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- Literature Cited
Array comparative genomic hybridization (aCGH) is a method for comparing copy number of genomic loci between test and reference samples. Many types of genomic copy number variation cause recognizable syndromes ranging from imbalance of entire chromosomes (aneuploidy), as in Down syndrome, to submicroscopic imbalance, as in 22q11.2 deletion causing DiGeorge/velocardiofacial syndrome (VCFS). Detection of genomic copy number variation is now considered the standard of care in the evaluation of children with unexplained developmental delay, intellectual disability, autism spectrum disorders, or congenital anomalies (Manning and Hudgins, 2010; Miller et al., 2010; Shen et al., 2010). Submicroscopic genomic imbalance is a frequent cause of idiopathic intellectual disability, with microscopically visible abnormalities accounting for at least 3% to 4% of cases (Shevell et al., 2003), and many of these cases are recognizable syndromes such as Down syndrome. In cases with a normal karyotype, the addition of subtelomeric (ST) FISH testing achieves a diagnosis in another ∼2.4% of cases (Ravnan et al., 2006). Traditional cytogenetic methods are labor intensive, especially when multiple genomic regions are interrogated by ST-FISH, and cannot approach the coverage and yield of aCGH. Studies based on earlier BAC aCGH and higher density oligo aCGH suggest that the yield is generally in the range of 10% to 15% (Stankiewicz and Beaudet, 2007; Baldwin et al., 2008; Sagoo et al., 2009, Miller et al., 2010).
aCGH provides a way to interrogate many sites in the genome in a single experiment on a standard 1 × 3–in. glass slide. In clinical use, the widest application has been in the diagnostic evaluation of children with developmental delay/intellectual disability, dysmorphic features, multiple congenital anomalies, and autism (Vissers et al., 2003; Shaw-Smith et al., 2004; Jacquemont et al., 2006; Sebat et al., 2007; Shaikh, 2007; Weiss et al., 2008; Shen et al., 2010). Regardless of content, most aCGH platforms rely on the general principles of (1) fluorescent labeling of sample and reference DNA, (2) hybridization of labeled DNA to the array, (3) scanning to detect fluorescence intensity at all sites of hybridization, and (4) data analysis. This protocol is specific to Agilent aCGH using a two-color system to compare sample and reference DNA. Several other commercially printed oligonucleotide arrays are available and can be used with proper optimization of conditions.
Two types of oligonucleotide probe designs have been typical for aCGH. Agilent and NimbleGen platforms use long (50 to 70 mer) oligonucleotide CGH arrays and focus on high-quality copy number detection. Affymetrix and Illumina genotyping arrays use short (25 mer) oligonucleotide probes for genome-wide SNP genotyping, as well as copy number detection (Shen and Wu, 2009). The former use a two-color system to label DNA prior to hybridization, while the latter use a one-color system and infer copy number by comparing to a reference dataset. Long-oligo and short-oligo platforms showed functional convergence in recent years, as exemplified by the introduction of Affymetrix's hybrid array (Affy 6.0 array with both SNP and CNV probes, and the more recent Cytoscan HD) and Agilent's CGH+SNP array. Now both types of array platforms can detect copy number variation, as well as copy neutral zygosity status for any chromosome segment (a.k.a. LCSH: long contiguous stretches of homozygosity). This facilitates clinical applications beyond constitutional copy number analysis of patients with developmental disabilities, including analysis of loss of heterozygosity (LOH) in tumors and constitutive absence of heterozygosity (AOH) that occurs with consanguinity or uniparental disomy (UPD).
The first step in aCGH is labeling of genomic DNA from test and reference samples with different color fluorophores (e.g., Cy5 and Cy3). Labeled DNA from test and reference samples is combined in an equimolar ratio and co-hybridized to an array of DNA probes corresponding to informative regions of the genome. During hybridization, sequences from the test and reference DNA compete for probe binding. All probes are validated empirically to hybridize under the same conditions so that any differences in signal intensity are reflective of differences between test and reference DNA copy number. After washing to eliminate nonspecific hybridization, the relative intensity of each fluorophore at a particular probe coordinate is read by a laser scanner. Software applications for imaging (Feature Extraction) and informatics (CGH Analytics, now part of CytoGenomics) quantitate the color ratio (Cy5/Cy3) for each probe coordinate. This ratio reflects the relative copy number of the corresponding genomic DNA between test and reference samples. Each array feature is mapped to the human genome reference sequence and the results are indicated in a graphical display.
