            DATA PREPARATION AND THE CONVERT PROGRAM


ACQUIRING RAW DATA

DNA SIMDEX uses the (X,Y) coordinates of the bands and lane origins
(wells) to perform its functions.  There are several ways in which
(X,Y) coordinate data can be generated for use in DNA SIMDEX.  The
fastest method is to use an image analysis system that can
determine the centers of gravity of the DNA bands in an
electrophoretic gel or autoradiogram.  Autoradiograms and gels
often have stray marks and varying background tints that make them
difficult to analyze directly in an image analysis system.  To
overcome this problem, we suggest marking the location of the bands and lane origins on an overhead transparency.  The transparency can
be scanned using an image analysis system.  After the image
has been captured, the background is erased, and the centers of
gravity for each band and lane origin are calculated by the image
analysis system.

NOTE:  Each image analysis system is unique.  As a result,
some alterations in the (X,Y) coordinates may be necessary for use
in DNA SIMDEX.  Please read the guidelines for (X,Y) coordinates.

(X,Y) coordinates can also be determined manually.  The centers of
gravity of bands and lane origins can be approximated by overlaying gels or autoradiograms with a fine grid or by measuring the distances with a ruler.   Several rules must be followed when determining (X,Y) coordinates.  Please read the guidelines on (X,Y) coordinates below.






(X,Y) COORDINATES

DNA SIMDEX uses the (X,Y) coordinates of the bands and lane origins
(wells) to perform its functions.  There are three rules that must
be followed in assigning (X,Y) coordinates to bands and lane
origins.

1) GEL OR AUTORADIOGRAM ORIENTATION.  DNA SIMDEX is designed to
analyze electrophoretic gels and autoradiograms with the DNA lanes
in a horizontal position.  The lane origins (wells) should be on
the right.  In this orientation, the DNA runs from right to left.

 -----------------------------------------------------------------
|  |  |   |   |                                       | Origin 1  |
|   |  |  |  |                                        | Origin 2  |
|   |   |     |      |                                | Origin 3  |
|    |         |                                      | Origin 4  |
|_________________________________________________________________|

2) (X,Y) ORIGIN.  DNA SIMDEX places the (X,Y) origin in the top
left corner of the gel or autoradiogram.  X coordinates increase to
the right and Y coordinates increase in descending order as shown
below.


(0,0)-------------------------> increasing X values
|   |  |  |   |   |                                      | Origin 1
|    |  |  |  |                                          | Origin 2
|    |   |     |      |                                  | Origin 3
|     |         |                                        | Origin 4
|
|
|

Increasing Y
values

3) VALUE LIMITS.  X values can range from 1 to 500.  X values
greater than 500 will result in errors.  However, there is no limit
to Y values which are used primarily for lane identification.







LANE IDENTIFICATION

DNA SIMDEX has an automatic lane identification algorithm.  A band
or lane origin that has a Y value within 5 pixels of the previous
band will be assigned to the same lane as the previous band.  For
example, if the following coordinates were loaded into DNA SIMDEX

10,6
20,8
30,13
40,11
50,20

the bands at X=10, 20, 30 and 40 would all be grouped into one lane
while the band at X=50 would begin a new lane.  The 5-pixel
tolerance will accommodate slight variations in the center of
gravities calculated using an image analysis system.

DNA SIMDEX uses migration distance to determine matching and non-
matching bands.  As a result, each lane must have a lane origin.
The lane origins are usually the (X,Y) coordinate of the DNA wells.
DNA SIMDEX will automatically assign the pair of coordinates with
the greatest Y value in each lane to be the lane origin.


PREPARING (X,Y) COORDINATE FILES

(X,Y) coordinate files can be prepared on an image analysis system
or a word processor.  There are a few important points to remember
when creating an (X,Y) coordinate file.

1)  (X,Y) coordinate files should contain ONLY the (X,Y)
coordinates of the DNA bands and lane origins.

2)  The coordinates for each lane must be grouped together.

3) Each lane must be given a lane origin.  The lane origin usually
corresponds to the (X,Y) coordinates of the DNA well.

An example of an (X,Y) coordinate file is shown below.

