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Overview of array-based Copy Number
analysis for Cytogenetics
Topics in Copy Number variation analysis
October 2009
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Contents
Overview ................................................................................................................................................................................. 2
Tools ........................................................................................................................................................................................ 2
An array-­‐based Cytogenetic Test ............................................................................................................................................ 2
Using parental samples ........................................................................................................................................................... 4
Utilizing accumulated patient data ......................................................................................................................................... 4
Gene significance .................................................................................................................................................................... 6
Conclusion ............................................................................................................................................................................... 8
Overview
Array CGH technique is going through a transition from being purely a research tool to being used in clinical Cytogenetic
tests on a daily basis. Such transition calls for a new generation of analytical solutions capable of semi-­‐automated and
reliable copy number reporting.
Tools
oneClickCGH® is a unique software package that facilitates automated analysis of array CGH data and user-­‐friendly copy
number reporting for individual samples. Cytogenetic labs can use this package to support identification of clinically
relevant copy number changes and to build robust and comprehensive reports. It is capable of analyzing medium-­‐ and
high-­‐resolution data from various array CGH and SNP platforms. Statistical algorithms used in this software ensure
reliability of aberration detection, which assists Cytogenetic users in producing their final report documents.
CGH Fusion® is a leading commercially validated package used for analysis of array CGH data across multiple samples. It
is capable of building copy number profiles for large sets of samples within a specific biological context. CGH Fusion can
facilitate constriction of libraries of copy number profiles characterizing specific disorders or cohorts of healthy patients
in a simple user-­‐friendly manner. It can also provide valuable biological insights into regions of frequent copy number
change by incorporating various gene annotations and known CNVs.
An array-based Cytogenetic Test
Usually analysis of test data extracted with array CGH or SNP array starts with detection of suspected regions of copy
number changes. Several basic operations are performed on raw data (depending on the actual array platform used):
background subtraction, normalization, filtering of outlier replicates and actual detection of regions of abnormal log-­‐
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ratio values. All aforementioned operations can be performed automatically and almost no manual intervention is
needed at that stage.
However, not all automatically detected chromosomal aberration regions need to be reported as relevant to the test.
Some of them can be simply experimental defects and some come from regions of known genetic variance, which do not
contribute to the clinical condition under study.
Therefore a manual procedure of aberration approval and reporting is needed. Such procedure usually is centered
around building a final report with manually approved copy number changes based on references to various knowledge
databases (public or internal). Such aberration reporting procedure can be streamlined with oneClickCGH, which allows
creation of comprehensive report documents in various formats and facilitates references to publicly available
databases such as the Data Base of Genomic Variants, UCSC Genome Browser, OMIM, Gene Ontology database etc.
First, all detected copy number changes need to be reviewed and gene and/or known CNV annotations can be used to
After all detected aberrations have been approved and necessary comments have been added, report section of
oneClickCGH contains only relevant regions of copy number change that can be saved as a report document
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Using parental samples
ones.
ical. However,
when such data are available, CGH Fusion can provide a user-­‐friendly environment for identification and reporting
User can simply load all available samples into CGH Fusion, analyze them simultaneously and review them side by side.
Plot below illustrates this operation.
It is easy to see from this plot that two out of three mother s) demonstrate similar aberrations,
which also coincide with a region of known CNVs from Toronto DGV highlighted with orange at the top of the plot.
a known CNV inherited from one of
parent genomes.
Utilizing accumulated patient data
Ultimately, every diagnostician would like to be able to understand clinical relevance of a chromosomal anomaly under
scope by comparing frequency of its occurrence in patients with a particular disorder vs. healthy patients. This can be
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done if array CGH data for large cohorts of patients have been accumulated by the lab. If sufficient number of patients
with the disorder were tested, such data can be used to build an aberration frequency profile for the disease. On the
other hand, you can use data from healthy patients to assess frequency of normal CNV occurrence, which may explain
some of the anomalies initially thought to be related to the disorder under study.
CGH Fusion can help users build a comprehensive and informative aberration frequency profile for every patient group
that lab has accumulated array Copy Number data for. Publicly available datasets can also be used for such purpose.
You can easily load hundreds or thousands of samples into CGH Fusion and perform batch copy number identification on
them. Output of that procedure can then be used to build an aberration frequency profile illustrated below.
Such profile clearly indicates regions of frequent copy number anomalies for the patient cohort under study and
provides a quantitative measure of aberration frequency in the cohort.
By re-­‐using profiles constructed with CGH Fusion in oneClickCGH during a routine Cytogenetic test, one can
quantitatively estimate probability of an aberration region contributing to a particular disorder.
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Illustration above demonstrates how a gain and a loss detected in a sample (upper plot) coincide with regions of high
CNV frequency highlighted by green and red peaks of aberration frequency profile for healthy individuals (lower plot).
Aberration frequency profile was constructed using data from HapMap samples extracted using Affymetrix SNP 6.0 chip.
Gene significance
Finally, gene annotations for a chromosomal region of interest and any biological information associated with those are
probably the most informative pieces of the puzzle when it comes to interpreting detected aberrations. Of course, one
can always manually review sets of genes from suspect regions of copy number change and skim through various
publications and biological information associated with those genes. This procedure can be semi-­‐automated with
oneClickCGH or CGH Fusion, where gene annotations are conveniently located alongside log-­‐ratio plot.
Picture below demonstrates how users can review genes within a region of interest and identify conditions genes under
scope have been associated with in available publications. This example illustrates how Angelman syndrome can be
added to the list of conditions a copy number loss on chromosome 15 may be related to.
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This procedure can be further automated by short-­‐listing genes affected by copy number changes along genome for
current sample. By a click of a button users can display a table listing affected genes and corresponding significance p-­‐
values ordered from most statistically significant to least statistically significant. The table also lists conditions cited in
association with the genes and corresponding Ontology terms. Below you can see how such list also highlights gene
UBE3A identified in the previous paragraph.
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Conclusion
oneClickCGH in combination with CGH Fusion provide a powerful suite of DNA Copy Number analysis tools for array
users. These software products provide the new level of in-­‐depth array data interpretation and comprehensive Copy
Number reporting that can be beneficial for an individual user and for the laboratory workflow as a whole. It is a truly
universal software suite that is capable of handling array data of any resolution obtained with any hardware platform:
Affymetrix, Agilent, Illumina, PerkinElmer, Roche-­‐Nimblegen and more.
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