The new Agilent CGH+SNP array uses a similar protocol to earlier Agilent arrays, with the additional step of DNA sample digestion with restriction enzymes, which allows genotyping of SNPs located at the enzymes' recognition sites. The procedure is outlined in Figure 8.12.1. For genotyping arrays, such as Affymetrix or Illumina, only a single test DNA is labeled and hybridized to the array. The genotypes are determined by evaluating the signal intensity between alternative probes. The copy number variations are inferred from comparing the signal intensity to signal of previous run controls (McCarroll et al., 2008), but detailed workflows for these arrays are not covered in this unit. The Agilent CGH+SNP arrays use the same Agilent CGH workflow as a CGH-only array. The copy number status is determined by the log2 ratios of long oligonucleotide CGH probes between sample and reference DNAs. In addition, a set of SNP probes (a total of 60 K SNP probes on Agilent 4X180K CGH+SNP catalog array) is selected for genotyping purposes. Probes are designed to include restriction digestion sites (AluI/RsaI) that overlap with known SNP sites. Both sample and reference DNAs are digested by the restriction enzymes before labeling. If both alleles in the target DNA are cut (0 uncut alleles), the resulting signal of the SNP probe will be low. If both alleles are uncut, the signals of the SNP probe will be high. For a heterozygous SNP locus, there will be one cut and one uncut allele, and the resulting signal intensity of the SNP probe will be intermediate.
Figure 8.12.1. Basic steps of oligo array CGH with timeline.
The copy number of one allele at each SNP site is determined by measuring the relative signal intensity between the sample and a reference. The zygosity status is determined by the number of uncut alleles: 0 uncut (AA); 1 uncut (AB); 2 uncut (BB). Regions of homozygosity are located by finding genomic regions with a statistically significant scarcity of heterozygous calls. Since the genotype of the reference is known, the SNP calls can be made for each individual SNP locus.
In a clinical diagnostic setting, data reliability can be ensured through several quality control steps. There are several data quality parameters, such as DLR spread (discussed below) that help determine overall data quality. Even if overall data quality is good, characteristics of each aberration must be considered. For example, clinical guidelines require that a certain number of successive probes show a consistent signal in order to make a call, such as five probes for a deletion and ten for a duplication. These requirements are generally more stringent for gains versus losses because the probe ratios are closer to the reference for a single copy gain as compared to a single copy loss (Kearney et al., 2011a). As a result, aCGH resolution depends on feature density. Whole genome high-resolution arrays are capable of detecting very small aberrations throughout the genome (e.g., <10 kb), but current clinical guidelines suggest that detection of all copy number gains and losses >400 kb is sufficient (Miller et al., 2010; Kearney et al., 2011a). Because the whole genome is not evenly covered by the oligo probes, the actual detectability depends on the location of the imbalance events in the genome and the performance of the hybridization (i.e., the quality of the dataset). In some cases, a second hybridization with dye reversal to eliminate false-positive results from differences in fluorophore intensity can be considered.
Interpreting aCGH data for clinical diagnosis is challenging because copy number variants (CNVs) are now recognized as an extremely common type of genetic variant. Some behave in a Mendelian fashion, while others may contribute to complex traits or have no effect at all. The ability to distinguish pathogenic from benign copy number variation is a challenge for any clinical laboratory, and key factors are discussed in Background Information. The reader is referred to recent expert reviews and published guidelines addressing issues related to clinical aCGH interpretation (Lee et al., 2007; Vermeesch et al., 2007; Kearney et al., 2011b).
100 to 150 ng/µl genomic DNA in TE buffer:
Sample (test) DNA (isolated from blood, see unit 14.4)
Control (reference) DNA (e.g., normal male or female):
NA12878 (European Female)
10× reaction buffer C (provided with Rsa I; Promega)
QIAprep Spin Miniprep Kit (Qiagen; cat. nos. 27104, 27106) with Buffers PB, PE, and EB
Ethanol (denatured; Fisher)
BioPrime Array CGH Genomic Labeling System (Invitrogen; cat. no. 18095011), with:
2.5× Random primers solution (octamers)
40 U/µl exo-Klenow fragment
10× dUTP (nucleotide mix; PerkinElmer)
2′-Deoxyuridine conjugated with cyanine 3 and cyanine 5 (Cy3-dUTP and Cy5-dUTP; PerkinElmer)
1× TE buffer, pH 8.0 (Promega)
Oligonucleotide aCGH Hybridization kit, large volume (Agilent, 5188-5380), containing:
Oligonucleotide aCGH wash buffers 1 and 2 (Agilent)
Spectrophotometer (e.g., NanoDrop ND-1000, Thermo Fisher Scientific)
Microcon YM-30 Centrifugal Filter Kit (Millipore; cat. no. 42410)
Oligonucleotide microarray, e.g., Human Genome 244 k array (Agilent Technologies, G4411B)
Hybridization Oven (Agilent Technologies)
Ozone detector (e.g., Teledyne Model 400E), optional
Ozone converter (e.g., Ozone Solutions “Ozone Interceptor”), optional
Array scanner (e.g., Agilent G2565AA or 2565 BA)
Computer workstation (e.g., Dell Optiplex GX745)
Feature Extraction software (included with purchase of array scanner)
CGH Analytics software (Agilent)
Digest DNA samples
Obtain test and reference DNA samples and check purity on a NanoDrop ND-1000 UV-VIS spectrophotometer (appendix 3D).