30,4
70,5
50,4
460,6
30,21
55,20
459,22


This set of numbers would be read by DNA SIMDEX as two lanes.  Lane
1 would have bands at X=30, 50 and 70 with a lane origin at 460.
Lane 2 would have bands at X=30 and 55 and a lane at 459.  DNA
SIMDEX will automatically arrange the bands in increasing X value.
(X=30, 50, and 70.)




THE CONVERT PROGRAM

DNA SIMDEX is designed to use files created from an Olympus CUE 2
image analysis system (Olympus, Lake Success, New York).  To allow
DNA SIMDEX to work with any image analysis system, or without one,
we have written a simple program called CONVERT.  The CONVERT
program will load a set of (X,Y) coordinates into a file that can
be used by DNA SIMDEX.

To start the CONVERT program, change the computer prompt to the
corresponding drive and type "CONVERT".  Convert's main menu will
appear.


 -------------Main Menu--------------
|                                    |
| (1) File to File Conversion        |
| (2) Hand Entry to File Conversion  |
| (3) Quit                           |
|____________________________________|


CONVERT is designed to create a DNA SIMDEX file from a file of
(X,Y) coordinates, or from (X,Y) values entered manually.  Each of
these options is described below.




(1) FILE TO FILE CONVERSION

This option is designed to convert a file containing (X,Y)
coordinates into a file that is readable by DNA SIMDEX.  (See
Preparing (X,Y) Coordinate Files)

To convert an (X,Y) file to DNA SIMDEX file, select (1) File to
File Conversion on the main menu.  This will pull up the File to
File Conversion Screen.

 ------File to File Conversion Screen-------
|                                           |
| File Name For Conversion:                 |
| Total Number of Bands and Lane Origins:   |
| New File Name:                            |
|___________________________________________|

You will be asked to enter the name of the (X,Y) coordinate file to
be converted.  Make sure that you enter the complete name of the
file including any extensions.  Failure to enter the complete name
will result in an error.

The program will then ask you the total number of bands and lane
origins contained in the (X,Y) coordinate file.  This number is
needed by DNA SIMDEX and will become part of the new DNA SIMDEX
file.  For example, if you were loading the following file,

30,4
70,5
50,4
460,6
30,21
55,20
459,22

you would tell the computer that there are 7 bands and lane origins
in the file.  (5 bands, 2 lane origins.)

The program with then ask for a new file name which will act as the
name for the DNA SIMDEX file.

NOTE:  IF A FILE ALREADY EXISTS BY THAT NAME IT WILL BE ERASED AND
THE NEW DNA SIMDEX WILL BE WRITTEN IN ITS PLACE.


After the computer has created the new file, press ENTER to return
to the main menu.


(2) HAND TO FILE CONVERSION

This option allows you to create a DNA SIMDEX file based on (X,Y)
coordinates entered manually.  The Hand Entry to File Conversion
screen will appear.

 ---------Hand Entry to File Conversion Screen------
|                                                   |
| New File Name:                                    |
| Total Number of Bands and Lane Origins:           |
| X Coordinate for #___                             |
| Y Coordinate for #___                             |
|___________________________________________________|

The computer will ask you for a file name.  This file name will be
the name of the DNA SIMDEX file containing your data.  The computer
will then ask you how many bands and lane origins will be entered
into the new file.

The computer will then ask you to enter the individual coordinates.
There are a few rules to remember in entering the (X,Y)
coordinates.

1) The coordinates for each lane must be entered in order.

2) Every lane must have a lane origin.  This lane origin should be
entered as a regular band.  DNA SIMDEX will automatically assign
the band with the greatest X value in each lane to be the lane
origin.

3) The X coordinate must have a range between 1 and 500.

When all coordinates have been entered, a DNA SIMDEX file will be
created.  When prompted, press ENTER to return to the main menu.






(3) QUIT

This option in the main menu will terminate the CONVERT session and
exit you to the system.






                            DNA SIMDEX 2.0


STARTING DNA SIMDEX

To start DNA SIMDEX, change the computer prompt to the
corresponding drive and type "SIMDEX".  The title screen will be
displayed.  Press any key to move to the main menu.

 -------------Main Menu--------------
|                                    |
| 1) Load A Gel                      |
| 2) Compare Lanes Directly          |
| 3) Compare Lanes Using Standards   |
| 4) Manipulate Values               |
| 5) Band Flagging Setup             |
| 6) Quit                            |
| 7) Parameter Set                   |
| 8) Gel Setup                       |
| 9) Designate Output File           |
|____________________________________|

Each of these options is described below.


