases in which the “design
information” tab contains no information, please recheck
your design input. Common input errors include: 1) design
of an allele probe pair with no variation specified in
the position/range cell, 2) the minimum amplicon length
is longer than the length of the actual target. Please
contact us if you are having problems finding the troublesome
setting.
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ASSESSING DESIGNS
7. Assessing Primers and Probes |
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The best prediction of the success
of an assay is how well your primer/probe binds to your
target, under the solution conditions of your assay. Your
primer/probe has competing secondary optimal and suboptimal
structures. So does your target.
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Although our primer/probe design
module does consider a large number of advanced parameters
when predicting best potential probes and primers, it does
not simulate each of the primer/probe candidates against
the target automatically. This would be computationally
too extensive. Thus, here is how you should assess these
primer/probe candidates to make sure they are indeed good
ones:
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Run Design for Probe/Primer
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Copy several back into experiment.
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Simulate one at a time to assess
percent bound and good eff Tm.
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High percent bound and ideal
eff Tm for your assay predicts a good primer or probe.
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8. Primer/ Probe positions from Design Results |
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MOVING FILES
9. Moving an Experiment |
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go to the directory you saved your
experiment in
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sort the directory for file name
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select all files starting with your
experiment name. If my experiment is called "test"
for example, select the following files (if present):
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test.nal (contains numerical
analysis information)
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test.odf (contains design information)
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test.odf.backup
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test.oef (contains experiment
and simulation information)
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test.oef.backup
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test.oof (contains results of
a simulation)
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test.osf (contains results of
a design)
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test_....tbs (contain secondary
structure information)
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right-click and select "cut"
if you want to also remove the experiment from its current
directory or "copy" if you want to also keep copies
there
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go to the directory you want to
move your experiment to, right-click and select "paste"
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double-click on "test.oef"
and open it in for example notepad
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go to the line "OUTPUT_DIRECTORY=
" and change this to the directory you copied your
experiment to
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if you now open up your experiment
from this new directory everything should be pointing to
the right directory when opening different parts of the
experiment (e.g. the results summary or the design results)
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10. Moving an Entire Project |
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move every experiment which you
want to be in the project to the correct directory as described
above in part A
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open up Visual OMP and add the experiment(s)
you want to be in the project
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select "save project as"
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save it to the intended directory
(can be different from the directory/directories your experiment(s)
are in)
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CUSTOMIZING
VISUAL OMP
11. Custom Solution Conditions on the
Experiment Conditions Tab |
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When one or more of the Solutions
Conditions on the Experiment Conditions tab are changed
from a default solution, this custom solution can be
saved by clicking on the “Save Current Conditions as
Setting” button, located below the solution conditions.
Visual OMP will ask for a name of the custom solution.
After clicking on the “add” button these conditions
will be saved and can be used in other experiments.
They are accessible via the drop-down list below the
solution conditions. Solutions may be deleted from this
list by using the “Delete Current Conditions from Settings”
button.
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12. Custom Advanced settings for Experiment
Conditions on the Experiment Conditions Tab |
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Changes in the settings on the
right hand side of the Experiment Conditions tab, the
“Advanced Settings”, can be saved as the default settings
for all other experiments by using the “Save current
Settings as Default” button. When changing some settings,
these defaults can be retrieved by the “Restore Default
Settings” button. The original defaults of Visual OMP,
which come with it when one first downloads the program
can be retrieved through the “Restore Factory Settings”
button.
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Please note that these 3 buttons
apply to the slidebar and checkboxes on the Experiment
Conditions tab and to the values of the parameters under
the Structure, NumAnaly, Extensibility and Custom Stats
Settings buttons.
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13. Custom Strategies for Design on
the Probe/Primer Design Tab |
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Custom Strategies for designs can
be saved on the Probe/Primer Design tab. If after opening
up the Advanced Parameters for a design (right-clicking
and selecting “Advanced Parameters”) changes are made
to default strategies, such as Balanced or By Tm (last
column on the Probe/Primer Design tab), this custom
strategy can be saved. This can be done by either right-clicking
and selecting “Save Custom Strategy” or by clicking
on the “Save Custom Strategy” icon in the toolbar (a
knight with a floppy disk). After entering a name and
clicking “ok” the custom strategy will be added to the
drop-down list of strategies under the “Strategy” column
and will be available in all other experiments.
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14. Using Blast while designing probes or
primers |
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15. Stand-alone Blast-utility |
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Hitting the “B”-icon in the toolbar will
bring up the form for the stand-alone Blast, which can be
used for any sequence in a project. For the stand-alone
Blast, the database can be either local or over the internet,
i.e. NCBI direct link.
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16. Ways to Obtain a Database (in FASTA
format) on a Local Drive |
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If you have the sequences you want
to Blast against in a Visual OMP experiment file, you can
export this file as a FASTA (.fas) file via “Export” under
“File”.
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If you have a custom collection
of sequences you would like to use, you may paste them into
a notepad file and then save it as “database.fas” instead
of a .txt file. A description of the fasta-format can be
found at http://ngfnblast.gbf.de/docs/fasta.html.
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Download the appropriate fasta-file
from (ftp://ftp.ncbi.nlm.nih.gov/blast/db/FASTA). Since
these files can be large (i.e. human genome), it is required
to unzip these .gz-files, see point 17 below.
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17. Unzipping Instructions |
Go to www.gzip.org to the executables section.
Choose the executable which fits your OS and download. You
can use this program through the command prompt:
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Open up the command prompt (from
the start-menu choose “run”, type “cmd”)
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Go to the directory of your gzip.exe.
For example, if it is on my desktop, I type: “cd C:\Documents
and Settings\User\Desktop”.
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Use the “gzip –d” command to unzip
the .gz file:
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if the downloaded database (ecoli.nt.gz
for example) is also on the desktop, I type “gzip –d
ecoli.nt.gz”
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if the database is in a directory
other than the one I have gzip.exe in, I give the total
path where the zipped database is. For example, if I
saved ecoli.nt.gz to C:\User, then I type: “gzip –d
C:\User\ecoli.nt.gz”
- Rename the unzipped file to ecoli.fas,
now it’s ready to BLAST against
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18. Formatting |
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SCIENCE
| 19. The effect on Tm by Target and Oligo Concentrations |
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