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.