(1) LOADING A GEL

A DNA SIMDEX session is initiated by loading a gel into the
computer's memory.  This is done by selecting the (1) LOAD A GEL
function from the main menu.  The program then asks for the file
name to be loaded.  The complete name including extension must be
entered.  If the data is located on another drive, a drive
identification and subdirectory must also be included.  (For
example:  A:/DNAGELS/GEL1.DOC).  To function properly, files should
be created on a Olympus Cue 2 image analysis system (Olympus, Lake
Success, New York) or be files created using the CONVERT program.
(See Data Preparation and the Convert Program for details.)

Once you have indicated the file to be loaded, the computer will
ask you for a Gel Identification.  This identification can be a
name, number, or code by which the gel will be recognized
throughout the remainder of the session.

Once the Gel I.D. has been entered the computer will load the (X,Y)
coordinated into memory.  DNA SIMDEX is designed to store up to 60
lanes at a time.  Loading more than 60 lanes at a time will cause
the program to malfunction.

The computer assigns each lane a number starting with the first
lane in the file.  (See Data Preparation and the Convert Program
for details on lane identification.)  When more than one gel is
loaded, the computer will continue numbering the lanes starting
with the last value on the previous gel.  For example, if the first
gel loaded has 14 lanes, the computer will begin numbering the
second gel starting with 15. These computer assigned lane numbers
can be used to identify lanes for comparison and manipulation
throughout the program.

In addition to the computer assigned lane numbers, each lane is
given an in-gel lane number.  This number is only a function of the
lane order within the gel.  In other words, the first lane in a gel
will have always have an in-gel number of 1.  The second lane in a
gel will always have an in-gel number of 2.  This is true even if
other files have been previously loaded.  For example, if Gel #1
was loaded before Gel #2, the lanes would be assigned the following
numbers.

GEL #      Computer-Assigned    In-Gel
              Lane  Number    Lane Number

  1              1               1
  1              2               2
  1              3               3
  1              4               4
  1              5               5

  2              6               1
  2              7               2
  2              8               3
  2              9               4
  2             10               5


These in-gel lane numbers are use to identify lanes when using a
Gel I.D. Lane option.

While the computer is downloading the information from the data
file, it will display the computer assigned number for each lane.
The program will then indicate how many lanes are contained in the
files.  An example screen is shown below.

 __________________________________
|                                  |
| FILE NAME:?  Gel 2               |
| GEL I.D.:?  Gel 2                |
| GENERATING LANE    24            |
| THIS GEL HAS A TOTAL OF 3 LANES  |
|__________________________________|










2) COMPARE LANES DIRECTLY

Once a gel has been loaded, lanes can be compared using the (2)
COMPARE LANES DIRECTLY command from the main menu.  After selecting
this option, the program will ask how you would like to select the
lanes for comparison.


 ------Lane Selection Window---------
|                                    |
| Lane Selection By...               |
| 1) Lane Number                     |
| 2) Gel I.D. and In-Gel Lane Number |
|____________________________________|


Lanes can be selected according to the computer assigned lane
number or according the gel I.D. and in-gel lane number. (See
LOADING A GEL for details on computer assigned and in-gel lane
numbers.)

Once a selection has been made, the computer will prompt you for
the necessary information.  If the Gel I.D. and In-Gel Lane option
is being used, please type the gel I.D. exactly as you did when
loading the gel.  Please note that DNA SIMDEX is case sensitive.
In other words DNA SIMDEX considers "GEL 1" and "gel 1" two
different titles.

Once the necessary information has been entered, the computer
will make a direct comparison of the two lanes.  The Results Screen
will then be displayed.

