199 lines
8.9 KiB
Plaintext
199 lines
8.9 KiB
Plaintext
GIP
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A digitizer is a two dimensional surface which is such that
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if a special pen is pressed onto it, the pens coordinates can be
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recorded by a computer. These coordinates can be interpreted by a
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program.
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The digitizing device we use works by the pen emitting a high
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frequency sound which is picked up by two microphones positioned at
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the rear of the working area. The pen position is determined by
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triangulation and the digitizing device sends the coordinates to the
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computer. As no special surface is required the device can
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conveniently be positioned on a light box giving the sequencer an
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unobscured view of the autoradiographs.
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The digitizer is called a GRAPHBAR MODEL GP7 made by Science
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Accessories Corp, 970 Kings Highway West, Southport, Connecticut
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06490, USA.
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The program uses a menu to allow the user to select commands
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or to enter the uncertainty codes for areas of the gel that
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are difficult to interpret. A menu is simply a series of boxes drawn
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on the digitizing surface that each contain a command or
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uncertainty code. When the user puts the pen down in these special
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regions the program interprets the coordinates as commands and acts
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appropriately. A copy of the menu should have been sent to you. It
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should be stuck down on the surface of the light box in the
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digitizing area. For convenience it is best to position it to the
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right of the digitizing area, but in practice as long as its top edge
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is parallel to the digitizer box, it can be put anywhere in the
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active region.
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Entering gel readings using a digitizer
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The autoradiograph should be stuck down on the light box with
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the lanes running, as near is as possible, at right angles to the
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digitizer. To read an autoradiograph placed on the light box the user
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need only define the positions of the four sequencing lanes and the
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bases to which they correspond and then use the pen to point to
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each successive band progressing up the gel. The program examines
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the coordinates of each pen position to see in which of the four
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lanes it lies and assigns the corresponding base to be stored
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in the computer. Each time the pen tip is depressed to point to a
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position on the surface of the digitizer the program sounds the
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bell on the terminal (a different sound for each of the four bases on
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the microcomputer version of the program) to indicate to the user
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that a point has been recorded. As the sequence is read the
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program displays it on the screen.
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The program uses a menu to allow the user to select commands
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or to enter the uncertainty codes for areas of the gel that
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are difficult to interpret. A menu is simply a series of boxes drawn
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on the digitizing surface that each contain a command or
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uncertainty code. When the user puts the pen down in these special
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regions the program interprets the coordinates as commands and acts
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appropriately. As well as the uncertainty codes
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A,C,G,T,1,2,3,4,B,D,H,V,R,Y,X,-,5,6,7,8 the following commands are
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included in the menu: DELETE removes the last character from the
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sequence; RESET allows the lane centres to be redefined; START means
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begin the next stage of the procedure; STOP means stop the
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current stage in the procedure; CONFIRM means confirm that the last
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command or set of coordinates are correct.
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The digitizing device also has a menu of its own. This lies in
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a two inch wide strip immediately in front of the digitizing box. Pen
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positions within this two inch strip are interpretted as commands to
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the digitizer and are not sent to the GIP program. In general the
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only time users will need to use the device menu is when they tell
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GIP where the program menu lies in the digitizing area. This is done
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by first hitting ORIGIN in the device menu and then hitting the
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bottom left hand corner of the program menu. The program menu can
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hence be positioned anywhere in the active region but should be
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arranged parallel to the digitizer.
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The user should try to hit the bands as near as possible to
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the centre of the lanes because the program tracks the lanes up the
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film using the pen positions. By using this tracking strategy the
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user only has to define the centres of the bottom of the lanes before
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starting to read the film. The program can correctly follow quite
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curved lanes and constantly checks that its lane centre coordinates
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look sensible. If the lane centres appear to be getting too close the
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program stops responding to the pen positions of bands and hence does
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not ring the bell. If this occurs users must hit the reset box in the
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menu and the program will request them to redefine the lane centres
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at the current reading position. Then they can continue reading. As a
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further safeguard the program will only respond to pen positions
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either in the menu or very close to the current reading position.
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Running the gel reading program
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The autoradiograph should be firmly stuck down on the light box and
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the program started by typing GIP. It will ask the first question.
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" ? FILE OF FILE NAMES="
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Type the name for the file of file names and then follow the
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instructions.
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" HIT DIGITIZER MENU ORIGIN"
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" THEN PROGRAM MENU ORIGIN"
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" THEN HIT START IN PROGRAM MENU"
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If the bell does not sound after you hit start try hitting metric in
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the device menu (the program uses metric units, and some digitizers
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are set to default to use inches; hitting metric switches between
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the two).
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After the bell has sounded the program will give the default lane
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order.
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" LANE ORDER IS T C A G"
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" IF CORRECT HIT CONFIRM, ELSE HIT RESET"
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If the lane order, reading from left to right is correct hit confirm
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in the program menu. If you are using a different order hit reset
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and you will be asked to define the lane order from left to right
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using the program menu (as follows).
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" DEFINE LANE ORDER (LEFT TO RIGHT) USING MENU"
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Hit the boxes in the menu that contain the symbols A,C,G,T in the
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left-right order of the lanes. The program will respond with the
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lane order as above and ask for confirmation. When this is received,
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the next task is to define the start positions of the next four
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lanes.
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" HIT START, THEN HIT (LEFT TO RIGHT)"
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" THE START POSITIONS FOR THE NEXT FOUR LANES"
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Hit the centres of the four lanes at a height level with the first
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band that is going to be read. The program will report the mean lane
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separations and asks for confirmation that they are correct.
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" MEAN LANE SEPARATION IS XX"
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" HIT CONFIRM TO CONTINUE"
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Users will become familiar with the values from their films and will
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spot any unusual numbers. Asking for confirmation allows users to
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try again if they had made a mistake, but generally the lane
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separation values can be ignored. Hit confirm, and the program will
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give the message
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" HIT START WHEN READY TO BEGIN READING"
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Hit start and the program will give the message
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" HIT BANDS, UNCERTAINTY CODES, RESET OR STOP"
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Hit the bands, interpretting the sequence progressing up the film.
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If necessary use the uncertainty codes. If the pen stops responding
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hit reset and follow the instructions as above. When the sequence
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becomes unreadable hit stop and the program will ask for a file name
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for the gel reading just read.
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" ? FILE NAME FOR THIS GEL READING="
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Type the file name observing the rules about legal gel readings
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names. The program will ask if you wish to read another sequence.
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" TO ENTER ANOTHER GEL READING TYPE 1"
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To enter another type 1 and you will be back to the step of defining
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the lane order. Typing anything else will stop the program.
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Running the microcomputer version of the gel reading program
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The microcomputer version of GIP is slightly different and is called
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GIPB. The BBC micro does not have the capacity to process the gel
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readings beyond the reading stage. This means that users of this
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program would need to transfer their gel readings from the micro to
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another machine using a terminal emmulator. Transferring many files
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is tedious and so the microcomputer version of the gel reading
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program stores all the gel readings for each run of the program in a
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single file. This special file contains both sequences and file names
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and can be moved in a single transfer to another machine. Once on the
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other machine the single file must be split into separate gel reading
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files and a file of file names. This is done using the program
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BSPLIT. As far as using the microcomputer version of GIP, the only
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difference is that the first file name the program requests is not a
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file of file names, but a name for the single file to contain all the
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gel readings and their names.
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