GELLAB-II - NCI/IPS 2D Electrophoretic Gel Analysis System

A Short Description Of The GELLAB-I/-II System

Peter F. Lemkin

Image Processing Section

Laboratory of Experimental and Computational Biology, DBS

National Cancer Institute, NIH/FCRDC,

Building 469 Rm 150

Frederick, MD 21702

lemkin@ncifcrf.gov

REVISED August 17, 1993 - PF Lemkin

D R A F T

GELLAB-II is an integrated collection of programs for the analysis of 2D electrophoretic gels. Currently, it runs on UNIX systems. The display of gel images is available if the hardware supports a color display under X-windows. These programs were developed at the Image Processing Section of the Laboratory of Experimental and Computational Biology, DBS in the National Cancer Institute/FCRDC.

Programs are invoked by naming the program and specifying optional arguments. All programs have several common UNIX-style command-line switches to facilitate learning the consistent user interface. This subset of switches is useful in learning how to run particular programs. Descriptions of the algorithms used in these programs are given in many of the GELLAB-I papers listed in the references [4,7-13,17,20,29-30]. Good introductory papers which describe the basic GELLAB analysis are [4] and [10] with [12] being a more general and detailed summary. Extensions to the early system are discussed in [11,13,17-18,20,29-30]. A comparison of aspects of 2D gel data base analysis systems is given in [29]. This document lists the GELLAB-II programs and describes the basic steps in performing a 2D gel analysis. Full documentation of GELLAB-II is given in the book [31]. A few selected examples of running some of these programs are included at the end of this document.

In general, almost all GELLAB-II programs require one or more arguments so you should read the individual programs' documentation (using -info as suggested above) prior to attempting to run them. GELLAB-II programs use a resource file called gel.rc in the users current path to provide state information. This includes various directories for image files, gel data base files and other intermediate files (see DIRECTORIES AND SUPPORT FILES). If you do not have this file in your path, running any GELLAB program (such as pgelrc - which prints a user friendly form of gel.rc) can be used to prompt the user in defining the initial gel.rc file. A batch script generation facility which is part of GELLAB (makjob) can greatly automate running these programs. Then minimum investigator intervention is required for major parts of its operation in composite gel database prepration.

The GELLAB-II programs are listed below. Corresponding literature references are given in [...] which describe the algorithms in more detail. No individual UNIX man pages are available for these programs. The equivalent information is available with -info switch options instead and in the book [31].

1.The GELLAB-II programs include:

accppx - display gel image(s) given accession number(s).

camera - capture DataCopy camera image into PPX file.

cgelp2 - interactive Paged Composite Gel database analysis. uu

cmpgl2 - paired 2 GSF spot lists using landmark DB=>GCF.

dendrogram - Rspot, gel expression profile cluster analy.

dwrmap - draw Rmap numbered plot of GSF spot list.

getacc - multiple gel accession: images, info., calibration.

landmark - interactive graphics LandMark Set DB acquisition.

makjob - create GELLAB-II scripts for batch processing gels.

markgel - generate Rmap image from cgelp2 (.sps) data file.

mosaic - generate mosaic image from cgelp2 (.sps) file.

pgelrc - "pretty print" the gel.rc GELLAB-II state file.

plotn - plot GELLAB-II Universal Graphics Files (.ugf).

ppxcvt - cvt. foreign format image to Portable PiXture file.

ppxodt - Portable PiXture (.ppx) file image debugger.

ppx2ps - convert image file to Postscript for laser printer.

sg2gii - segment gel image to Gel Segmentation File (.gsf).

tek2psG - convert tektronix 4010 input to PostScript.

Xpix - with X11 display & manipulate PPX images.

2. PERFORMING AN ANALYSIS

A typical GELLAB analysis of a set of 2D gels is a data reduction process. It analyzes a set of gels of the same material but with different experimental conditions to produce lists of spots with similar specific attributes. These subsets of spots are clustered using statistical techniques. The major steps of an analysis are: (1) accessioning (gel scanning), (2) spot quantitation, (3) gel pairing, (4) composite gel DB construction, and searching and display of search results of different views of the DB. Briefly, this data reduction is acheived as follows:

