staden-lg/help/GIP.RNO

.NPA
.left margin1
.CENTER
GIP
.LEFT MARGIN1
.PARA
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. 
.para
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.
.LEFT MARGIN1
The digitizer
             is called a GRAPHBAR MODEL GP7 made by
             Science Accessories Corp,
             970 Kings Highway West,
             Southport,
             Connecticut 06490,
             USA.

.para
               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.
.sk1
.left margin1
 Entering gel readings using a digitizer
.left margin1
.para
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.


.para
               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.
.para
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.
.para
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.
.sk1
.left margin1
 Running the gel reading program
.left margin1
The autoradiograph should be firmly stuck down on the light box and the 
program started by typing GIP. It will ask the first question.
.left margin2
" ? FILE OF FILE NAMES="
.left margin2
Type the name for the file of file names and then follow the instructions.
.left margin2
" HIT DIGITIZER MENU ORIGIN"
.left margin2
" THEN PROGRAM MENU ORIGIN"
.left margin2
" THEN HIT START IN PROGRAM MENU"
.left margin2
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).
.left margin2
After the bell has sounded the program will give the default lane order.
.left margin2
" LANE ORDER IS T C A G"
.left margin2
" IF CORRECT HIT CONFIRM, ELSE HIT RESET"
.left margin2
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).
.left margin2
" DEFINE LANE ORDER (LEFT TO RIGHT) USING MENU"
.left margin2
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.
.left margin2
" HIT START, THEN HIT (LEFT TO RIGHT)"
.left margin2
" THE START POSITIONS FOR THE NEXT FOUR LANES"
.left margin2
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.
.left margin2
" MEAN LANE SEPARATION IS XX"
.left margin2
" HIT CONFIRM TO CONTINUE"
.left margin2
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
.left margin2
" HIT START WHEN READY TO BEGIN READING"
.left margin2
Hit start and the program will give the message
.left margin2
" HIT BANDS, UNCERTAINTY CODES, RESET OR STOP"
.left margin2
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.
.left margin2
" ? FILE NAME FOR THIS GEL READING="
.left margin2
Type the file name observing the rules about legal gel readings names.
The program will ask if you wish 
to read another sequence.
.left margin2
" TO ENTER ANOTHER GEL READING TYPE 1"
.left margin2
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.
.left margin1
.sk1
Running the microcomputer version of the gel reading program
.left margin1
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.