 _________________________________________________________________
|  Similarity Index = .5575                                       |
| _______________________________________________________________ |
| Gel 1, Lane 1                          Gel 1, Lane 2            |
| Matching Bands = 5                     Matching Bands = 5       |
|                                                                 |
| 1 (1)              2(3)                1 (1)            3 (2)   |
| 3 (4)              4(5)                4 (3)            5 (4)   |
| 5 (6)                                  6 (5)                    |
|                                                                 |
| Non-Matching Bands = 1                 Non-Matching Bands = 3   |
|                                                                 |
| 6                                      2                7       |
|                                                                 |
 -----------------------------------------------------------------
| (1) Return to Main Menu  (2) Print  (3) Save  (4) Match Edit    |
| (5) Perform Another Comp.  (6) Up  (7) Down (8) Notes (9) Map   |
 -----------------------------------------------------------------


On the top right corner of the Results Screen is the computed
similarity index.  The main body of the Results Screen shows the
individual matching and non-matching bands for each lane. Matching
bands are followed by a number in parenthesis which corresponds to
the matching band in the other lane.  For example   7(8) means that
band 7 in one lane matches with band 8 in the other.

Several options are available from the results screen.  These
options are explained below.

1) RETURN TO MAIN MENU.  This option returns you to the main menu.

2)  PRINT RESULTS.  This option will print a hard copy of the
results on a laser jet or line printer.

3) SAVE RESULTS.  This option allow you to save the results as a
file.  If a designated output file has not been assigned (See
Designate Output File) the computer will prompt you for an output
filename.  The results of this comparison will be saved onto the
end of the file.   Output files can then be loaded and manipulated
with a word processor.  Output files will contain extra quotation
marks and commas.  These extra characters can be easily deleted
with a word processing program leaving a clean output file.


4) MATCH EDIT. This option allows you to match or unmatch bands.
First, the computer will ask if you would like to match or unmatch
bands.

 --Match Edit Menu --
|                    |
| 1) Match           |
| 2) Unmatch         |
|____________________|

If you select to Match Bands the program will request the band
number from the first and second lane.  These bands will be matched
and a new similarity index will be calculated.

If you select to unmatch bands, the computer will request the band
number from the band in the first lane.  This band will be
unmatched along with its partner.  A new similarity index will be
calculated.

5) PERFORM ANOTHER COMPARISON.  This function will execute another
direct comparison.

6 & 7)  UP, DOWN.  The Matching and Non-Matching Band Windows can
be scrolled up and down by pressing "6" or "7".  This allows for
all bands to be displayed despite the restrictions of the screen
size.

8) NOTES.  This option allows you to prepare explanatory notes that
can be saved in a file.  The computer will ask for 10 lines of
text. If you do not require that much room, simply press enter for
the remaining lines after your message.  If a designated output
file has not been declared (See Designate Output File), the program
will prompt you for an output file.  The note will be saved at the
end of specified file.

9) HIGH RESOLUTION MAP.  Perhaps the most valuable tool that can be
accessed from the Results Screen is the High Resolution Map. This
map shows the location of the matching and non-matching bands in
both lanes.  The matching bands are shown in pink while the non-
matching bands are shown in white.  The first lane is displayed
above the second lane.












































(3) COMPARE LANES USING STANDARDS

Once one or more gels have been loaded, lanes can be compared using
standard lanes as references.  This is especially useful for
comparisons between gels that have been run under different
conditions.

When comparing lanes using standards, two lanes with IDENTICAL
BANDS must be declared as standards or reference lanes.  If either
of the two standard lanes contain unique bands, DNA SIMDEX's
algorithm will malfunction giving inaccurate results.

Comparisons with standards can be made by selecting the COMPARE
LANES USING STANDARDS (3) command from the main menu.  The program
will ask your lane selection preference.

 ------Lane Selection Window---------
|                                    |
| Lane Selection By...               |
| 1) Lane Number                     |
| 2) Gel I.D. and In-Gel Lane Number |
|____________________________________|

The lanes can be selected according to the computer assigned lane
number or according to the Gel I.D. and In-Gel Lane Number (see
LOADING A GEL).

Once a selection has been made, the program will prompt you for the
necessary information.  If Gel I.D. and In-Gel Lane Number option
is being used, please type the gel I.D. exactly as you did when
loading the gel.  DNA SIMDEX is case sensitive.  In other words DNA
SIMDEX considers "GEL 1" and "gel 1" two different titles.

