Copy Number and Cytogenetics Support—Getting Started

CytoScan™ Cytogenetics Suite consists of CytoScan™ arrays and CytoScan™ Reagent Kit, GeneChip™ Command Console™ Software (AGCC), and Chromosome Analysis Suite (ChAS). It enables the performance of high-resolution, genome-wide DNA copy number analysis and also provides genotyping information for the detection of copy neutral loss/absence of heterozygosity (LOH/AOH), which can be used to detect uniparental isodisomy (UPD). The combination of high-resolution DNA copy number data and the ability to detect gains, losses, and UPDs on a single array makes CytoScan™ Cytogenetics Suite ideal for cytogenetics studies. 

It is recommended to run no more than 24 samples at a time. The assay is a four-day protocol; however, the technician can use an optional three-day protocol after becoming comfortable with the assay. 

The Clontech Titanium™ Taq PCR Kit (300 reactions, Cat. No. 639240 or 400 reactions, Cat. No. 639243) and absolute ethanol are also required. All other reagents are included in CytoScan™ Reagent Kit.

After any stage in the assay (including fragmentation) you can store the samples at -20˚C if you are not proceeding directly to the next step. However, once you have initiated a stage, you must complete it before storage of the samples.

The DNA is digested with Nsp I restriction endonuclease and ligated to adaptors that recognize the cohesive four base pair overhangs. All fragments resulting from restriction enzyme digestion, regardless of size, are substrates for adaptor ligation.

Using two separate rooms greatly reduces the risk of samples being contaminated by previously amplified PCR products. If only one room is available, designate one area of the room as the pre-PCR clean area and a separate area as the post-PCR clean area. If you are using a one-room configuration, we highly recommend using a laminar flow cabinet for the pre-PCR clean area. See the CytoScan™ Assay User Manual for more details about the recommended laboratory setup.

The minimum yield recommended is 2.5 µg/µL. Yields can range from 3.0-4.5 µg/µL  and the average yield for seven or more samples processed in a run (not including the negative control) should be greater than 3.0 µg/µL. If the average yield is below this, consult the troubleshooting section of the CytoScan™ Assay User Guide (Cat. No. 901808).

MAPD: Median of the Absolute values of all Pairwise Differences is a per-microarray estimate of variability, like standard deviation (SD) or interquartile range (IQR). It measures the variability in log2 ratios by looking at the pair difference of all probes and taking a median value. The effect of an occasional big difference in log2 ratios between probes is removed by taking a median value and not a mean. This variability can come from different sources:
Intrinsic variability in the starting material, hybridization cocktail preparation, microarray, or scanner.
Apparent variability induced by the fact that the reference may have systematic differences from the sample on this microarray.
Regardless of the source of variability, increased variability decreases the quality of the CN calls. A high MAPD can be attributed to any of the above factors and indicates that CN calls may be inaccurate, leading to a higher false positive/negative rate.

SNPQC: This is a measure of how well genotype alleles are resolved in the microarray data. In other words, it estimates the distributions of homozygous AA, heterozygous AB, and homozygous BB alleles and calculates the distance between them. The better the separation of these distributions, the better the ability to identify a genotype based on its cluster position. The larger the difference between the peaks and the troughs, the better the resolution of homozygotes and heterozygotes and the higher the SNPQC metric is. If the three peaks are not well resolved, the difference between peaks and troughs will be low, resulting in a lower SNPQC value. A low SNPQC value indicates that quality of the SNP allele data is compromised, due to higher noise within the array, which compromises the overall quality and clarity of results.

Waviness-SD is a global measure of variation of microarray probes that is insensitive to short-range variation and focuses on long-range variation. Based on an empirical testing dataset, array data with Waviness-SD >0.12 has either sample or processing batch effects that will reduce the quality of the copy number calls. Elevated Waviness-SD is not always an indication of too much noise. Elevated Waviness with good MAPD and SNPQC metrics can occur in samples with many copy number changes or very large regions of change. It is therefore advised to check the data when observing elevated Waviness with good MAPD and SNPQC. The Waviness-SD metric is applicable to blood and cell line data. The Waviness-SD metric is not intended for alternative sample types such as solid tumor or FFPE samples in which the results may vary as a result of the biological complexity. For these sample types, it is recommended to use the ndwavinessSD.

