Chromosome Aberration Simulator (CAS): User's Guide

Go To Abbreviated Version

Authors: A.M. Chen, P. Hahnfeldt, and R.K. Sachs

Version: 1.8

Release Date: January 24, 1998.

This is a test version. This user's guide was last modified 7/18/98 by A. Chen, P. Hahnfeldt and R.K. Sachs. For updated CAS information and Fortran source files or UNIX executables contact Ray Sachs. You can also download a Windows version of the software.

1. Introduction

This software simulates chromosome aberrations produced by ionizing radiation during the G0/G1 phase of the cell cycle. It uses the classic random breakage and reunion model, modified to incorporate proximity effects. The basic assumptions are randomness of break induction, localization of chromosomes, and randomness of misrejoining for free ends from breaks within one interaction site (Sachs et al. 1997). The model is implemented via Monte-Carlo simulations (Chen et al. 1996). All reactions are assumed complete, i.e. there are no unrejoined free ends. Chromosome arm lengths for the human genome or for non-human genomes can be systematically taken into account. Scoring appropriate for conventional staining (Savage 1976), or for fluorescent in situ hybridization (FISH) experiments involving whole-chromosome painting with additional colors (Savage and Simpson 1994, Tucker et al. 1995), is included. The program gives a cell-by-cell record of simulated rearrangements, much as in an actual experiment. Simulated relative frequencies and simulated dose-response curves for different types of aberrations can also be obtained.

1.1 Input.

In order to perform simulations, the user must specify the genome and the chromosome staining/painting system being simulated (Section 3). The user also provides numerical input (Section 2), including the average number of reactive double strand breaks per cell (a parameter proportional to the dose) and the number of interaction sites in one cell (an integer related to the magnitude of proximity effects). These two parameters must be adjusted to the experiment being simulated by trial and error or by using values in the literature. Then frequencies of all kinds of simple or complex aberrations can be simulated in detail.

Input data can be entered using the dialog-based interface. However, the specification of the genome and the staining/painting system can also be done by editing the appropriate file using a text editor (see Section 3).

1.2 Output.

 

Output is in three files: "numbers.txt", "other.txt", and "details.txt". For FISH experiments the "details.txt" file contains cell by cell records of visibly rearranged chromosomes, up to the first 1,000 cells.

 

For three selected staining/painting protocols the software supplies, in the file "numbers.txt", simulated numbers of certain aberration types (see Section 4 below). This output can be obtained for up to a few million simulated cells on a fast PC. Then the "other.txt" file contains aberrations too complex to coincide with any of listed types, up to the first 1,000.

 

For other staining/painting systems, the file "numbers.txt" contains:

the total number of rearranged chromosomes which have one and only one color junction;

the total number of FISH-painted centric rings.

1.3 Terminology

Biological terminology used in this guide is mainly taken from Savage (1976), Savage and Simpson (1994), Tucker et al (1995), and Chen et al (1996).

2. Running the Program.

To run the program you must have the dialog.exe and chrom.exe files in your current working directory. The sample below assumes that the _male.txt file shipped with the software is placed in the same directory too (if it was deleted or modified some values prompted by the program may be different).

 

Prompts include examples, such as "last value: 2000" in 2.1 below. These examples are supplied by the program for the first simulation and thereafter are the answers the user gave in the previous simulation. In each case the user can accept the prompted value just by pressing "Enter" or can type a new value.

 

2.1 Run the dialog.exe program.

The program prompts:

Enter total cell number ( last value: 2000); to accept press <Enter>

Type 10000 and press "Enter"

[You have specified 10,000 as the total number of cells to be simulated].

 

2.2 The program responds by the following prompt:

Enter the number of reactive DSBs (last: 8.5)

Press "Enter" to accept this value

[This adjustable parameter, for the average number of reactive DSBs per cell, i.e. DSBs not rapidly restituted, corresponds to the dose; in some simulations for x-ray or gamma-ray irradiation of human lymphocytes or fibroblasts in vitro, a value of about 2.5 times dose in Gy, was appropriate (Chen et al. 1996). The value should be adjusted for each experiment; for high LET larger values are needed (Chen et al. 1997).]

 

2.3 The program prompts:

Enter a positive integer site number (last value: 12)

Type 25 and press "Enter"

[This integer specifies the number of interaction sites in one cell nucleus. It controls the magnitude of proximity effects (Sachs et al. 1997); the more sites, the stronger are the proximity effects. Using just one site corresponds to no proximity effects (Chen et al. 1996). In experiments with human fibroblasts or lymphocytes values from about 12, for x-ray irradiation, to about 25, for alpha-particle irradiation, were found appropriate.