The program will make a comparison of the two lanes using the
standard lanes as references and the Results Screen will be
displayed.
 _________________________________________________________________
|  Similarity Index = .5575                                       |
| _______________________________________________________________ |
| Gel 1, Lane 1                          Gel 1, Lane 2            |
| Matching Bands = 5                     Matching Bands = 5       |
|                                                                 |
| 1 (1)              2(3)                1 (1)            3 (2)   |
| 3 (4)              4(5)                4 (3)            5 (4)   |
| 5 (6)                                  6 (5)                    |
|                                                                 |
| Non-Matching Bands = 1                 Non-Matching Bands = 3   |
|                                                                 |
| 6                                      2                7       |
|                                                                 |
 -----------------------------------------------------------------
| (1) Return to Main Menu  (2) Print  (3) Save  (4) Match Edit    |
| (5) Perform Another Comp.  (6) Up  (7) Down (8) Notes (9) Map   |
 -----------------------------------------------------------------
On the top right corner of the Results Screen is the computed
Similarity Index.  The main body of the Results Screen shows the
individual matching and non-matching bands for each lane.  Matching
bands are followed by a number in parenthesis.  The number in
parenthesis corresponds to the matching band in the other lane.
For example   7(8) means that band 7 in one lane matches with band
8 in the other.

Several options are available from the results screen.  These
options are explained below.

1) RETURN TO MAIN MENU.  This option returns you to the main menu.

2)  PRINT RESULTS.  This option will print a hard copy of the
results on a laser jet or line printer.

3) SAVE RESULTS.  This option allow you to save the results as a
file.  If a designated output file has not been assigned (See
Designate Output File) the computer will prompt you for an output
filename.  The results of this comparison will be save at the end
of the output file.  Output files can then be loaded and
manipulated on a word processor.  Output files will contain extra
quotation marks and commas.  These extra characters can be easily
deleted on a word processing program leaving a clean output file.

4) MATCH EDIT. This option allows you to match or unmatch bands.
First, the program will ask if you would like to match or unmatch
bands.

 --Match Edit Menu --
|                    |
| 1) Match           |
| 2) Unmatch         |
|____________________|


If you select to match bands, the program will request the band
number from the first and second lane.  These bands will be matched
and a new similarity index will be calculated.

If you select to unmatch bands, the program will request the band
number from the band in the first lane.  This band will be
unmatched along with its partner.  A new similarity index will be
calculated.

5) PERFORM ANOTHER COMPARISON.  This function will execute another
comparison using standards.

6 & 7)  UP, DOWN.  The matching and non-matching band windows can
be scrolled up and down by pressing "6" or "7".  This allows for
all bands to be displayed despite the restrictions on screen size.

8) NOTES.  This option allows you to prepare explanatory notes that
can be saved in a file..  The computer will ask for 10 lines of
text. If you do not require that much room, simply press enter for
the remaining lines after your message.  If a designated output
file has not been declared (See Designate Output File), the
computer will prompt you for an output file.  The note will be
saved at the end specified file.

9) HIGH RESOLUTION MAP.  Perhaps the most valuable tool that can be
accessed from the Results Screen is the High Resolution Map. This
map show the location of the matching and non-matching bands in
both lanes.  The matching bands are shown in pink while the non-
matching bands are shown in white.  The first lane is displayed
above the second lane.










































(4) MANIPULATE VALUES

This option allows the user to manipulate the data that is
presently in the computer's memory.  The changes made to the data
will be effective until the end of the session.  However, no
changes will be made to data files.  The Manipulate Values Menu is
shown below

 --Manipulate Values Menu --
|                           |
| 1) Shift Values           |
| 2) Delete Values          |
| 3) View Lanes             |
| 4) Main Menu              |
|___________________________|

The options available from the manipulate values menu are explained
below.

1) SHIFT VALUES.  This function allows you to shift the bands in a
lane to the right or to the left.  All of the bands within a lane
will be shifted by the same amount.  Lanes can be selected
according to the computer assigned lane number or according the Gel
I.D. and In-Gel Lane (See LOADING A GEL)

To shift bands to the right, enter a positive number when prompted.
To shift bands to the left, enter a negative number when prompted.

 -------------------PROMPT---------------------
|                                              |
| Enter Amount to Shift (- Left, + Right) :?   |
|______________________________________________|

For example, if you enter  -3 all bands will be shifted to the left
3 pixels.  Coordinate (10,3) will then become (7,3).  On the other
hand, if you enter a +3, all bands will be shifted to the right 3
pixels.  Coordinate (10,3) will then become (13,3).

After a shift has been requested, the computer will ask you to
confirm the change.