The CytoScan™ arrays use the following QC metrics:
SNPQC ≥15
MAPD ≤0.25
Waviness-SD ≤0.12

These QC metrics have been fine-tuned for blood-derived constitutional samples. The SNPQC and Waviness-SD metrics are based on an assumption of a relatively normal diploid genome for which the majority of the genome is not mosaic. For hematological malignancy samples, the baseline assumption pertaining to constitutional samples does not meet the normal aberration frequency pattern and high levels of mosaicism, which will likely trigger the SNPQC and Waviness-SD metrics to fail. Only the MAPD metric should be considered for non-constitutional samples. A failure of any one of these metrics for constitutional samples is a failure for that array result. There is no direct correlation between the absolute passing numeric value for any one of the metrics and the quality of a sample. For example, a sample that has an SNPQC=30 is not necessarily a better quality sample than one that has an SNPQC=20.

Yes, genotypes can be generated in ChAS. The software uses BRLMM for genotyping, which uses a Bayesian model based on prior clusters. This product is not intended for genome-wide association studies

AA markers would be distributed about +1.
AB markers would be distributed about 0.
BB markers would be distributed about –1.

The Reference Model file in the CytoScan™ HD Array and CytoScan™ 750K Array set includes 380 microarrays, which were run as part of a larger set of microarrays by nine operators. These operators processed ~48 unique samples in two rounds each, with random placement of sample DNAs across the PCR plates and with random use of reagents and instruments. The source DNA includes the following:

• 284 HapMap samples including at least one replicate of each of 270 HapMap samples: 90 from each of the Yoruban, Asian, and Caucasian ethnic groups, from cell-line derived DNAs from the Coriell Institute of Medical Research
• 96 DNA samples from blood of phenotypically healthy male and female individuals obtained from BioServe Biotechnologies

The CytoScan™ HD Array samples that were used to create the Reference Model file were chosen to be run by different operators and with different kits and reagents, while still covering all the HapMap cell line ethnic groups, plus the normal bloods of both sexes.

Yes, refer to the CAGdb database, ICCG, and DECIPHER for additional public databases that may assist in data analysis, and for which we do not have a direct link within ChAS.

They can export the Whole Genome View as a PNG file. To do this, click on File → Export window PNG

Out of the six data files mentioned in the preceding question, it is important to backup and archive the ARR, CEL, CYCHP, and CHPCAR files at a minimum. This will allow someone to maintain the ability to either reanalyze from the CEL file or re-visualize the results using the CYCHP/CHPCAR files.

• Applied Biosystems™ GeneAmp™ PCR System 9700 (silver or gold-plated silver block only; do not use an aluminum block)
• Applied Biosystems™ Veriti™  96-Well Thermal Cycler
• Bio-Rad™ DNA Engine™ PTC-200 Thermal Cycler
• Eppendorf™ Mastercycler™ Pro S Thermal Cycler 
• Applied Biosystems™ 2720 (pre-PCR room only)

CytoScan™ array CEL files are processed and analyzed in ChAS. This is a free download from our website. For processing the CEL files, a 64-bit computer with a minimum of 8 GB of RAM, is required. For viewing the resulting CYCHP files, either a 32-bit or 64-bit computer is required. For 32-bit computers, it is important to note that the minimum amount of RAM needed is 3 GB. The recommended system specifications are included in the Chromosome Analysis Suite User Manual (Cat. No. 702943).

For successful execution of the CytoScan™ assay, it is important that equipment be properly maintained and calibrated per the manufacturers’ specifications. Please be sure that the centrifuges, pipettes, thermal cyclers, and equipment, including the 645 hybridization oven, fluidics station(s), and scanner, have had their recommended maintenance prior to running the assay. 

