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THE TYCHO SYSTEM : COMPUTER ANALYSIS OF TWO DIMENSIONAL GEL ELECTROPHORESIS
Antoine Merle, I. Taibi, O. Valiron, Ph. Garderet
To cite this version:
Antoine Merle, I. Taibi, O. Valiron, Ph. Garderet. THE TYCHO SYSTEM : COMPUTER ANALYSIS OF TWO DIMENSIONAL GEL ELECTROPHORESIS. Journal de Physique Colloques, 1986, 47 (C5), pp.C5-95-C5-100. �10.1051/jphyscol:1986512�. �jpa-00225829�
JOURNAL D E PHYSIQUE
Colloque C 5 , suppl6ment au n o 8, Tome 47, aoOt 1986
THE TYCHO SYSTEM : COMPUTER ANALYSIS OF TWO DIMENSIONAL GEL ELECTROPHORESIS
A . MERLE*, I. TAIBI', 0. VALIRON" and Ph. GARDERET*
* C E N / G , IRDI/LETI, 85 X , F-38041 Grenoble Cedex, France
" I N S E R M U217, Laboratoire d'hematologie, DRF, 85 X , F-38041 Grenoble Cedex
Rdsumg
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En collaboration avec:Argome National Laboratory, le LETI a impl6- mentg le systime TYCHO d'aide au d6pouillement de gels d'6lectrophorise bi- dimensionnelle sur son dquipement informatique. Ce systime contient des outils g6nGraux de traitement d'images p&sentant des performances intgres- santes, et, en outre, la majeure partie de ce logiciel n'est pas spdcifique B l'application d'6lectrophoris.e.Le LETI dgveloppe Bgalement m e instrumentation specifique B partir des principes utilisds dans TYCHO.
Cet article donne m e vue g6n6rale des deux syst6mes et d6crit plus prgci- s6ment les techniques utilisges par le traitement d'image et les performan- ces des systimes.
Abstract - In collaboration with Argonne National Laboratory, the TYCHO sys- tem, developed at Argome by the group of Professor Anderson to assist two- dimensional electrophoresis gel analysis, has been implemented on the LETI computer equipment.
This system contains high performance general images processing programs with interesting performances and furthermore much of this software is not res- tricted to electrophoresis application.
The LETI has also been developing specific instrumentation based on the same principles.
This paper gives a general overview of the two systems, with their hardware and software components, and then describes in greater detail the techniques used in image processing and the performances of the systems.
Two dimensional electrophoresis /1/ is a sample separation method of proteins based on two physical characteristics : isoelectric point and molecular weight.
Separations are performed successively in perpendicular directions on polyacrylamide gels. Proteins are revealed either by staining or autoradiographic methods and appear as spots. A stqdard gel (15 by 15 cm) can contain up to 2000 spots.
The TYCHO system /2/ was developed at Argonne National Laboratory (US department of Energy) to process two dimensional. gel images and to allow a quantitative analysis of gels.
Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:1986512
C5-96 JOURNAL DE PHYSIQUE
1 - BASIC PRINCIPLES OF TREATMENT
A gel image is made up of a locally constant background on which proteins spots appear. The background noise is composed on the one hand of background inherent to the method (polyacrylamide gels for staining, film for autoradiographies), on the other hand of streaks produced by the diffusion in the gel. The streaks, even though they represent actual proteins present in the gel, are considered as insignificant and are subtracted because they are frequently not reliably attributable to any particular spot.
Spots are modelled by two-dimensional gaussian shapes, with different half- widths in the two dimensions, in order to reduce data and maintain high quantita- tive accuracy on the spot volumes (protein quantities).
A biological experiment needs the creation of a set of gels, the system includes a set of tools for interactive handling and comparing sets of spotlists.
That is to say :
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an experiment is composed of a set of gels- a gel is an image
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an image is a set of spots and non-significant background- a spot is a two dimensional gaussian shape.
1.1. Image processing
In TYCHO, the image processing has two main goals :
- to produce a "cleaned" image by background subtraction and smoothing
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to detect spots present in the image.This part of the system is composed of several filters parameterized to fit the specificities of each gel.
It provides :
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digitization correction (densitometer lighting)- correction of film responses for autoradiographies
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background detection and subtraction (with streaks)-
thresholding (if needed)- smoothing (median filter)
- convolution of the image (for detection)
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detectionSome classical filters like Gradian, Laplacian filters or two-dimensional convolu- tion are implemented in TYCHO, but not used for two dimensional electrophoresis analysis.
The general approach adopted for the problem of background and streak subtraction is to find the minimum element in some selected region surrounding each pixel, for each pixel of the image. Convenient regions to choose are vertical or horizon- tal line segments as "+" or "X" shapes. Here vertical and horizontal line segments are employed because they allow detection and subtraction not only of general background, but of streaks as well. Because of the erosion inherent in this method, a second pass is necessary in which the maximum is t e n over the same kernel.
Detection is based on the detection of local maxima after convolving the tvcleaned"
image with a "+" shaped kernel consisting of centered cosine curves. Such a convolution produces a sharp spike wherever the image has a peak or shoulder approximately the shape of the kernel's central peak. Here spots can be easily detected as local maxima. The set of local maxima whose pixel values in the original image are above certain thresholds constitutes the starting spotlist.
With the mathematical modelisation used, each spot is represented by a two- dimensional gaussian shape. Each spot is thus characterized by five parameters (X and Y location, amplitude, half-width in the two dimensions). Spots are fitted to the processed image by a least-squares method /3/. In order to take into account the problem of overlaps, all the parameters of all the spots which overlap in a region are fitted in the same time.