(1) Accession a set of gels by scanning them with the getacc program which: (a) assigns an accession number to each gel, (b) scans the gels into Portable PiXture (.ppx) files, and (c) requires the experimenter to enter associated experimental study accession information, and (d) calibrate the ND step wedge scanned with the gel and define an active region in the gel image called the computing window. The information from (c) and (d) is entered into an accessiion file. This accession file typically has a gel prefix and a .id file extension. Eg. gelts3.id. During a data acquisition session, one would enter a number gels and at the end of the session getacc would prompt you for a few pieces of information necessary for further processing. These includes (a) the name of the Reference gel or Rgel, (b) a three character project prefix used for all files associted with the project. and (c) the names of the different experimental classes to which the different gels belong. It will then generate UNIX batch scripts to: interactively landmark these gels; segment or extract quantitated lists of spots from the gels; pair N-1 of the N gels with the selected Reference gel; and construct the composite gel database and perform some initial statistical tests. The makjob program also lets you generate these batch scripts for different sets or subsets of gels which have been previously accessioned.

(2) Spot-list extraction and quantitation is performed by the sg2gii program which results in a Gel Segmentation File (.gsf) and an optional extracted spot image file. The GSF file contains position and quantitation information for all spots in a single gel and must be further processed to compare it with other gels.

(3) Pairing of GSF spot lists from two gels (one of which is the Rgel) is performed by the cmpgl2 program. The output is called a Gel Comparison File (GCF) and is a file with a .gcf file extension. It consists of the pairing of spot data from the two GSF input files. The pairing program also requires a list of a small number of corresponding landmark spots for the two gels being paired. This is stored in the LandMark Set (LMS) data base file which typically has a lms prefix and .lm file extension. LMS data can be acquired several ways: (a) using the landmark interactive graphics program running under X-windows, (b) using program dwrmap to draw Rmaps from GSF data which can be plotted with plotn. The landmark numbers can be read manually from the plots and then entered via a terminal session using the landmark program. A third method involves using the Xpix program in its "compare" mode to interactively generate the landmark coordinate pairs which can then be text edited into the proper LMS DB format. Needless to say, the use of the landmark program is to be encouraged as it is by far the easiest and most accurate.

(4) Construction of the Paged Composite Gel DB (PCG DB) is performed by cgelp2. Cgelp2 requires a set of N-1 GCF files for N gels since the Rgel is included in each GCF. By "paged" we mean that as the data base is too large to fit in memory. Pieces of it are paged in and out of memory from the actual PCG DB disk file (which has a .pcg file extension). The accession file is also accessed to extract the "study" information for each gel in the PCG DB. This information is used for automatic classification of gels into the current experimental classes of gels and other analyses. Exploratory data analysis really starts once the PCG DB is constructed. The particular strategy to follow is outside the scope of this document but is discussed in many of the papers listed in the REFERENCES on cgelp2 and papers on particular biological problems.

The initial batch script generated by getacc or makjob: (a) constructs the PCG DB file, (b) constructs an initial experimental gel classification based on accession file study information, (c) normalizes the protein concentration values between gels using the Ratio-List method and reorders spots in all Rspots sets in the PCG DB based on this normalization. It (d) then performs an initial F-test and t-test at p-values of 0.90, 0.95 and 0.99 for all of the experimental classes. It performs a Wilcoxon-rank-sum test of classes 1 and 2 at 0.90, 0.95 and 0.99 for classes 1 and 2 as well as a missing-class test. It also computes and displays histograms of various Rspot set spot features for the entire PCG DB in order to aid in setting the initial prefilter parameters. Subsequent analyses consist of changing the view of the PCG DB, performing searches in the new view and displaying this transformed data as images, plots, tables, lists, etc. See [12-13,17,29-30] for more discussion or using GELLAB for exploratory data analysis.

3. LIST OF PROGRAMS

camera - runs the NCI/IPS DataCopy 612F CCD camera to scan an image generating a .ppx image. It is may be used with or without the rest of GELLAB. When used with GELLAB, it is called by getacc. Note that images scanned on other scanners may be used instead such as the Molecular Dynamics scanners, etc. and used by the getacc program described below.

