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