Figure 1 gives an example of TYCHO image processing applied to a small region of a gel.
1.2. User environment
TYCHO also includes tools to define an efficient user environment : synthetic image generation, interactive spotlists handling,
...
Spotlists can be stored in a simplified data base system where the biologist can add biological information to spotlists or spot sets (protein names, biological properties of a spot set,
...
). This part is linked to the editing part to access information via an image environment.Images and spotlist can be plotted on a BENSON plotter.
To use quantitative data obtained for comparative studies, one must establish a correct correspondence between spots on different gels. TYCHO matching component /4/ enables common spots in the two lists to be found. To do that, starting with initial identifications (less than 10 per cent) , local distortions (transla- tions, rotations) are estimated and applied to one list to link the coordinate systems of the two lists. Identifications are then carried out with some distance criterions.
This program uses the idea of a "reference gel" (named MASTER). An "OBJECT"
gel will be compared with a master which will contain all the spots present on at least one gel. This reference is built incrementally with successive matches, adding at each step all the unmatched spots of the object to the master.
1.3. Hardware
The TYCHO system was developed to allow a real exploitation of the two dimensional electrophoresis method, thus, the peformance aspect was important.
The system runs on a VAX 11/780 computer (Digital Equipment Corporation) under the VMS system. It is based on intensive use of an array processor AP 120B AFLoating Point System) both for the computation itself and for interactive handling of spotlists. It supports different image display systems (Grinnell, Ramtek, De-Anza).
1.4. Performance
All the times given here are elapsed time. The image processing is composed by about 10 passes (depending on the digitization method) which work from disk to disk.
The computation times, measured on the LET1 installation for standard electropho- resis images (1500 x 1500 pixels with approximately 1000 spots) are about :
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Image processing : 15 mn (1 to 2 nm by step)-
Fitting : from 50 to 150 mn depending on gels-
Automatic matching : 2 mnJOURNAL DE PHYSIQUE
Fig. 1 : Example of TYCHQ image processing
4 - The raw image shows spots on a noisy background, with big horizontal and small vertical streak
B - During the processing, the vertical streak is subtracted
C
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After background and streak subtraction, smoothing and thresholding (with a threshold at 2), the processed image is the reference for spot detection and fittingD
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The synthetic image built from the fitted spotlist.2 - MODULAR INSTRUMENTATION
The LETI, with the collaboration of the INSERM, has also been developing a specific instrumentation for two dimensional electrophoresis. The guidelines behind the research were to ensure that the functions required by the use of this technique were provided for while at the same time taking into account certain cost imperatives.
This instrumentation, defined as a tool for research, has also to be opened so as not to restrict further evolution required by the users. It offers, besides the conventional attributes of a micro-computer, specific system functions for two-dimensional gel analysis.
The selected hardware is composed of a micro-computer INTEL 8086 running iRMX86 with the classical peripherals (alphanumeric terminal, 10 Mb disk, 640 Kb RAM, floppy disk) and, for image display and acquisition, of IMAGING TECHNOLOGY boards, of a video camera and of a color monitor.
Acquisition and image processing programs work on 512 x 512 pixels images.
The interactive handling program allows any image size to be treated. Thus, images with a size greater than 512 x 512 pixels, can be initially processed on another system (TYCHO for example) and, then, handled interactively on the modular system.
With the basic configuration, the full processing time of one gel (from the acquisition to the achievement of a fitted spotlist) is about 4 hours.
3
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CONCLUSIONThe TYCHO system is an efficient tool enabling two-dimensional electrophoresis techniques to be used in biology laboratories. It also allows quantitative analysis on gels, practically impossible to do without computer help.
In fact, TYCHO is not really specific to electrophoresis application. Image processing is a general tool. At the same time, the modelling by two-dimensional gaussian shapes and all the tools linked to it, are, also exploitable for other applications.
This realisation is a good example of an efficient tool using standard high- level computer equipment. It also provides a good example of an intensive use of an array processor.
The modular instrumentation is able to solve problems of biology laboratories using two-dimensional electrophoresis which have not the means to develop their own analysis system.
For the future, if the use of the two-dimensional electrophoresis method continues to develop at the same speed, a good solution to the problem of computer analysis of gels, could be to create a computer center equiped with a high level system, such as TYCHO. Biological laboratories, equiped with a workstation, could then have considerable computer facilities available while having local low cost tools for local treatment.
THANKS
This work was supported by the LETI laboratories (CEA-IRDI) and the INSERM under contract no 137 001.
The transfer of the TYCHO system onto our computer equipment was only possible through the friendly collaboration of the group of Norman and Leigh Anderson.
C5-100 JOURNAL DE PHYSIQUE
We wish to thank particularly John Taylor for his sound advice. The presence at Argonne of our colleagues of the INRA, particularly JP. Hofmann, was an appreciated help.
REFERENCES
/1/ High resolution two-dimensional electrophoresis of proteins.
P.H. O'Farrel
J. Biol. Chemistry 250, 4007-4021 (1975)
/2/ The TYCHO system for Computer Analysis of Two-Dimensional gel Electrophoresis patterns.
N.L. Anderson, J. Taylor, A.E. Scandora, B.P. Coulter and N.G. Anderson.
Clinical Chemistry, 27/11, 1807-1820 (1981)
/3/ Estimation of two-dimensional electrophoresis spot intensities and positions by modeling.
J. Taylor, N.L. Anderson, B.P. Coulter, A.E. Scandora and N.G. Anderson.
Electrophoresis 1979.
/4/ A computerized system for matching and stretching two-dimensional gel patterns represented by parameter lists.
J. Taylor. N.L. Anderson and N.G. Anderson.
Electrophoresis 1981.