All aCGH platforms are sensitive to the quality and quantity of prepared DNA. For clinical samples, DNA extracted from peripheral blood will yield the highest and most reproducible quality of DNA. Buccal swab samples are not as reliable for this application and should be discouraged.
Prepare a master mix (50 µl/reaction) for DNA digestion. Combine reagents in the order indicated:
30 µl nuclease-free water
10 µl 10× reaction buffer C (provided with Rsa I)
Prepare separate tubes containing 3 µg test DNA and 3 µg reference DNA in 50 µl TE buffer. Add 50 µl digestion master mix (final 100 µl/reaction), mix thoroughly, and incubate for 2 hr at 37°C in a thermal cycler. Place the tubes on ice following digestion.
Clean up digested DNA
Add 500 µl of 5× Buffer PB to each digested 100 µl sample.
Buffers listed in this section refer to manufacturer's protocol.
Transfer to a QIAprep Spin Miniprep column and centrifuge 60 sec at 17,900 × g, room temperature. Discard flowthrough.
Add 750 µl Buffer PE (with ethanol added; see QIAprep Miniprep Handbook). Centrifuge 60 sec at 17,900 × g, room temperature, and discard flowthrough.
Repeat spin for 60 sec at the same speed and temperature and transfer the column into a clean 1.5-ml tube.
Elute DNA with 50 µl buffer EB, let sit 60 sec, and then centrifuge 60 sec at 17,900 × g, room temperature.
Concentrate the DNA to a volume of <21 µl using a Speedvac Concentrator.
Store the samples at –20°C or continue with labeling.
Avoid drying the sample completely during concentration. Samples can be stored for several weeks at –20°C prior to labeling.
Label genomic DNA
Dispense 20 µl of 2.5× random primers solution into the 21 µl DNA sample.
Perform this and all subsequent work in a room without an outside window or cover any outside windows. Standard-intensity fluorescent room lighting will decay the fluorophore signal within several seconds. Samples are most vulnerable when not covered (e.g., while pipetting hybridization solution onto microarray slides). These steps should be performed as quickly as possible. Samples should not be susceptible to photobleaching while in the covered water bath or in the hybridization oven.
Denature at 95°C in a heat block for 5 min and then set on ice for 5 min.
Prepare the exo-Klenow master mix in the order shown (9 µl/reaction):
3 µl Cy3-dUTP (for reference sample) or Cy5-dUTP (for test sample)
1 µl exo-Klenow fragment.
The exo-Klenow fragment of DNA polymerase I is a mutant of the large fragment of the DNA polymerase I holoenzyme that has both 5′-to-3′ and 3′-to-5′ exonuclease activity removed. Random octamers provide priming sites for the exo-Klenow enzyme. Fluorescently modified nucleotides are incorporated as the polymerase extends from the priming sites.
Add 9 µl mix to the DNA samples and incubate at 37°C in a water bath with a stainless steel cover for 2 hr.
Add 5 µl stop buffer. Store reactions at –20°C or continue with clean up.
The labeled samples must be purified to remove contaminants prior to denaturing and hybridization to a microarray.
Although labeled samples can be stored for several days at –20°C, proceed to the next step as soon as possible to achieve the highest signal strength during scanning. In typical laboratories, labeled samples would be stored overnight in the dark at –20°C.
Clean up labeled DNA
Combine Cy5 and Cy3 samples (control and test) for a total of 110 µl.
Add 400 µl of 1× TE, pH 8.0, and transfer to a MicroCon YM-30 filter in a 1.5-ml collection tube.
Centrifuge 7 min at 7000 × g, room temperature, and discard flowthrough.
Add 480 µl of TE, centrifuge again (use same conditions from step 18), and discard flowthrough.
Invert the filter into a fresh 1.5-ml tube and centrifuge 1 min at 700 × g, room temperature.
Bring the total volume of the sample to 70 µl with 1× TE, pH 8.0.