cgelp2 - runs the Paged Composite Gel database analysis system. This builds a PCG DB file from a set of Gel Comparison Files (GCF)s produced by the cmpgl2 program. When running cgelp2 additional information is available on top level commands by typing HELP to list all of the top level commands or HELP specific-command. For example, type HELP HELP to get more information on the HELP command. There is also an ?APROPOS facility for finding relevant commands. [4,9-10,12-13,17-18, 20-22,29-30]

cmpgl2 - runs the gel pairing program which generates a Gel Comparison File (GCF) (.gcf) from two Gel Segmentation Files (GSF) (.gsf) produced by the sg2gii segmentation program. It also requies a landmark set DB (LMS) entry for the two gels being paired. [4,8,10,12]

dendrogram - generates a dendrogram cluster analysis plot. It uses cgelp2 oduced SPSS (.sps) or INQUIRE (.inq) files. It can cluster a set of Rspots as a function of density of a set of gels or cluster a set of gels as a function of the density profile of a set of Rspots sets. It also plots the results after they are generated and/or makes an optional .ugf plot file. A data file (.dgm) is also produced which contains numeric cluster analysis information. [24]

dwrmap - given a Gel Segmentation File (GSF) Plots a Rmap from the .gsf file. It plots a Rmap with spots labeled by their GSF spot number. [13]

getacc - in a data acquisition session, acquire 2D gel images and their related accession information which is appended to the accession file. At the end of the session, ask a few questions reguarding the type of experiment to be performed. Then generate UNIX batch scripts to a) interactively landmark the set of gels, b) segment the gel images into GSF spot lists, c) pair GSF spot lists into GCF paired spot lists, d) merge the GCF files by constructing a composite gel PCG DB file and e) perform an initial statistical analysis of the PCG DB. [4,7,12]

landmark - is an interactive X-windows graphics program to landmark two gels. This process defines a small set of corresponding spots (10 to 20) upto to 52 in each of the two gels. These spot postions are used to update an entry in the LandMark Set (LMS) data base file which is used by other programs including the spot pairing program cmpgl2. It can also edit previously defined LMS entries. Landmarks entered when the first gel is landmarked with the Rgel are used when landmarking the remaining gels. This makes landmarking much easier and more reproducible. [4,8,10,12]

makjob - generates GELLAB-II UNIX scripts. It requests a list of gel accession numbers for a subset of gels previously accessioned. It then asks a few questions reguarding the type of experiment to be performed and generate UNIX batch scripts to a) interactively landmark the set of gels, b) segment the gel images into GSF spot lists, c) pair GSF spot lists into GCF paired spot lists, d) merge the GCF files by constructing a PCG DB file and e) perform an initial statistical analysis of the PCG DB. A makjob run can be customized to perform some and not other specific analyses (see -info switch for makjob). See example of running makjob in the EXAMPLES. [12,13]

markgel - generates a Rmap image having specified a gel accession number and a SPSS (.sps) file generated by the cgelp2 program. The Rmap is the synthetic image generated by the projection of the set of spots specified by the SPSS file onto a copy of the gel image associated with the gel accession number. [4,9,12]

mosaic - generates one or more mosaic images having specified a particlar Rspot number and a SPSS (.sps) file generated by the cgelp2 program. image or plot of a Rspot for a set of gels is a composite image or graphic formed from panels from each gel arranged in a regular checkerboard pattern ordered by minimum spot density (protein concentration). The panels are taken from a subregion of each gel surrounding a particular Rspot. [4,9,12]

pgelrc - prints a user friendly form of the gel.rc GELLAB state file. This file contains the default names of various data base files, directories, segmentation parameters, and information on the last data processed. It is used by all GELLAB-II programs upon startup If the file does not exist, then running pgelrc will run an interactive Question and Answer session to generate the initial gel.rc file and gellab directory tree. You can also find the status of the next free accession number and PPX file, which accession numbers are in the system and what PPX files from the database are out on the disk. [12]

plotn - reads Universal Graphics Files (.ugf) produced by various GELLAB-II programs cgelp2, dwrmap, dendrogram. It is able to replot a .ugf on the same or different type of display as well as to plot the file on other devices (such as a Postscript laser printer). Used with tek2psG it can print plot files on a laser printer.

ppxcvt - convert different input picture file formats into the GELLAB Portable PiXture file format (.ppx). It can convert ASCII hex, decimal or octal numbers as well as other binary formats with variable header, leading and trail number of pixels/lines and variable number of lines, TIFF, BioImage, Elsie, etc.. It also has input header optical density calibration conversion to PPX header format for selected input types (such as BioImage). Input images may be sampled. Data may also be transformed by complementing, linear scaling or performing a log transform to produce the 8-bit data required for the PPX file.

ppxodt - is a picture debugger for opening, reading pixels, 3x3 neighborhoods, and 18x18 windows of a .ppx file. Data may be viewed in hex, octal or decimal. Individual pixels may be changed and the edited picture file saved.

ppx2ps - converts a .ppx image file to Postscript which can then be printed on a laser printer.

sg2gii - is a gel spot list segmentation program which generates a Gel Segmentation Files (GSF) from the image file associated with the gel accession number. Soots are found and then quanitfied. [4,7,10,12,13]

tek2psG - converts Tektronix 4010/4015 input (such as is produced by plotn) to PostScript which is suitable for printing on a PostScript laser printer.

