Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a diagnostic service laboratory.
Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a clinical diagnostic service laboratory. DNA extracted from a patient’s sample (blood, saliva or other tissue types) is labeled with a fluorochrome (either cyanine 5 or cyanine 3). A reference DNA sample is labeled with the opposite fluorochrome. There follows a cleanup step to remove unincorporated nucleotides before the labeled DNAs are mixed and resuspended in a hybridization buffer and applied to an array comprising ~60,000 oligonucleotide probes from loci across the genome, with high probe density in clinically important areas. Following hybridization, the arrays are washed, then scanned and the resulting images are analyzed to measure the red and green fluorescence for each probe. Software is used to assess the quality of each probe measurement, calculate the ratio of red to green fluorescence and detect potential copy number variants.
It has been known for many years that deletions or extra copies of chromosomal segments cause intellectual disability, dysmorphism and congentical malformations, and in some cases cause genetic syndromes1. However, the only technology available until the mid-2000s for the genome-wide detection of these changes was G-banded chromosome analysis, which has a resolution of around 5-10Mb, depending on the region and nature of the imbalance, and which cannot detect abnormal chromosomes where material has been replaced by a region from a different chromosome with the same banding pattern. Ancillary cytogenetic techniques such as fluorescence in situ hybridization and multiplex ligation-specific probe amplification have been available for the interrogation of specific loci, in cases of suspected specific syndromic imbalance, but it was not until the introduction of array comparative genomic hybridization (array CGH) into routine clinical diagnostic service2-5 that genome-wide detection of copy number variants (CNVs) became possible at a greatly increased resolution (typically around 120kb). Clinical service work alongside research studies have shown that CNVs for some regions are widespread in the normal population6-7, whilst other CNVs, previously undetectable, are associated with neurodisabilities such as autism and epilepsy8-11.
The protocol described in this paper is used in our UK National Health Service (NHS) clinical diagnostic laboratory; we use novel hybridization strategies, batch testing and robotics to minimize cost in this state-funded service.
Prior to the protocol detailed below, high quality DNA should be extracted from the appropriate starting material, commonly blood, cultured cells or tissue samples. Spectrophotometry can be used to measure concentration (should be >50 ng/ul) and check 260:280 absorbance ratios (should be 1.8-2.0). Gel electrophoresis can be used to check that the DNA is of high molecular weight without significant degradation.
This protocol is designed for higher throughput laboratories that are labeling 96 samples per run using automated liquid handling robotics. However, it may be adapted for lower throughput labs without automation.
1. Labelling Reaction
2. Removal of Unincorporated Nucleotides
3. Combine Hyb Pairs
4. Hybridization
5. Washing and Scanning Slides
Each probe on a hybridized array is visualized as a mixture of red and green fluorochromes (see Figure 1). The ratio of red to green fluorescent signal for each probe is quantified by the scanner and the associated software plots these as log2 ratios according to their genomic position, and identifies regions falling outside preset boundaries. The resulting array traces allow interpretation of regions identified as genomically unbalanced. For instance, the trace from a child with Williams syndrome, a recurring microdeletion syndrome mediated by low copy repeats in the proximal region of chromosome 7, is illustrated in Figure 2. This imbalance was identified by the software and indicated by a red line.
Probe fluorescent log ratios should cluster closely around 0, as shown in Figure 2, indicating a green/red ratio of 1:1 for normal regions of the genome. Scattered array traces may result in inaccurate calling of abnormal regions, or failure to identify genomic imbalance (see Figure 3). Such scatter may be caused by a number of factors, including poor DNA quality, or the presence of raised levels of ozone in the atmosphere. Monitoring of ozone levels is recommended, and where elevated ozone levels are problematic, dedicated ozone exclusion cabinets are available; use of these cabinets generally results in markedly improved array quality.
Figure 1. Image of an array following hybridization. The white box shows a magnified area, where red probes and green probes can be visualized (indicating imbalance for the regions represented by these probes), against a background of yellow probes (indicating balanced genomic regions).
Figure 2. (A) Example trace for chromosome 7 in a sample with a 1.7Mb deletion, shown by an average ratio of -0.8 for 43 consecutive probes. The deleted region in this case is associated with Williams-Beuren syndrome, consistent with the referral indication of dysmorphic features, a heart defect and intellectual disability. (B) The deleted region above displayed in the USCS genome browser, showing the genes within the deleted region. Please click here to view a larger version of this figure.
Figure 3. Example of a scattered array trace for chromosome 7 in a sample with poor hybridization. Please click here to view a larger version of this figure.
Reagent | Volume (µl) |
Primers & reaction buffer | 20 |
DNA (1µg) + water | 20 |
Klenow Exonuclease DNA polymerase | 10 |
Table 1. Summary of the reagents used for the labeling reaction.
Array CGH will not detect balanced rearrangements or ploidy abnormalities such as triploidy. Furthermore, low level mosaic imbalances may not be detected. However, array CGH has a higher resolution for CNV detection than G-banded chromosome analysis which it has replaced in many cytogenetics laboratories. It is therefore the current gold standard for genome-wide CNV detection. It may be replaced by next-generation sequencing technologies in the future but currently, their cost and the technical complexities associated with using short reads for CNV detection mean that this is not yet a good fit for clinical use.
The protocol described here is in routine use in our clinical service laboratory. Two runs of 96 samples each are processed each week using a liquid handling robot and a dedicated silica membrane spin column processing robot. Automation is highly recommended to improve consistency and maintain quality. DNA extracted from blood, saliva, prenatal samples (e.g., chorionic villi or amniotic fluid) or tissue samples can be, and routinely is, used with this protocol.
Ozone is known to degrade cyanine dyes and therefore ozone monitoring and protection is recommended. Seasonal variation in ozone levels is also common. Our laboratory monitors and records ozone levels continuously. A wall-mounted ozone removal unit is used to reduce ozone levels and particularly sensitive parts of the protocol (i.e., washing and scanning arrays) are performed in ozone-free hoods. If possible, ozone levels are kept below 5 ppb.
Most reagents are bought as kits from manufacturers to effectively outsource quality control; this has proved to be an effective strategy. However, this does not mean that quality control is not required. Indeed, quality metrics are carefully monitored for any anomalies: derivative standard deviation of log2 ratios (DLRS) should be below 0.2, cyanine 3 and 5-signal intensities should be greater than 500 and cyanine 3 and 5-signal to noise ratios should be >30. These metrics are generated as part of the scanning protocol by the scanning software and are recorded for long term monitoring.
The authors have nothing to disclose.
The authors have no acknowledgements.
Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
CGH microarray 8 x 60K | Agilent | G4126 | |
Array CGH wash buffers | Agilent | 5188-5226 | |
Array CGH hybridisation buffer | Agilent | 5188-5380 | |
Minelute purification kit | Qiagen | 28006 | |
Array CGH labelling kit | Enzo | ENZ-42672-0000 |