2.4 The program prompts:

Enter any positive integer (last value: 3455)

Press "Enter" to accept this value

[This "seed" starts the random number generator. If you want to duplicate previous results exactly, use identical input, including this seed. Changing the seed, leaving all other input values the same, gives a randomly different simulation of the same situation.]

 

2.5. The program prompts:

Enter chromosome data file (default _male.txt)

Press "Enter" to accept the _male.txt file.

[Note that this file happens to start with an underscore, i.e. _male.txt not just male.txt. The user also can specify another file containing the chromosome coloring, e.g. by typing newcolor.txt, or type new to create a new file (See Section 3 for a discussion of chromosome data files).]

 

2.6. The program prompts:

Would you like to modify the file (y/n)?

Press "n" .

[User also can press "y" to modify the coloring scheme. Modified data may be placed into a new file or override the existing one].

 

2.7. The program performs calculations and asks if the user want to see the results. The output data are also placed into the numbers.txt, detail.txt, and other.txt files.

3. The Chromosome Data File

The chromosome data file contains the following information:

1. The number of FISH colors plus one.

2. For each chromosome, the lengths of both arms and the color.

This file can be created or modified by using any text editor (e.g. notepad. If you use a powerful editor like Microsoft Word, make sure to save it as a "text only" (ASCII) file ) or during the dialog-based data entry process. Some samples of this file are shipped with CAS (_male.txt, _female.txt, and a sample in each of the four directories COLOR01 - COLOR04.

3.1 Chromosome Data File Details

The first line of the chromosome data file is a whole-number representing the number of FISH colors plus one. Examples are "1" for conventional staining; or "2" for one counterstain (blue) and one FISH color; etc. Centromeres are usually assumed identifiable, and a special color for a pan-centromeric probe does not count. In this software the color blue (abbreviated "b") is always used for the background color, i.e. the stain in the case of conventional staining or the counterstain for FISH experiments.

 

The rest of the chromosome data file is a four-column table representing the lengths of both arms of the chromosome and its color. Each row corresponds to one chromosome.

 

The columns are separated by the blanks. The first column contains a "label".

 

The second and third columns represent the lengths of long and short arms.

Arm lengths can be in any units. Best results are obtained if the arm lengths are scaled in such a way that the largest chromosome is a few hundred units. If you do not know the lengths of the chromosome arms for non-human cells being simulated, approximate results can be obtained by making all arm lengths equal, i.e. by entering the same number (e.g. "100") for each arm length.

 

The last column contains one color. Using the first letter of the word denoting this color (e.g. r - red) is normally recommended, but two points should be taken into account:

different colors must correspond to different letters.

‘b’ always denotes ‘background’ and this color plays a special role in the calculations. At least one chromosome must be colored b in the file.

 

This chromosome data file can be created or modified by using any text editor or during the dialog-based data entry process. Some samples of this file are shipped with the software.

3.2 Examples

1. The following chromosome data file would be used for human male cells with chromosomes 1, 3, and X painted green ("g"), all other chromosomes counterstained blue ("b"), and centromeres recognizable. In this example, lengths of the long arm (e.g. 135) and short arm (e.g. 128) are in Mb but any consistent units would work.

Chromosome data file contents:

2

1 135 128 g

1' 135 128 g

2 156 97 b

2' 156 99 b

3' 115 99 g

3' 115 99 g

... [38 more lines here for 19 blue homologue pairs. Data on human chromosome arm lengths can be found in the _male.txt file shipped with the program].

X 102 62 g

Y 46 13 b

2. Another (hypothetical) example.

The following file could be used for painting of a genome with only 10 chromosomes, where the lengths of the chromosome arms are unknown to the user. The first 4 chromosomes are painted different colors and the next 6 are counter-stained:

5

1 50 50 g

2 50 50 y

3 50 50 r

4 50 50 p

5 50 50 b

6 50 50 b

7 50 50 b

8 50 50 b

9 50 50 b

10 50 50 b

3.3 Human Data

If the user prints "new" for the chromosome data file the dialog-based creation of the new file starts. "Creation from the beginning" within the program is allowed only for human data. In this case the program asks for the sex, number of colors and color for each chromosome. CAS uses the pre-defined data on human chromosome length. The same effect can be achieved by modifying the _male.txt or _female.txt files shipped with CAS.

The program asks for the number of colors and for each color and prompts the corresponding values from the "based" file as a default. As usual, the user may change any value or accept the default by pressing "Enter".