Store the sample at –20°C or continue with hybridization.
Again, although labeled samples can be stored for several days at –20°C, proceed to the next step as soon as possible to achieve the highest signal strength during scanning.
Pay attention to the size and color of the pellet. The pellet should have a purple hue, which indicates balanced labeling between Cy5 and Cy3. An off-color pellet (too blue or too red) may suggest labeling failure.
Add 1250 µl water to lyophilized Blocking Agent to make a 10× solution, and let sit at room temperature for 60 min to reconstitute.
The 10× Blocking Agent can be prepared in advance and stored at –20°C.
Add the following components to each tube containing 70 µl purified labeled sample (total 300 µl/reaction):
30 µl Agilent 10× blocking Agent
150 µl Agilent 2× hybridization buffer.
Transfer the tubes to 95°C for 3 min for denaturation.
Transfer the tubes to 37°C for 30 min for prehybridization.
Microcentrifuge the tubes for 5 min at top speed, room temperature, to pellet any precipitates.
CAREFULLY collect the supernatant without disturbing the bottom of the tube and apply the supernatant onto the gasket of the microarray. Assemble the gasket.
Make sure the active side of the chip (labeled “Agilent”) is used for hybridization. The numeric barcode is on the inactive side. The hybridization mixture should be applied directly to the gasket slide and not to the active side of the array slide. Tighten the gasket without allowing any liquid to leak out.
After assembling the gasket, rotate the unit to make sure all areas are covered by the hybridization solution and all bubbles move freely. It is acceptable to have some bubbles in the hybridization, provided that the solution covers the entire surface area of the printed array. The array must not be allowed to dry out during hybridization or subsequent washes.
Hybridize at 65°C for 40 hr at rotation setting 20 in an Agilent hybridization oven.
Samples at this stage can be stored for several days at –20°C, but proceeding to the next step as soon as possible will achieve the highest signal strength during scanning. Any remaining sample stored in this manner could be used for a repeat experiment if problems were encountered during scanning. However, samples showing low signal intensity during scanning would require repeat labeling.
Wash the samples
Disassemble the gasket in wash buffer 1.
Wash the array 5 min in wash buffer 1 in a Coplin jar with stirring.
Wash the array 1 min in prewarmed wash buffer 2 at 37°C in a Coplin jar with stirring.
Slowly take the slide out of wash buffer 2 and immediately scan the slide.
Limit light exposure to the slide during and after washing and proceed to scanning as soon as possible after washing to avoid dye decay.
After washing, the slides are very sensitive to photobleaching from ozone exposure. Ozone levels are higher in the daytime and during hot weather. Try to avoid prolonged exposure of the slides (<1 min) to room air when ozone concentration is >10 ppb. Washing the slides in batches of four slides helps to avoid delays between the washing and scanning steps.
The standard protocol involves acetonitrile washes in a vented hood. Acetonitrile creates a physical barrier over the slide. The need for a vented hood can be circumvented by monitoring ozone levels and using an ozone converter.
Scan array and perform feature extraction
Perform scanning according to the manufacturer's protocol with the following settings:
Save the image in the appropriate folder as raw data.
Perform feature extraction according to manufacturer's instructions, using TIFF image files.
Exact procedures for feature extraction vary according to the manufacturer's software. Feature extraction requires comparison of data points on the assay to a design file containing coordinates for all features (probes) on the array. Processing of this data will follow a standard protocol designed by the array manufacturer. Perform manual gridding if necessary, and repeat feature extraction using the corrected grid file. Review the QC report generated by the feature extraction software. Using the derivative log ratio spread (dLRsd) as the main quality index, a dLRsd measure of <0.25 can be considered acceptable.
Analyze the data
Import the FE data file (.txt) to CGH analytics for data visualization and aberration detection.
We use Aberration Detection Method algorithms (ADM-2) as the statistical method for aberration detection and use an aberration filter to identify aberrations covered by more than a predetermined number of probes.
Visually inspect any detected potential imbalance loci. Report the size and location of the imbalanced region.
Determine if the imbalance events are true for both forward (Cy5 for test sample, Cy3 for reference) and reverse (Cy3 for test sample, Cy5 for reference) labeling.
Determine if the imbalance is a reported copy number variant by comparison to publicly available databases and the internal laboratory database.
Determine if the imbalance event is associated with any reported genetic disorder.
Depending on the results, consider FISH or other forms of confirmatory testing.
Recommendations may include parental testing to determine if the identified variant is a de novo imbalance event. Subsequent testing may include standard cytogenetics to look for a balance rearrangement in a parent.
Review and amend the original report when results of additional testing become available.