The use of the following magnetic racks is supported:

• DynaMag™-2 Magnet (Cat. No. 123-21D)
• MagnaRack™ (Cat. No. CS15000)

• DNA must be double-stranded genomic DNA.
• DNA must be free of PCR inhibitors.
• DNA must not be contaminated with other human genomic DNA sources or with genomic DNA from other organisms.
• DNA must not be degraded.
• DNA should have an A260/A280 between 1.7–2.1 (numeric rounding allowed).

The recommended starting amount is 250 ng (5 uL with 50 ng/uL) dissolved in low EDTA TE buffer. High EDTA concentration may negatively impact the downstream enzymatic reactions. It is recommended to run the gDNA samples on a 0.8-1% agarose gel for side-by side comparison with a control DNA (included in the kit). High quality genomic DNA will run as a major band at approximately 10-20 kb on the gel. For more detailed information please refer to Genomic DNA General Requirements in the CytoScan™ Assay User Guide (Cat. No. 901808)

Both blood and cell line sample sources have been tested with the CytoScan™ assay.
Methods that include boiling or strong denaturants are not acceptable because the DNA would be rendered single-stranded. Genomic DNA extracted using the following methods have been tested:

• QIAGEN – Gentra Puregene™ Kit
• 5 PRIME – PerfectPure™ DNA Blood Kit

The CytoScan™ assay requires genomic DNA concentration ≥50 ng/µL. Therefore, the elution volumes for each of the kits will need to be adjusted accordingly to achieve the desired concentration.

It is highly recommended not to mix different sub kit lots from different modules because functional testing is done based on sub kits from the same lot.

No. The CytoScan™ assay has been optimized for performance with only CytoScan™ Reagent Kit.

No, the reagents and arrays are sold as bundles for 24 reactions (Cat. No. 901835).

Positive control: This sample has prior proven performance and represents a pristine sample, meeting all genomic DNA input requirements. The performance of this sample helps to assess the quality of the assay steps and final product. In troubleshooting cases, performance of this control sample versus that of non-control samples can help to parse out possible assay failure culprits.

Negative control: For this assay, the negative control is simply reduced EDTA TE Buffer and acts as a control for the process up to the PCR quantitation and PCR gel. If no smear is seen on the PCR gel, it is considered passing, indicating that there is no DNA contamination in any of the reagents that could lead to amplification of other targets. A failing result will have a smear in the PCR gel. Note that the presence of PCR primer dimers is expected.

The post-PCR lab contains amplicons, whereas the pre-PCR area should not. These amplicons can be inadvertently transferred back to the pre-PCR area, thus contaminating the lab and posing a contamination threat to all reactions set up in the pre-PCR lab. Because all samples are amplified using the same primers and adaptors, contaminating DNA can also be amplified.

The 10 assay processing steps, as listed in the CytoScan™ Assay User Manual (Cat. No. 703038) are the following:

1. Genomic DNA Preparation

2. NSP1 Restriction Enzyme Digestion

3. Ligation

4. PCR

5. PCR Product Purification

6. Quantitation

7. Fragmentation

8. Labeling

9. Hybridization

10. Wash, Stain, Scanning

The purification process is required to remove all the non-amplified DNA after the PCR process.

Probes on the array are 25-mers. The fragmentation step reduces the purified PCR product size (150– 2,000 bp) to an even smaller size range (25–125 bp), making them more optimal for hybridization to the array probes. Ideally, 25 bp-sized PCR products hybridize to 25-mer probes on the array; however, optimal hybridization can be achieved with 25 to 125 bp-sized PCR products.

No, the routine QC includes running samples only on a gel. Some samples will be saved for additional analysis on the Bioanalyzer, should that become necessary

The labeled products can be stored at –20°C for no more than 10 days

The hybridization time is 16–18 hours. If there are a limited number of fluidics stations, the hybridization process can be staggered by two hours. For example, if there is only one fluidics station and eight arrays, the hybridization for four arrays can be started at one time, and then the hybridization for the other four arrays started two hours later. After 16 hours, the first four arrays can be placed on the fluidics station, and then the next four arrays placed on the fluidics station two hours later. Thus, all eight samples will have hybridized for 16 hours. Another option is to wash the first set of samples at 16 hours and the second set at 18 hours.

