Visualization, Analysis, and Design of
COMBO-FISH Probes in the Grid-Based
GLOBE 3D Genome Platform
Nick KEPPERa,b,c*, Eberhard SCHMITTc*, Michael LESNUSSAa,
Yanina WEILANDc, Hubert B. EUSSENd, Frank G. GROSVELDe,
Michael HAUSMANNc,1, and Tobias A. KNOCHa,b,1
aBiophysical Genomics, Dept. Cell Biology & Genetics, Erasmus MC,
Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands.
bGenome Organization & Function, BioQuant & German Cancer Research Center,
Im Neuenheimer Feld 267, 69120 Heidelberg, Germany.
cKirchhoff Institute of Physics, University of Heidelberg, Im Neuenheimer Feld 227,
69120 Heidelberg, Germany.
dDept. Clinical Genetics & Genetics, Erasmus MC, Dr. Molewaterplein 50,
3015 GE Rotterdam, The Netherlands.
eDept. Cell Biology & Genetics, Erasmus MC, Dr. Molewaterplein 50,
3015 GE Rotterdam, The Netherlands.
Abstract. The genome architecture in cell nuclei plays an important role in
modern microscopy for the monitoring of medical diagnosis and therapy since
changes of function and dynamics of genes are interlinked with changing
geometrical parameters. The planning of corresponding diagnostic experiments
and their imaging is a complex and often interactive IT intensive challenge and
thus makes high-performance grids a necessity. To detect genetic changes we
recently developed a new form of fluorescence in situ hybridization (FISH) –
COMBinatorial Oligonucleotide FISH (COMBO-FISH) – which labels small
nucleotide sequences clustering at a desired genomic location. To achieve a unique
hybridization spot other side clusters have to be excluded. Therefore, we have
designed an interactive pipeline using the grid-based GLOBE 3D Genome Viewer
and Platform to design and display different labelling variants of candidate probe
sets. Thus, we have created a grid-based virtual “paper” tool for easy interactive
calculation, analysis, management, and representation for COMBO-FISH probe
design with many an advantage: Since all the calculations and analysis run in a
grid, one can instantly and with great visual ease locate duplications of gene
subsequences to guide the elimination of side clustering sequences during the
probe design process, as well as get at least an impression of the 3D architectural
embedding of the respective chromosome region, which is of major importance to
estimate the hybridization probe dynamics. Beyond, even several people at
different locations could work on the same process in a team wise manner.
Consequently, we present how a complex interactive process can profit from grid
infrastructure technology using our unique GLOBE 3D Genome Platform gateway
towards a real interactive curative diagnosis planning and therapy monitoring.
Keywords. Genome organization, Globe 3D Genome Platform, COMBO-FISH,
interactive extreme grid visualization, grid and GPU computing.
* Equal contribution
1 Corresponding authors: email: Hausmann@kip.uni-heidelberg.de, TA.Knoch@taknoch.org
In the last decade diagnosis and therapy monitoring has been more and more based on
the investigation of the nanostructure of the cell nucleus in context of its functional
dynamics [1-5]. The ability of modern high-resolution microscopy down to the nano
level has allowed structural biology and medicine to develop new biochemical methods
for visualization of genetic changes. During the development of many diseases,
relevant changes of distinguished nuclear and genetic parameters like the number of
chromosomes, multiplicities of genes , or rearrangements of chromosomes  can
be relatively easy observed. But also on a finer genetic scale, mutations, loss or gain of
parts of genes like exons or introns, and other rearrangements of genetic elements are
indicators of malignant changes . These changes can nowadays be quantified: On a
global level, geometric descriptors of the architecture of the nucleus like e.g. nuclear
diameter, eccentricity of its ellipsoidal form, density of chromatin, or positioning of a
gene have been shown to indicate diagnostically relevant changes [9-12]. Also on the
local level such changes can be monitored, although appropriate biochemical and
nanoscopical imaging and analysis techniques, which are needed to generate
experimentally valid quantifications, are still under development.