3.4 Non-human Genome

 

The only way to modify the number of chromosomes and/or the lengths of the arms is by using a text editor. Afterwards additional files with the same number of chromosomes and arm lengths may be created using a dialog-based procedure similar to the one for human data. Thus the first time a non-human genome is simulated, a text editor is needed for the chromosome data file.

4. Output Information

4.1 details.txt file.

This file contains cell by cell information, similar to that which would be seen during an actual experiment. Rings are denoted by @. Here are some examples:

 

4.1.1 Suppose the painting system is that some chromosomes are painted yellow and the rest are counterstained blue. Suppose that some lines in the file "details.txt" read:

...

******* cell # 65

2 by

0 by

******* cell # 66

...

This means that in the 65th cell simulated, there is a blue-yellow rearranged fragment with 2 centromeres and a blue-yellow acentric fragment. In this cell there is no other damage to yellow chromosomes that can be identified via colors or centromeres (there could be, e.g., a cryptic yellow inversion). Rearrangements of blue chromosomes by themselves, without participation by yellow chromsomes, is ignored.

4.1.2. Similarly, the information

...

******** cell #712

1 yby

2 by

0 by

...

can refer to a cell where a blue fragment, not containing a centromere, has been removed from a blue chromosome and inserted into a yellow chromosome, with the remaining two pieces of the blue chromosome undergoing illegitimate reunion with two pieces of a different yellow chromosome, forming one acentric fragment and one rearranged chromosome with two centromeres.

4.1.3.

...

******* cell #963

1 @y

 

...

refers to a yellow centric ring; an accompanying acentric fragment, 0y, is implied in this special case (in all other cases all painted components are explicitly listed).

The program does not produce this details.txt file for conventional (one color) staining.

4.2 other.txt file

This file contains the same information as the details.txt file but includes only the cells that have aberrations too complex to coincide with any of the types listed in the numbers.txt file.

4.3 numbers.txt file

This file contains total numbers of aberrations of certain types, depending on the painting system used.

4.3.1 One color:

All chromosomes are stained blue and centromeres are identifiable. The _numbers.txt file shows the totals for the following types:

True Dicentric: a rearranged, non-ring chromosome with two centromeres, accompanied by one acentric fragment (Savage 1976).

Acentric Ring: may show up as interstitial deletions, or as excess acentrics.

Generalized Dicentric: true dicentrics + 2* tricentrics + 3* tetracentrics + ...(Savage and Papworth 1972).

Generalized Centric Ring: true centric ring accompanied by one acentric fragment +2* bicentric rings accompanied by two acentric fragments + ...

Dicentric Dispersion Ratio: cell-to-cell dicentric variance divided by average dicentrics per cell; Here, a tricentric is equivalent to two true dicentrics, etc.

4.3.2 Two colors:

Some chromosomes are counterstained blue (b), others are painted (e.g. yellow, y).

 

PAINT is the terminology proposed by Tucker et al. (1995). S&S is the CAB terminology used by Savage and Simpson (1994). The CAB patterns assume that only one painted chromosome is involved in any aberration; if both homologues are involved, Simpson and Savage would classify them twice, (Simpson and Savage 1996).

 

If the painted chromosomes are not visibly involved with other chromosomes, the cell is classified as "Normal", i.e. 1A in S&S. This class includes inversions, which are assumed to be not recognizable by the whole chromosome painting method.

 

The following four Apparently Simple Aberration Groups are defined in Savage and Simpson (1994, S&S), where "Simples" are defined as exchanges involving 2 reactive DSBs on 1 or 2 chromosomes:

Apparently Simple Dicentric: dic(AB)+ace(ab) in PAINT; 2A in S & S.

Apparently Simple Translocation: t(Ab)+t(aB) in PAINT; 2B in S & S.

Apparently Simple Centric Ring: r(B) in PAINT; CR1 in S & S.

Apparently Simple Acentric Ring: r(b) in PAINT; 2C in S & S.

The following eight Visibly Complex Aberration Groups are defined in Simpson and Savage (1995, S&S), where "Complexes" are defined as exchanges involving 3 or more reactive DSBs on 2 or more chromosomes:

AG 1. Single Insertion: abA or baB in PAINT or any similar pattern with the same colors but different centromere placement: 1B-C, 2D-E etc. in S & S.

AG 2. Double Insertion/Tiger: e.g. abaB+bA or abA+baB in PAINT, 2L-2O, 2S-2AA etc. in S & S.

AG 3. Painted Ring + Other: e.g. r(b)+Ab+aB, r(B)+Ab+ab in PAINT; 2P-R, 3A-I, 4A-B, CR2-15 etc. in S & S.

AG 4. Monocentric + Fragment: aB+ab in PAINT; 2F in S & S.