It is not recommended to stagger more than three wash/stain cycles in an eight-hour workday. Please consult the local Field Application Specialist for more details on the hybridization process.

The hybridization temperature is 50°C. The rotation speed is 60 rpm

No, this has not been validated at this time.

Deionized (DI) water should always be used on the fluidics stations for all protocols, including the shutdown and bleach protocols.

Yes, it is recommended to run two water shutdown protocols after bleaching the fluidics station.

The correct fluidics script is the CytoScan750K_Array_450.

The CytoScan™ 750K Array uses a 64 format and a 5 µm feature size.

The CytoScan™ 750K Array covers both constitutional and cancer genes with:

• Overall intragenic coverage at 1 marker / 1,737 bases
• ClinGen (previously ICCG and ISCA) constitutional coverage at 1 marker / 1,099 bases
• Complete cancer gene coverage at 1 marker / 1,269 bases
• 12,000 OMIM genes at 1 marker / 2,204 bases
• >36,000 RefSeq genes at 1 marker / 1,737 bases
• Backbone (non-gene) coverage at 1 marker / 6,145 bases across genome for breakpoints
• Overall (gene and non-gene backbone) coverage at 1 marker / 4,127 bases

The CytoScan™ 750K Array includes 200,000 genotype-able SNPs and 550,000 non-polymorphic probes.

Each array takes approximately 7 minutes to scan.

The CEL file is ~46 MB, and the CYCHP file is ~33 MB.

All genotyping and copy number analysis is done with the Chromosome Analysis Suite (ChAS) Software
v.1.2 or higher. Additionally, there is an update installer to install the 750K-specific files. That installer is downloadable from the Affymetrix website and is named “CytoScan750K_ChASSupportUpdate.exe”. This product is not intended for genome-wide association studies.

The CytoScan™ 750K Array uses the following QC metrics:

• SNPQC>=15
•  MAPD<=0.25
• Waviness SD<=0.12

These QC metrics have been fine tuned for blood-derived constitutional samples. The SNPQC and Waviness SD metrics are based on an assumption of a relatively normal diploid genome which for which the majority of the genome is not mosaic. For hematological malignancy samples the baseline assumption pertaining to constitutional samples is violated with regard to of aberration frequency and high levels of mosaicsm which will likely trigger the SNPQC and Waviness SD metrics to fail. Only the MAPD metric should be considered for non-constitutional samples. A failure of any one of these metrics for constitutional samples is a failure for that array result. There is no direct correlation between the passing numeric value for any one of the metrics and the quality of a sample. For example, a sample that has an SNPQC=30 is not necessarily a better quality sample than one that has an SNPQC=20.

Yes, genotypes can be generated in the Chromosome Analysis Suite (ChAS) Software v.1.2 or higher. The software uses BRLMM for genotyping, which uses a Bayesian model based on prior clusters. This product is not intended for genome-wide association studies.

A total of 5 files are produced that are key to the process:

• ARR file—this file includes sample information
• AUDIT file—this file is a log of the sample history
• DAT file—this file is the raw data from the scanner
• CEL file—this file is the gridded and processed data
• CYCHP file—this file is the output of ChAS and contains all of the analysis data

CytoScan™ HD Array uses a 49 format and a 5-µm feature size.

CytoScan™ HD Array covers both constitutional and cancer genes with:

• Overall intragenic coverage at 1 marker/880 bases
• ClinGen (previously ICCG and ISCA) constitutional coverage at 1 marker/384 bases
• Complete cancer gene coverage at 1 marker/553 bases
• 14,000 OMIM genes at 1 marker/723 bases
• >36,000 RefSeq genes at 1 marker/880 bases
• Backbone (non-gene) coverage at 1 marker/1,737 bases across genome for breakpoints
• Overall (gene and non-gene backbone) coverage at 1 marker/1,148 bases

CytoScan™ HD Array includes 750,000 genotype-able SNPs and 1.9 million non-polymorphic probes.