Especially the planning of diagnostic experiments and their imaging is a complex
and often interactive IT intensive challenge requiring high-performance. High-
performance grid infrastructures are due to their scalability and variety especially
suited for analyses in the life sciences . E.g. the capability of instant access to
computing resources makes the grid interesting for online planning of complex
experiments. The combination with visualization resources [14, 15] in the grid makes it
highly flexible, to run computational expensive calculations in combination with
demanding visualizations exported to a laptop computer in the laboratory. A prominent
solution for the genomic field from the DNA sequence to the morphologic level is the
grid-based GLOBE 3D Genome Platform : It is a flexible and easy to use software
package, which supports computational aspects of different clinical and molecular
biological methods. Beyond, even several people at different locations could work on
the same process by a distributed team of scientists in a telemedicine approach.
A prominent technique that has been established for standard diagnosis and
treatment monitoring is fluorescence in situ hybridization (FISH). Here, long
fluorescently labelled polynucleotides are hybridized to their complementary DNA
sequence. Thereafter, the targeted loci can be imaged by appropriate microscopic
setups. Thus, e.g. their position can be located quantitatively in their geometric nuclear
environment. For high-resolution experiments with small regions to be targeted we
developed the COMBinatorial Oligonucleotide FISH (COMBO-FISH) method .
Here short oligonucleotides of 15 to 25 bp, which uniquely localize at the given
sequence of interest (in general a part of a gene), are combined. Due to its specificity
COMBO-FISH requires an integrated and interactive analysis and planning to achieve
high quality quantitative results. Therefore, we have integrated a sequence of genome-
wide acting algorithms [16-18] with the grid-based GLOBE 3D Genome Viewer and
Platform : It allows a distributed team of specialists to interactively design and
display different labelling variants of candidate probe sets in their 3D architectural
embedding [1-5] of the respective chromosome region important to estimate
hybridization dynamics. Consequently, we show how a complex interactive process
can profit from grid technology using our unique GLOBE 3D Genome Platform
gateway towards a real interactive curative diagnosis planning and therapy monitoring.
1. COMBinatorial Oligonucleotide Fluorescence in situ Hybridization
In FISH, fluorescent ligand carrying polynucleotide probes are hybridized to their
complementary DNA counterpart. Probes stretch over usually thousands of base pairs
sometimes up to the entire chromosome for complete chromosomal labelling. Usually
the cells or tissues have to undergo a severe and complex preparation, often modifying
nuclear architecture: after chemical fixation of the material, the DNA is denatured so
that the single stranded probe DNA can bind by Watson-Crick pairing. Thereafter, the
so labelled region can be imaged by e.g. 2D or 3D microscopy – due to the region size
in at least one single spot or an entire chromosome part. By subsequent image analysis
(geometrical) information as e.g. gene copy number or general topological relations are
extracted and used for biological or medical applications. To label very small and non-
overlapping targets especially for genomic architecture studies under native or even in
vivo conditions, COMBinatorial Oligonucleotide fluorescence in situ hybridization
(COMBO-FISH) was developed [16-18].
1.1. The general concept of COMBO-FISH
In COMBO-FISH, short oligonucleotide probes of 15-25 bp are used in a combinatorial
set of 20-40 probes – statistically a single probe has many binding sites throughout the
genome – so that they colocalize on the genetic element to be targeted uniquely and
thus produce a microscopically nicely to image spot. I.e. that clusters of more than 3 to
6 oligonucleotides colocalizing within 250 kb should not occur. COMBO-FISH probes
can be designed for arbitrary sequences but are especially suited for oligopurine and
oligopyrimidine probes, which allow binding to the DNA double helix via Hoogsteen
pairing in a triple helical manner without any structure/morphology disturbing DNA
denaturation. Thus, COMBO-FISH can in principle also be applied to vital cells .
Since each oligoprobe carries only one or two fluorescent ligands each colocaliziation
spot is detectable well by fluorescence imaging against the background of minor
binding (single hybridization or small cluster) events and general background
fluorescence which can be easily filtered by appropriate software tools.
1.2. Algorithms for COMBO-FISH
First COMBO-FISH requires that the corresponding genome is completely sequenced
to design oligonucleotides combinations colocalizing at the genetic target of interest
while excluding further clustering elsewhere (conditions of 1.1.). To ease probe design
we either calculate or use a data base of very well binding arbitrary or oligopurine/
oligopyrimidine sequences. Then candidate probes within the genetic region of interest
are selected. On average, 30-100 such probes are found for polypurines on a whole
gene. Thereafter, the location of the candidate probes is rechecked in the entire genome
by an exact search and highly frequent sequences – very often-repetitive ones – are
removed from the candidate set. Finally, the candidate set is iteratively (with automatic
suggestions) reduced until no further clusters of the specified size exist. This usually
results in half of the number of the original probe set. The whole process takes around
15-30 min using the Globe 3D Genome Viewer and Platform and using either external
or grid resources for the calculations. Especially in highly-repetitive genome regions it
seems to be an intriguing genetic feature that such uniquely colocalizing probe sets
exist (e.g. for the SNRPN-SNURF gene region: Figure 2A).