AG 5. Dicentric + Monocentric: AB+Ab in PAINT; 2G in S & S.

AG 6. 3 Chromosomes/4 Breaks: aberrations which apparently involve a minimum of 4 breaks on 3 chromosomes (e.g. AAB+ab, Aba+ab+AB in PAINT; 2AB-AC, 3J-P etc. in S & S).

AG 7. 4 Chromosomes/5 Breaks: aberrations which apparently involve 5 breaks on 4 chromosomes (e.g. AAB+Ab, ABa+ab+ab in PAINT; 2AD-AE, 3Q-T, 3AF-AH etc. in S & S).

AG 8. Other Complexes: apparently involving 6 or more breaks in S & S.

4.3.3 Three colors

In this experiment some chromosomes are counterstained blue, a subset of chromosomes are painted, e.g., red; and another subset of chromosomes are painted another color, e.g. green Centromeres are assumed not to be recognizable. Each cell with painted visible aberrations is classified as one of the following (Chen et al. 1996):

Normal: cells with no visible painted damage, which includes all inversions.

Apparently Simple (Twin): cells with two bicolored chromosomes; further classified into three color combinations, e.g.: bg twins, br twins and gr twins.

Double Twin: cells with two sets of twins.

Three-color, Three-way Interchange: cells with three rearranged chromosomes, the color patterns for them are ab+bc+ca in PAINT (centromeres not recognizable).

Simple Painted Ring: cells with acentric or centric painted rings, they are further classified as green or red rings.

Painted Ring + Other: cells with rings plus some other painted aberrations.

Insertion: cells with aba or bab type aberrations in PAINT (centromeres not recognizable).

Insertion + Twin: cells containing exactly one set of twins and one insertion.

Other Complex: cells with more complicated aberrations than above.

4.3.4 Four (or more) colors

In all other cases, the numbers.txt file contains two results. One is "simple bicolored", the total number of bicolored rearranged chromosomes which have one and only one color junction. For example, a bicolored dicentric with its accompanying acentric fragment, formed from a painted and a counterstained chromosome in the simplest reactions, give rise to two bicolored rearranged chromosomes which have exactly one color junction, i.e. contribute two to the entry for "simple bicolored". The same is true for translocations in simple cases. When several different FISH colors are used, "simple bicolored" is a sum, e.g. yellow-blue + red-blue + yellow-red. At low or moderate doses, "Simple bicolored" is an index of total damage. To increase it in the simulations increase the adjustable parameter for number of reactive DSBs, or decrease the adjustable parameter for site number.

 

The other number in the numbers.txt file is then the total number of painted centric rings (bicentric rings are counted twice, tricentric ones three times, etc.). To increase this number relative to "simple bicolored" in the simulations, increase the proximity effects by increasing the site number.

4.4 Other output.

Once the adjustable parameters are determined by trial and error, various other quantities of interest can be simulated, by using the details.txt file. Examples include: frequencies for those aberration types whose frequency is not in the numbers.txt file; the average number of color junctions per cell; dose response curves for various categories of aberrations; etc. Users who modify the source code slightly, can get additional results automatically, for example: the total number of dicentrics (painted or not) in the whole genome; the number of cells which are "non-transmissible", i.e. have damage putatively fatal at mitosis (Savage, 1995); etc.

5. Distribution disk

The distribution disk contains the following files:

dialog.c - C source code of the dialog-based user interface ;

dialog.exe - executable file for this program;

chrom.exe - executable file for the calculation program (source code is available from A. Chen upon request);

_chrom.ini - sample initialization file;

_male.txt, _female.txt - sample chromosome data files;

CAS.doc - this file

and the following directories containing some sample files (chromosome data file (color.txt), initialization file (_chrom.ini) and results of calculations with these files: numbers.txt, details.txt, and other.txt (if these files are produced for the given example) ):

COLOR01 - sample files for 1-color;

COLOR02 - sample files for 2-color staining/painting;

COLOR03 - sample files for 3-color staining/painting;

COLOR04 - sample files for 4-color staining/painting;

6. Migration

All the executable files were created under the Windows95 operating system. For other platforms, e.g. UNIX, SunOS, and Macintosh, they should be created using the appropriate C and FORTRAN compilers (see also our ftp site).

 

Both the dialog and chrom program are written using the standard C and FORTRAN languages, so they are as platform-independent as possible. Only ten lines of code are known to be system-dependent:

- Lines 8-9 of the dialog.c source code:

char* show = "notepad ";

char* cop_command = "copy _fort.tmp _chrom.ini";

Here "notepad" and "copy" should be replaced by system-dependent viewer/editor

and copying command (e.g. "more" and "cp" for the UNIX platform).