 -----Confirmation Menu-----
|                           |
| (A) Accept Change         |
| (X) Cancel change         |
| (M) See Map of Change     |
|___________________________|

Before accepting or canceling the change, you can see a map of the
change by pressing "M" for map.  The old lane will be shown on the
top of the screen while the new shifted lane is shown on the
bottom.

2) DELETE VALUES.  This function allows you to delete bands in a
lane.  Lanes are selected according to their computer assigned lane
numbers.  Once a band is deleted, the bands within the lane are
renumbered according to their X coordinate values.  In other words,
if you delete band 7 in a 10 band lane, bands 8, 9, and 10 will now
be recognized as bands 7, 8 and 9.

After a deletion has been requested, the program will ask you to
confirm the change.

 ----Confirmation Menu----
|                         |
| (A) Accept Change       |
| (X) Cancel Change       |
| (M) See Map of Change   |
|_________________________|

Before accepting or canceling the change, you can see a map of the
change by pressing "M" for map.  The old lane will be shown on the
top and the new lane with the deletion will be shown on the bottom.


3) VIEW LANES.  This function allows you to view two lanes at a
time.  This can be useful when you want to quickly review the
location of bands within lanes.  When prompted, enter the computer
assigned lane numbers of the lanes to be viewed.

4) Main Menu.  This function returns you to the main menu.


























(5)  BAND FLAGGING SETUP

Band flagging is a way of marking special bands, and their matching
counterparts, for easy detection.  To mark bands, you must first
select the lane to be flagged.  Lanes are selected according to the
computer assigned lane number.

After selecting a lane, the program will then ask you how many
bands you would like to mark within the lane.  The program will
then ask for the band numbers or the bands you wish to mark.

When a marked band in the FIRST lane of a comparison is matched
with a band in the second lane, a star will appear by the matching
pair in the Results Screen.  In the high resolution map of the
Results Screen, a blue marker is placed over all bands that might
qualify as matching pairs of flagged bands in lane #1.




(6)  QUIT

This function will terminate the session and exit you to the DOS
system prompt.



(7) PARAMETER SET

DNA SIMDEX compares the migration distances of bands to determine
matching and non-matching bands.  There is a margin of variation
allowed for matching pairs.  This margin of variation is based on a
percentage of the migration distance of the bands being compared.
The default value is 2.0 percent of the total migration distance.
For example, if a band has migrated 100 pixels, the computer will
search for possible matching bands, in the other lane, with
migration distances of 98 to 102 pixels.  By using the PARAMETER
SET function you can set new variation values.


 ----------Parameter Set Screen--------------
|                                            |
| Percent Diff. Allowed for Match =2.0       |
| New Percent Diff. Allowed for Match:?_____ |
|____________________________________________|


The old variation value is displayed.  The computer then asks for a
new value.  YOU MUST ENTER A VALUE.  If you simply press ENTER the
value will be set to 0.0.




(8)  GEL SETUP

This function allows you to give individual names, numbers, or
codes to each lane.  The program will ask for the beginning and
ending lane numbers.

 -------Gel Setup Screen------------
|                                   |
| Enter Number of First Lane _____  |
| Enter Number of Last Lane _____   |
| Name Lane ____                    |
|___________________________________|

For example, if we have 20 lanes entered into the computer and we
want to assign special names to lanes 1 to 10, we enter 1 for the
beginning lane number and 10 for the ending lane number.  These
lane numbers must be the computer assigned lane numbers for each
lane.

The program will then ask for the individual names for each lane.
These names will appear on the Results Screen and in printed or
saved files.  However, names assigned in gel setup can not be used
to select lanes for manipulation or comparison.







(9)  DESIGNATE OUTPUT FILE

This function allows you to designate a specific file to which all
of the results and explanatory notes will be saved.  Before
prompting you for an output file title, the program checks to see
if an output file has already been designated.  If you have
designated an output file, the computer will automatically save
data to the designated file instead of prompting you for a file
name.

The designated file can be changed at any time during a session. If
you press ENTER when prompted to designate a file name, the program
will disable the function and you will be prompted for an output
file when saving results or explanatory notes.

The program will also ask you if you would like lists matching and
or non-matching bands printed and saved.  The program default is
set to print all band information including lists of matching and
non-matching bands.