The correct fluidics script is CytoScanHD_Array_450.

The CEL file is ~66 MB, and the CYCHP file is ~119 MB.

All genotyping and copy number analysis is done with ChAS. CytoScan™ Cytogenetics Suite is not intended for genome-wide association studies.

It is a 49 format array.

Sample must be double-stranded genomic DNA (gDNA) that is not degraded (size 10 kb by gel analysis), not contaminated, and free of PCR inhibitors. Sample should be diluted to 20 ng/µL in low-EDTA TE buffer.

Consumable and equipment requirements are the same for running both the CytoScan XON assay and the CytoScan assay.

Chromosome Analysis Suite (ChAS) Software version 3.3 is required to analyze CytoScan XON arrays.

CytoScan™ Optima Array content has been selected empirically from CytoScan™ HD Array and was designed for prenatal and miscarriage sample analysis. The functional resolution of CytoScan™ Optima Suite is 1MB for losses and 2MB for gains genome wide 5MB for LOH/AOH 100kb resolution in 396 relevant regions for prenatal research. 

CytoScan™ Optima Array has a total of 315,608 features covering control, CN, and SNP probes. There are a total of 18,018 non-polymorphic CN probes and 148,450 SNP markers on the Optima Suite.

The CEL file is ~3 MB, and the CYCHP file is ~18 MB.

The CytoScan™ Amplification Kit has been designed to be used at the PCR Amplification step of the CytoScan™ Assay (CytoScan™ HD and CytoScan™ 750K Suite). The CytoScan™ Amplification Kit (Cat. No. 902975) is manufactured by Takara Bio USA, Inc. and is equivalent to the Clontech Titanium™ DNA Amplification Kit (Cat. Nos .639240, 63924). The amplification reagents of the CytoScan™ Amplification Kit generate PCR products to be used in the preparation of the target DNA to be hybridized to CytoScan™ 750K and HD arrays. The kit contains enough reagents to prepare 400 PCR reactions, equivalent to 96-100 samples processed with the CytoScan™ Assay.

The only difference is that the CytoScan™ Amplification Kit is sold in a pouch, while the Clontech Titanium® DNA Amplification Kit (Cat. No. 639243) is sold in a box by Takara Bio USA. The Tubes contained in the Affymetrix branded pouch are the same part number and include the same reagents as the ones in Clontech Kit.

The CytoScan™ Amplification Kit has enough reagents to process 400 PCR reactions, equivalent to 96-100 samples. 

No, the kit is only sold as standalone. It can be included in the CytoScan™ Array and Reagent Bundles quote, order and shipment as 1 amplification kit per 4 CytoScan™ HD/750K Array and Reagent Bundles. 

No, because the Clontech™ Kit and CytoScan™ Amplification Kit contain the same reagents, there are no changes to the protocol. 

Note: an Update of User Guides and Site Preparation Guides is planned for 2017 that will contain several cosmetic updates and will also reflect the inclusion of the CytoScan™ Amplification Kit as the preferred kit in place of the Clontech kit. The Titanium® DNA Amplification Kit will still be supported.

No, because Clontech Kit and CytoScan™ Amplification Kit contain the same reagents, there are no changes to the protocol. 

Clontech Titanium® Taq polymerase does not freeze, so no freeze-thaw cycles are applicable when taken out of the freezer in the proper conditions (cooling block, etc.). dNTPs have been validated for 10 freeze-thaw cycles. 

Several of our CytoScan™ users aliquot the Clontech Titanium® DNA Amplification kit upon reception.
Aliquoting the kit is recommended but not mandatory.

The OncoScan™ CNV Plus Assaycontains copy number probes plus somatic mutation probes. The OncoScan™ CNV Assay has only the copy number probes.

Yes, OncoScan™ CNV Plus Assay and OncoScan™ CNV Assay OSCHP files can be opened and visualized at the same time in ChAS. The OncoScan™ CNV assay does not contain the somatic mutation probes that are on the OncoScan™ CNV Plus Assay.