- Lines 162-165 and similar lines 176-179 in the same program:

fn = _open ("_details.txt", O_RDONLY );

file_length = _filelength(fn);

_close (fn);

if( file_length > 0 )

These lines can be deleted for other platforms.

 

The authors cannot guarantee that these changes would be sufficient for any platform and compiler.

 

Calculation time depends on the cell number, average number of reactive DSBs and platform. Just for orientation, calculations with a Pentium PC may take less than a minute for 1,000 cells and 10 minutes - 1 hour for 1,000,000 cells (or even more for a large number of reactive DSBs)

7. Bibliography

 

Chen, A. M., Lucas, J. N., Hill F. S. Brenner, D. J. and Sachs, R. K., 1995, Chromosome aberrations produced by ionizing radiation: Monte Carlo simulations and chromosome painting data. Computer Application in Biosciences, 11, 389-397.

Chen, A. M., Lucas, J. N., Hill F. S. Brenner, D. J. and Sachs, R. K., 1996, proximity effects for chromosome aberrations measured by FISH. International Journal of Radiation Biology, 69, 411-420.

Chen, AM, Lucas, JN, Simpson, PJ, Griffin, CS, Savage, JRK, Brenner, DJ, Hlatky, LR, and Sachs, RK, 1997, Computer simulation of FISH data on chromosome aberrations produced by x-rays or a-particles. Radiation Research, 148, S93-S101.

Hahnfeldt, P., Sachs, R. K., Hlatky, L. R., 1992, Evolution of DNA damage in irradiated cells. Journal of Mathematical Biology, 30, 493-511.

Sachs, R. K., Yates, B. L., Tarver J., Morgan W. F. 1992, Modelling the formation of polycentric chromosome aberrations. International Journal of Radiation Biology, 62, 449-460.

RK Sachs, AM Chen & DJ Brenner, 1997, Review: proximity effects in the production of chromosome aberrations by ionizing radiation. Int. J. Radiat. Biol.71, 1-19.

Savage, J. R. K, 1976, Classification and relationships of induced chromosomal structural changes. Journal of Medical Genetics, 13, 103-122.

Savage, J. R. K. and Simpson, P. J., 1994, FISH ‘painting’ patterns resulting from complex exchanges. Mutation Research, 312, 51-60.

Savage, J. R. K., 1995, The transmission of FISH-painted patterns derived from complex chromosome exchanges. Mutation Research, 347, 87-95.

Simpson, P. J. and Savage, J. R. K., 1995, Estimating the true frequency of X-ray-induced complex chromosome exchanges using using fluorescence in situ hybridization. International Journal of Radiation Biology, 67, 37-45.

Simpson, P. J. and Savage, J. R. K., 1996, Dose-response for simple and complex chromosome aberrations induced by X-rays and detected using fluorescence in situ hybridization. International Journal of Radiation Biology, 69, 429-436.

Speicher, M. R., Ballard, S. G., Ward, D. C., 1996, Karyotyping human chromosomes by combinatorial multi-fluor FISH. Nature Genetics, 12, 368-375.

Tucker, J. D., Morgan, W. F., Awa, A. A., Bauchinger, M., Blakey, D., Cornforth, M. N., Littlefield, L.G., Natarrajan, A. T. and Shasserre, C., 1995, A proposed system for scoring structural aberrations detected by chromosome painting. Cytogenetics and Cell Genetics, 68, 211-221.

8. Acknowledgments

This software is partially based on previous versions by P. Hahnfeldt, J. Tarver and R. Sachs. (Hahnfeldt et al., 1992, Sachs et al., 1992). Funds for adding interfaces for Windows 95 and other platforms were supplied by NIH grant CA 63897-03. Revisions prepared under NSF grants DMS 9532055 and BIR 963-0735. The assistance of D. Brenner, L. Hlatky, J. Lucas and V. Kaganskiy is gratefully acknowledged.

9. Authorization

This software is freely available to all non-profit users. Users must check that it works for their purposes. Permission to use, copy, modify, and distribute this software and its documentation for any non-commercial purpose, without fee, and without written agreement, is hereby granted, provided that this paragraph and the preceding one appear in all copies. In no event shall the authors be liable to any party for direct, indirect, special, incidental, or consequential damages arising out of the use of this software and its documentation. We specifically disclaim any warranties, including, but not limited to, implied warranties of merchantability and fitness for a particular purpose. The software provided hereunder is on an "as is" basis, and we have no obligation to provide maintenance, support, updates, enhancements, or modifications.

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