Figure 1: Display of all 24 human chromosomes in the GLOBE 3D Genome Viewer: chromosome banding
(blue tones), centromers (green), and pericentromeric regions (red). At the bottom, the base pair position
(green, left) and an overview (right) of the selected chromosome with the selected area (purple box) is
displayed next to the frames displayed per second (yellow, left) and the magnification ratio (green, right).
Several control panels are displayed, which can be positioned independent of the main viewer frame.
2. The grid-based GLOBE 3D Genome Viewer and Platform
The GLOBE 3D Genome Platform is a grid-based integrative virtual “paper” tool, i.e. a
three-dimensional virtual desktop environment for genome research and exploitation of
genomic information in a service orientated and distributed/teamwork like manner .
It integrates three grid wise distributed resources via one single gateway: i) visual data
representation and graphical user access using the GLOBE 3D Genome Viewer module
of the platform, ii) data access and management via a file and web access system, and
iii) data analysis and creation on local or high-performance grid infrastructures.
The central part of the platform is the GLOBE 3D Genome Viewer module, which
is a unique grid and OpenGL 3D environment serving two main functions: i) display of
genomic data, and ii) being the graphical user interface for data access and
management as well as analysis and creation. It is optimized for the challenges of
research and health care to explore the genomic information in a holistic manner: i) the
representation of the real genome structure/architecture/organization, ii) the
representation of the various genomic experimental data and analysis types, and iii) the
extreme technological means to provide a reasonable viewing system to live up to the
Figure 2: (A) Detailed view of the SNRPN-SNURF gene region: the large horizontal bar (blue) represents
part of the 15q11.2 chromosomal ideogram band, location of the COMBO-FISH probes (orange), different
transcripts of the SNRPN-SNURF gene (yellow). The width of the horizontal yellow boxes is equivalent to
the length in base pairs. (B) DNA sequence of the COMBO-FISH probe 2711 (orange bar) of 20 bp length.
Since the SNRPN-SNURF gene transcripts are much larger only yellow bars are present. The displayed
region is marked by a purple box in the overview (bottom) of the selected chromosome 15.
The GLOBE 3D Genome Viewer consists of a main window for display of the data
(Figure 1-4). Several control panels can be positioned independently of the main
viewer frame. Within the main window three display categories exist: i) the actual data
to be visualized, ii) overview navigators concerning the actual data visualized, and iii)
supporting viewer status information (Figures 1-4). The actual data is obviously located
in the centre of the main frame in contrast to the supporting information, which is
displayed at the bottom or eventually at the other sides of the main window.
Concerning genomic data, every architectural level of one or several genomes (even of
different species) can be visualized simultaneously in a real (Figure 4) and in a
symbolic (mostly linear; Figure 1-3) representation and navigated by continuous scale-
free zooming from the entire cell nucleus or chromosome down to the base pair level.
Other data as e.g. microscopic images can also be displayed (Figure 4). Navigation
supports are e.g. 3D spatial orientation, depiction of a selected chromosome and
marking of a selected genetic region, zoom level, selected base pair position on a
selected chromosome. Status information is e.g. displaying frame rates, memory usage,
CPU load, internet connection status. The selected genetic region is marked in the
symbolic representation by a box (Figure 1-3) and in the real spatial or image data by
high-lighting (Figure 4). Additionally, annotations as e.g. chromosome numbers,
ideogram band numberings, gene names and locations are textually placed next to their
appearance. Genetic elements can be marked by a combination of different shapes,
textures, and colours. They can be correlated by different lines with similar properties
based on external or internal correlations. Consequently, for COMBO-FISH like
projects, sequential, structural or image data can be combined with external data (as
from the ENSEMBL  and UCSC  data bases) and displayed with annotations
and correlations in one single 3D viewing and desktop like working environment.