A minimum of 80ng of DNA is needed per sample for either the OncoScan™ CNV Plus Assay or the OncoScan™ CNV Assay.

Key opinion leaders in the oncology field as well as papers were consulted when choosing the 900 cancer-related genes for the array.

It is critical to have correct size of the fragments for an optimal hybridization. The fragmentation reagent is very viscous and it is easier to get a correct proportion of enzyme when a larger volume is used. We always recommend that you prepare a master mix for 24 samples even if a smaller number of samples are processed.

OncoScan™ CNV Plus Assay has 217,454 probes.

The OncoScan™ assay is enabled by the MIP (molecular inversion probe) assay. The MIP technology was an assay designed to differentiate between the A/T and G/C channel. Two chips are used to allow differentiation between the channels as the detection is single-color.

Virtually all probes in OncoScan™ sssay are SNPs. There are a few thousand probes that are not polymorphic.

OncoScan™ assays have a 50 kb–~100 kb resolution in ~900 cancer genes.

Outside of the cancer genes, 88% of the genome has 300 kb resolution and 97% of the genome has at least 380 kb resolution

The genome-wide resolution of LOH genome wide is ≤10 MB. If the aberrant cell fraction is high, the resolution increases to 3–5 MB.

OncoScan™ assays can detect mosaic aberrations found in 25% of the aberrant cells. TuScan operates in two modes:

• When the percentage of aberrant cell fraction is ≥40% in a homogeneous tumor, TuScan makes calls that reflect the integer copy number in the aberrant cells. 
• In all other cases, TuScan makes calls down to 25% aberrant cell fractions. These are “fractional” copy number calls (such as 2.3) reflecting the average CN across the aberrant and non-aberrant cell fraction.

OncoScan™ assays can detect copy number changes up to 50 copies. As the number of copies increases, the user should bin the calls. For example, copy numbers of 20−50 should be considered as one bin. The recommended bins are: 0, 1, 2, 3, 4, 5–6, 7–10, 11–20, 20–50.

The HapMap concordance of genotyping calls is greater than 99.5%.

Yes, if using our defined workflow and depending on the size of the exon. (Our algorithm requires 20 contiguous markers to make a call.) If using an “open” workflow, the user can define the number of probes they need to claim exon resolution.

We recommend the TuScan algorithm. This algorithm has fixed settings based on 19 probes/call to ensure high sensitivity and specificity at the stated resolution claims. Nexus Express Software for OncoScan™ assays has an additional algorithm for copy number calls from OncoScan™ assays: SNPFASST2 algorithm, developed and supported by BioDiscovery. This allows the user to adjust settings and make custom calls (e.g., with fewer probes). Use cases are when breakpoints are clear with fewer probes when probe re-centering is needed.

Log2 and B-allele frequency.

Linear copy number calls when you mouse over aberrations. In some cases, linear calls are calls in aberrant cell fraction only. In the software, when the percentage of aberrant cells has a percentage value, the linear call for aberrations in that sample is the call in the aberrant cells only. Values will be integers. When the percentage of aberrant cells is “N/A,” the linear call is an average call across the aberrant and non-aberrant cells, and values will be non-integers. You can estimate the copy number in aberrant cells if you have an independent tumor burden estimate.

This represents the percentage of aberrant cells in a sample:

When reported as “N/A,” it means that the percentage of aberrant cells could not be estimated.

When reported as a percentage (e.g., 60%), it means that 60% of the cells were aberrant.

This is possible when the tumor is nearly homogeneous (i.e., it has a major dominant clone), and the percentage of aberrant cell fraction is 40% or higher.

Ploidy refers to the baseline copy number observed in the tumor portion of the sample. Ploidy cannot always be estimated. For example, a sample with four copies of each chromosome and no other aberrations looks the same as a sample with two copies of each chromosome.

There are no officially recommended stopping points in the OncoScan™ assays. For additional information about specific parts of the assay, please contact support.