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.