3. Designing COMBO-FISH Probes for the SNRPN-SNURF Region of the Prader-
Willi/Angelman Syndrome with the GLOBE 3D Genome Viewer and Platform
To illustrate how the design pipeline of COMBO-FISH probes benefits from the
integration into the GLOBE 3D Genome Viewer and Platform – and thus high-
performance calculation and visualization using grid infrastructures – we show now in
detail the process of designing labelling probes for the SNRPN-SNURF gene region,
which plays an important role in the development of the Prader-Willi/Angelman
syndrome . PWS/AS is known for the various body deformations, physiological
changes, and especially also for the aggressive will to get and eat food. Genetically, it
is a prime example for a complex disease where i) DNA mutations and bigger DNA
rearrangements, ii) epigenetic modifications, and iii) structural/architectural changes of
the chromatin organization, individually or in combination play a role. PWS/AS is a
rare disorder (1:10,000-25,000 live birth), but especially the relatively small but
nevertheless even more important structural changes and the interplay with the other
causes remain unknown. Though progress has been made to unravel the dynamic
chromatin architecture on the nano level with sophisticated 3D high-resolution
microscopy [4, 5], the use of conventional FISH 16-600kbp probes pose severe
limitations. Thus, polypurine/polypyrimidine COMBO-FISH probes for different
regions of the SNRPN–SNURF gene are the method of choice for improved resolution
and use of Hoogsteen pairing in a triple helical manner avoiding any structure
disturbing DNA denaturation with the option of in vivo investigations.
The sequence of the SNRPN-SNURF gene has 13 exons according to the NCBI
human genome contig analysis file (based on the Human Genome Project ), which
need to be labelled evenly due to the many a combination of exons (leading to different
mRNA transcriptions) to achieve a proper structural investigation:
/note="synonyms: SMN, SM-D, HCERN3, SNRNP-N"
/product="small nuclear ribonucleoprotein polypeptide N,
transcript variant 4"
Based on this positional information, the COMBO-FISH candidate probe set for
SNRPN-SNURF is either calculated or chosen from the polypurine/polypyrimidine
database. After visual removal of repetitive and other unfavourable sequences this
results in 89 candidate probes (1st #: ordinal number; 2nd #: location within the gene
with reference to the contig base numbering; 3rd #: probe length; sequence):
2704: 4059501(15): agaggaaaaggagag
2705: 4061047(21): aaaggagaagagagaaaaggg
2706: 4061520(16): gaaggagagagaagaa
2708: 4062351(15): agaagagaggagagg
2710: 4064619(18): ggggggaggggagaggga
2711: 4066761(20): agagaaaagggaaaagaaga
2712: 4069722(16): aggaggaagggaaaag
Figure 3: Positions of the SNRPN-SNURF transcripts (yellow boxes) on chromosome 15 (top chromosome)
with COMBO-FISH probes in this region (orange). The >103 bp SNRPN-SNURF region has high
homologies on chromosomes 1, 2, 11, and 22, indicated by the lines to the corresponding sequence.
After locating all occurrences of each candidate sequence in the whole genome, the real
important part of the iterative process of reducing the probe set begins now in the
GLOBE 3D Genome Viewer. Especially in the case of SNRPN-SNURF in respect to its
function in PWS/AS it is important to have a precise visualization of the location of the
(candidate) probes within the gene and syndrome region. Though parts of the process
of probe set reduction is deterministic in so far as some of the probes have to be
eliminated to avoid the build up many a long cluster, there is astonishingly
considerable freedom of choice to eliminate clustering probes, especially towards the
end of the reduction process, which requires a dedicated and e.g. experiment based
individual choice. In that respect, visualizations as in the GLOBE 3D Genome Viewer
are especially well suited to guide this probe eliminating choice (Figure 2A), since one
can easily get an overview on the global level, but nevertheless can move by semantic
zooming fast to the base pair level as well. In particular, sequence reappearances with
high homologies of the SNRPN-SNURF gene and the PWS/AS region in general on
the global level within the same or other chromosomes are of major importance
especially during cluster elimination (Figure 3). E.g. one sees immediately that a 103 bp
long subsequence is duplicated on four other chromosomes. On the base pair level the
binding location and affinity of each single probe can be analyzed in detail (Figure 2B)
and choices can be made based on biochemical/physical considerations of the specific
binding process. Since here the calculation and analysis go hand in hand online, the
calculation performance can only be delivered via grid resources both in respect to
visualization and calculations. After selectively removing probes guided by the above
mentioned considerations, a set of 55 probes is obtained with no clusters with a size
larger than 4 oligonucleotides. The distribution is nicely covering the SNRPN-SNURF
gene region (Figure 2A). It is also clear that different subsets of the probe set can be
used to label different transcript regions or regions neighbouring the SNRPN-SNURF
gene region. With the GLOBE 3D Genome Viewer it is now very easy to find the
sequence of a specific region, e.g. probe number 2711 (Figure 2B).
