Overview Of Array Based Copy Number

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Overview of array-based Copy Number analysis for Cytogenetics

Topics in Copy Number variation analysis

October 2009


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-‐


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


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


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.


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.


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.


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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Overview Of Array Based Copy Number

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