206 lines
9.3 KiB
Plaintext
206 lines
9.3 KiB
Plaintext
.NPA
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.left margin1
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.CENTER
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GIP
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.LEFT MARGIN1
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.PARA
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A digitizer is
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a two dimensional surface
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which is such that if a special pen is pressed onto it, the pens
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coordinates can be recorded by a computer.
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These coordinates
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can be interpreted by a program.
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.para
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The digitizing device we use works by the pen emitting a high frequency
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sound which is picked up by two microphones positioned at the rear of the
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working area. The pen position is determined by triangulation and the
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digitizing device sends the coordinates to the computer. As no special
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surface is required the device can conveniently be positioned on a light
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box giving the sequencer an unobscured view of the autoradiographs.
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.LEFT MARGIN1
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The digitizer
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is called a GRAPHBAR MODEL GP7 made by
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Science Accessories Corp,
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970 Kings Highway West,
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Southport,
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Connecticut 06490,
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USA.
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.para
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The program uses a menu to allow the user to select commands or
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to enter the uncertainty codes for areas of the gel that are
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difficult to interpret. A menu is simply a series of boxes drawn on
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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.
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It should be stuck down on the surface of the
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light box in the digitizing area. For convenience it is best to position it
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to the right of the digitizing area, but in practice as long as
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its top
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edge is parallel to the digitizer box, it can be put anywhere in the active
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region.
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.sk1
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.left margin1
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Entering gel readings using a digitizer
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.left margin1
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.para
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The autoradiograph should be stuck down on the light box with the lanes
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running, as near is as
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possible, at right angles to the digitizer. To read
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an autoradiograph placed on the light box
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the user need only define the positions of
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the four sequencing lanes and the bases
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to which they correspond and then use the pen to point to each
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successive band progressing up the gel. The program examines the
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coordinates of each pen position to see in which of the four
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lanes
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it lies and assigns the corresponding base to be stored in the
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computer. Each time the pen tip is depressed to point to a position
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on the surface of the digitizer the program sounds the bell on the
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terminal (a different sound for each of the four bases on the
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microcomputer version of the program)
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to indicate to the user that a point has been recorded. As
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the sequence is read the program displays it on the screen.
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.para
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The program uses a menu
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to allow the user to select commands or
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to enter the uncertainty codes for areas of the gel that are
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difficult to interpret. A menu is simply a series of boxes drawn on
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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
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the sequence;
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RESET allows the lane centres to be redefined;
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START means begin the next
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stage of the procedure; STOP means stop the current stage in the
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procedure; CONFIRM means confirm that the last command or set of
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coordinates are correct.
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.para
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The digitizing device also has a menu of its own. This lies in a two inch wide
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strip immediately in front of the digitizing box. Pen positions within this
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two inch strip are interpretted as commands to the digitizer and are not
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sent to the GIP program. In general the only time users will need to use
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the device menu is when they tell GIP where the program menu lies in the
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digitizing area. This is done by first hitting ORIGIN in the device menu
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and then hitting the bottom left hand corner of the program menu. The
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program menu can hence be positioned anywhere in the active region but
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should be arranged parallel to the digitizer.
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.para
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The user should try to hit the bands as near as possible to the centre of
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the lanes because the program tracks the lanes up the film using the pen
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positions. By using this tracking strategy the user only has to define the
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centres of the bottom of the lanes before starting to read the film. The
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program can correctly follow quite curved lanes and constantly checks that
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its lane centre coordinates look sensible. If the lane centres appear to be
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getting too close the program stops responding to the pen positions of
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bands and hence does not ring the bell. If this occurs users must hit the
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reset box in the menu and the program will request them to redefine the
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lane centres at the current reading position. Then they can continue
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reading. As a further safeguard the program will only respond to pen
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positions either in the menu or very close to the current reading position.
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.sk1
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.left margin1
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Running the gel reading program
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.left margin1
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The autoradiograph should be firmly stuck down on the light box and the
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program started by typing GIP. It will ask the first question.
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.left margin2
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" ? FILE OF FILE NAMES="
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.left margin2
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Type the name for the file of file names and then follow the instructions.
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.left margin2
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" HIT DIGITIZER MENU ORIGIN"
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.left margin2
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" THEN PROGRAM MENU ORIGIN"
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.left margin2
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" THEN HIT START IN PROGRAM MENU"
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.left margin2
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If the bell does not sound after you hit start try hitting metric in the
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device menu (the program uses metric units, and some digitizers are set to
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default to use inches; hitting metric switches between the two).
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.left margin2
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After the bell has sounded the program will give the default lane order.
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.left margin2
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" LANE ORDER IS T C A G"
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.left margin2
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" IF CORRECT HIT CONFIRM, ELSE HIT RESET"
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.left margin2
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If the lane order, reading from left to right is correct hit confirm in the
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program menu. If you are using a different order hit reset and you will be
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asked to define the lane order from left to right using the program menu
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(as follows).
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.left margin2
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" DEFINE LANE ORDER (LEFT TO RIGHT) USING MENU"
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.left margin2
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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 lane order
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as above and ask for confirmation. When this is received, the next task is
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to define the start positions of the next four lanes.
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.left margin2
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" HIT START, THEN HIT (LEFT TO RIGHT)"
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.left margin2
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" THE START POSITIONS FOR THE NEXT FOUR LANES"
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.left margin2
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Hit the centres of the four lanes at a height level with the first band
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that is going to be read. The program will report the mean lane separations
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and asks for confirmation that they are correct.
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.left margin2
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" MEAN LANE SEPARATION IS XX"
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.left margin2
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" HIT CONFIRM TO CONTINUE"
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.left margin2
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Users will become familiar with the values from their films and will spot
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any unusual numbers.
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Asking for confirmation allows users to try again if they had made a
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mistake, but generally the lane separation values can be ignored.
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Hit confirm, and the program will give the message
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.left margin2
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" HIT START WHEN READY TO BEGIN READING"
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.left margin2
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Hit start and the program will give the message
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.left margin2
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" HIT BANDS, UNCERTAINTY CODES, RESET OR STOP"
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.left margin2
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Hit the bands, interpretting the sequence progressing
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up the film. If necessary use the uncertainty codes. If the pen stops
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responding hit reset and follow the instructions as above. When the
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sequence becomes unreadable hit stop and the program will ask for a file
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name for the gel reading just read.
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.left margin2
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" ? FILE NAME FOR THIS GEL READING="
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.left margin2
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Type the file name observing the rules about legal gel readings names.
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The program will ask if you wish
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to read another sequence.
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.left margin2
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" TO ENTER ANOTHER GEL READING TYPE 1"
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.left margin2
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To enter another type 1 and you will be back to the step of defining the
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lane order. Typing anything else will stop the program.
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.left margin1
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.sk1
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Running the microcomputer version of the gel reading program
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.left margin1
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The microcomputer version of GIP is slightly different and is called
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GIPB. The BBC micro
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does not have the capacity to process the gel readings beyond the reading
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stage.
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This means that users of this program
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would need to transfer their gel readings from the micro to another machine
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using a terminal emmulator. Transferring many files is tedious and so the
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microcomputer version of the gel reading program stores all the gel
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readings for each run of the program in a single file. This special
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file contains both sequences and file names and can be moved in a single
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transfer to another machine. Once on the other machine the single file must
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be split into separate gel reading files and a file of file names. This is
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done using the program BSPLIT. As far as using the microcomputer version
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of GIP, the only difference is that the first file name the program
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requests is not a file of file names, but a name for the single file to
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contain all the gel readings and their names.
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