Xpix - is a general purpose X-windows X11 or later .ppx file interactive display program. It is controlled by the user moving and clicking a mouse to get menus selections and interact with the image(s). It can manipulate one or two images on the screen at a time with each image having its own real-time small zoom window (additional images can be kept in memory and be alternately displayed). It can perform general image processing types of operations where you can save transformed image disk files for later recall [28].

4. EXAMPLES

The following is a short set of examples illustrating some of tyep types of operations possible with GELLAB-II. No examples of cgelp2 are given because that is beyond the scope of this brief document and is covered in the Reference Book [31]. See the detailed discussions in the papers listed in the REFERENCES.

Example 1: print GELLAB project current state file if it exists. If not, it will ask you if the default values are acceptable and then create a gel.rc file and gellab directory tree for the project in the current directory.

pgelrc

Example 2: generate batch scripts given a list of gel accession numbers in file tst3.ccl, a project prefix ts3, four experimental classes and the rgel.

makjob

which will prompt you with questions, or

makjob -rgel:0324.1 -class:AML:ALL:CLL:HCL:HL-60 -accs:ts3.ccl -prj:ts3

Example 3: given an accession number display the gel in a Xpix window.

accppx 324.1

Example 4: given two accession numbers display the corresponding gel image files in two Xpix windows.

accppx 324.1 369.1

Example 5: segment a gel into a GCF file and a 'z' segmented image spot file and then display it.

sg2gii 324.1

accppx 324.1 324.1 -P2:z

or

accppx 324.1 -P1:z -Laser

to print the segmented image to the laser printer

Example 6: segment a gel as above but also generate the 'c' central core image and the original less the segmented spots image 'y' files. Then display the these two derived images.

sg2gii 324.1 -ctlcoreimage -restofimage

accppx 324.1 324.1 -P1:c -P2:y

Example 7: pair GSF spot list files for two gels into a GCF file.

cmpgl2 324.1 369.1

Example 8: pair GSF spot list files for two gels into a GCF file and also generate 'u' and 'v' labled paired-spot image files. Then display the these two derived images.

cmpgl2 324.1 369.1 -MarkLabels

accppx 369.1 369.1 -P1:u -P2:v

or add the display switch to the same command:

cmpgl2 324.1 369.1 -MarkLabels -Xpix

Example 9: generate the two 'l' landmark set images for the two gels. but do not pair the gels.

cmpgl2 324.1 369.1 -onlyMarkLMSimages

accppx 324.1 369.1 -prefix:l

or add the display switch to the same command:

cmpgl2 324.1 369.1 -onlyMarkLMSimages -Xpix

Example 10: run cgelp2 on the command file batch script to create or explore a PCG DB.

cgelp2 -f[ile] ts3cgl.gdo

Example 11: start the CGL data base program on an existing data base file.

cgelp2 -d[atabase] ts3cgl.pcg

or start it with a graphical user interface:

cgelp2 -g[raphicalInterface] -d[atabase] ts3cgl.pcg

Example 12: generate an Rmap of gel 324.1 for SPSS file 'ts3s02.sps' proceduce from the cgelp2 PCG DB program and then display it.

markgel 324.1 ts3s02.sps -Xpix

Example 13: same as above but display the Rmap in an Xpix window window after it is generated.

markgel 324.1 ts3s02.sps -Xpix

Example 14: generate a mosaic image of Rspot 63 for SPSS file 'ts3s02.sps' produced from the cgelp2 PCG DB program.

mosaic 63 ts3s02.sps -Xpix

Example 15: same as above but display the mosaic in an Xpix window after it is generated.

mosaic 63 ts3s02.sps -Xpix : Display a previously computed Rmap and the mosaic image for a Rspot on the Rmap. Xpix window.

accppx 324.1 -P1:m w00064.ppx

REFERENCES