Beyond, the mere selection of probes, which could be done with much more effort
and not as intuitively also by using (paper) lists or with a 1D or 2D display system, the
GLOBE 3D Genome Viewer and Platform also allows investigation of a DNA sequence
in its spatial context (Figure 4), i.e. not only its spatial position within a structural local
or global embedding can be investigated (since the 3D structure of chromatin plays an
important role in gene regulation [1-5]), but more importantly estimates about probe
diffusion towards the target and chromatin dynamics obstructing binding can be made
just by visually inspecting the genomic architecture. Furthermore, also the relations of
e.g. the reappearing subregions can be reinvestigated in spatial terms and also other
spatial contact information within the same or to other chromosomes can be made.
Either structural predictions resulting from chromatin simulations or real experimental
data can be used and also directly compared. Relations to multi-dimensional
microscopy images (Figure 4) or to contact/interaction frequency maps  can also be
displayed and analysed. Again the enormous amount of data involved here (chromatin
approximation cell nucleus: 106-108 particles; image stack size: 102-103 MByte;
interaction maps: 1012 genome wide interactions) can only be analysed and displayed
using high-performance grid infrastructures and dedicated systems as the GLOBE 3D
Genome Viewer and Platform.
The function of genomes is closely connected to its organization and plays an
important role in modern microscopy for the monitoring of medical diagnosis and
therapy. The planning of corresponding diagnostic experiments and their imaging is a
complex and often interactive IT intensive challenge and thus makes high-performance
grids a necessity. To take full advantage of a recently by us developed innovative form
of cell or tissue labelling – COMBO-FISH – we have designed an interactive pipeline
for the grid-based GLOBE 3D Genome Viewer and Platform to design and display
different labelling variants of candidate probe sets. Thus, we have created a grid-based
virtual “paper” tool for easy interactive calculation, analysis, management, and
representation for COMBO-FISH probe design with many an advantage: Since all the
calculations, analyses and visualizations run in the grid, one can instantly and with
great visual ease locate duplications of gene subsequences to guide the elimination of
side clustering sequences during the probe design process, as well as get at least an
impression of the 3D architectural embedding of the respective chromosome region,
which is of major importance to estimate e.g. the probe dynamics. Beyond, even
several people at different locations could work on the same process in a team wise
manner. Consequently, we show how a complex interactive process can profit from
grid infrastructure technology using our unique GLOBE 3D Genome Platform gateway
towards a real interactive curative diagnosis planning and therapy monitoring.
Figure 4: Different representations of chromosome 15 in the GLOBE 3D Genome Viewer: It is possible to
locate any region of interest as e.g. here SNRPN-SNURF transcripts positions in a linear (bottom), twisted
(right), and even the location within the embedding into the genomic 3D architecture (here a simulated
chromosome [3-5]; left) of eventually the complete cell nucleus, as well as microscopic images (fibroblast
nucleus: DAPI Blue; chromosome 15 territory: Texas Red; SNRPN-SNURF region: Oregon Green).
Our special thanks go to C.-C. Seegler-Sandbanck, European Centre of Feasibility
Studies (ECFS), Strasburg, for stimulating and encouraging discussions. We would like
to thank Erasmus Medical Center, the BioQuant Centre/German Cancer Research
Centre, the German MediGrid and German D-Grid Initiative, the Erasmus Computing
Grid, and the Supercomputing Centre Karlsruhe (SCC; grant ChromDyn) for access to
their general and computing infrastructure. This work was partly supported by the
Bundesministerium für Bildung und Forschung (BMBF) under grants # 01 KW 9602/2
(Heidelberg 3D Human Genome Study Group, German Human Genome Project), # 01
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