Methods: REP-PCR Adding Gel Images to the Bionumerics Database (6-01-04)
Modified from Bionumerics v. 1.5 manual Applied Maths, Kortrijk, Belgium
Convert images to TIF format
Open Bionumerics program and click on analyze colony rep PCR for rep-PCR or click analyze Typhoon source tracking for HFERP
Drag the TIF images from the desktop to the data column in Bionumerics
Click on gel name so its highlighted, select file/open experiment file
(data)
Bionumerics will ask what fingerprint type to assign the gel, select
Box for rep-PCR, select Typhoon for HFERP
An image of the gel will appear in the fingerprint data window, if image
is upside down select file/tools/vertical mirror of tif image
If image is reversed select file/tools/horizontal mirror of tif image
Image on screen should now match thermal printer image of gel
The arrow keys in the toolbar allow you to move forward and backward
through the four steps involved in analyzing the gel images
Step 1. Strips (defining lanes)
Move green lines to size image by clicking on the boxes and dragging
the lines to frame the gel image
Go to Lanes / autosearch lanes, enter number of lanes on the image
Program will automatically put splines on gel image lanes
Molecular weight marker lanes should also have splines
Select a spline by clicking on it, can delete with toolbar - key, or
add a spline next to the selected spline with the toolbar + key
Write down the lane number assigned by the program to each lane with
a spline and the isolate associated with each particular lane, the isolates
in each lane will be entered in the data base after the image is analyzed
Choose Edit/ thickness (or edit key in the toolbar) to adjust thickness
of splines, The default value is 17, see p. 30 of BioNumerics manual for
a picture of optimal adjustment, the splines should enclose the lanes
but not overlap each other
Select number of nodes, the default value is 3
if lanes are bent due to electrophoresis can click on node and hold down
the shift key to bend the spline locally
Can choose Edit/ zoom in or Edit/ zoom out to more clearly see an image
when adjusting splines (or use the zoom keys on the toolbar)
Choose Edit / Edit Tone Curve
Choose linear, click on enhance weak bands 1-3X depending on the gel
image, make sure small molecular weight marker bands are visible
Click on disk image in toolbar to save image, press forward arrow key
to proceed to step 2
Step 2. Curves. (setting background and computing signal/noise ratio)
Select Edit / Settings
Nodes should be set to 2 which is the default value
Check averaging thickness, this refers to the thickness of the lanes
used for curve extraction, the lanes should be as broad as possible but
exclude smiling at the edges, see p. 35 of manual for picture of optimum
settings The default value is 11
Make sure apply background subtraction and apply least square filtering
boxes are not checked (computer will determine these values in the next
steps)
Select curves / spectral analysis
Write down signal/noise ratio Weiner cutoff scale (determines optimal
setting for least squares filtering i.e. 0.75%) and background scale percentage
(estimation of background disk size i.e. 10%)
Select Edit / Settings
Apply background (check box) and type in disk size (i.e. 10%), apply
least squares filtering and type in cutoff value (i.e. 0.75%), click OK
Click on disk icon to save file, click on forward arrow to proceed to
step 3
Step 3. Normalization (setting external and internal standards)
External reference standards will appear to the left of the gel image
corresponding to molecular weight standards 298-6108 bp, this same reference
system is used to normalize every gel that is entered into the data base
To use these standards for the present gel, click on the label of a reference
position to select, hold the CRTL key and click on the corresponding reference
bands in the three marker lanes, a green band will appear over the reference
bands (can right click on green bands to delete or see other options)
Repeat for all reference positions, green arrows will show adjustments
Select normalization/ show normalized view, (or shortcut button with
arrows) green arrows will disappear and gel will be aligned to the external
standards
Gel may still be wavy, therefore 4-6 bands must be selected as internal
standards, these bands should be present 3 or more lanes and both high,
middle, and low molecular weight bands should be selected
Click on a band to select it, go to references/ add internal reference
position, program will ask if you want to autosearch for similar bands,
select no
An unmarked arrow will appear to the left of the band to indicate the
position of the internal reference standard
Hold down the CTRL key and select the other lanes with the same band
Select normalization / show normalized view, identical bands across gel
should be perfectly aligned
Choose several additional bands to use as internal standards, gel should
now look great!
Select normalization and check the option to show distortion bars There
should be no dramatic color changes or a wrong assignment was made
Save file and proceed to step 4
Step 4. Bands (program finds bands on the gel)
Go to Edit / settings or edit key in toolbar, make sure min. profiling
is set at 3.00%, min. area is set at 0.00%, grey zone is set to 0.00%, and shoulder sensitivity = 0
(These settings allow most of the shoulder bands on shoulders and weak
bands to be found) press OK Go to bands / autosearch bands
Program will label selected bands on gel with green lines
- The program may omit faint bands and shoulders, therefore,
one must make the following corrections
-
delete green lines due to spots or other background noise on gel
(program cannot tell if the signal is from a spot or band), zoom in
and out on the image using the arrow keys
-
delete green lines on bands less than 300 bp (the lowest molecular
weight marker is 298 bp) these bands are faint and fuzzy
-
Check to see all bands are labeled with green lines, especially faint
high and low molecular weight bands and bands associated with shoulder
signals. Use arrow keys to zoom in (magnify) and zoom out (shrink)
gel image. Zoom in to compare the peak profiles to the right of the
images with the assigned band positions. Zoom out to see faint bands
more clearly. Right click on a band image to add a band or on a green
line, not associated with a band, to delete it. Zoom in order to add
green lines to the center of the band images. Program adds bands below
marks on opposite sides of the lane. A selected green line can be
repositioned by clicking on the middle box (located at the center
of the selected peak profile on the right hand side of the image)
and holding down the mouse. Bands may be weak and fuzzy so use your
best judgement.
Save file and close fingerprint image window
Now it is time to add new entries to the database corresponding to the
gel image and link them to the image file
Select database / add new entries from the main window
Enter the number of new isolates on the gel you just analyzed, these
entries will be added to the end of the database, the molecular weight
lanes are not included in the database
Double click on each new data base entry, the entry edit window appears
Fill in the information fields for each new entry in the database, to
speed entry use the copy and paste clipboard keys in the entry edit window;
and right click for entry options on each field in the entry window to
see available options
Make sure the gel file is selected in the main window under files, then
select File / open experiment file (entries) from the main window
A fingerprint entry window will appear listing the entries defined for
this gel Each entry corresponds to a lane in the gel image, one can also
see the gel image file by selecting File / open experiment file (data)
Next to entry defined in the entry window is a gray link arrow key, to
link each entry to the data base: click on the link arrow, drag it
onto the correct database entry, and release the mouse, the link arrow
key is now purple and the entry now has the same information fields as
the corresponding entry in the database (can right click on the purple
arrow to unlink it from the database)
A green dot is now to the right of the database entry, showing it is
now linked to an experiment.
Fingerprint Conversion Settings
These settings should be already set as the default values. This
menu is seen by clicking on the Edit settings icon or in the menu Edit
/ settings.
Raw Data:
- Thickness = 17 inverted values should be checked
- Nodes = 3 OD range = 255
- Background subtraction and spot removal should not be checked
Densiometric Curves:
- Avg. Thickness =11
- filtering = arithmetic average
- Nodes = 2
Background and least square filtering values are unique to each gel
The optimum values are calculated by the program under spectral analysis
See step 2: Strips above for instructions on setting these values
Normalization:
Resolution = variable, typically 460 - 475
Bands:
- Min. profiling = 3.00%
- Grey area = 0.00%
- Min. area = 0.00%
- Shoulder sensitivity = 0
|
