CytoScan™ HD Kit Plus 96, 96 arrays - FAQs

Here is the link to the CytoScan assay user guide: https://assets.thermofisher.com/TFS-Assets/LSG/manuals/703038_cytoscan_assay_UG.pdf
In Appendix D (starting on page 67), the manual outlines specifications for compatible thermocyclers. Any thermocycler that fits those specifications will work.

Note: Use the silver or gold-plated block only, do not use an aluminum block. In order to determine which block you have, look at the serial number on the block. An "A" in the serial number indicates that the block is aluminum; an "S" indicates that it is silver. Also, an aluminum block has a honeycomb appearance between the wells, whereas a silver block is smooth.

Methods that include boiling or strong denaturants are not acceptable, because the DNA would be rendered single-stranded. For genomic DNA extraction, we recommend using either the Qiagen Gentra Puregene Kit or the 5 Prime PerfectPure DNA Blood Kit.

Our CytoScan HD 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.

The presence of RNA and free nucleotides can interfere with some quantitation methods using a spectrophotometer or a NanoDrop instrument. To eliminate RNA contamination, perform RNase treatment during extraction as follows:
- For QIAGEN Gentra Puregene Kit: Perform RNase treatment as recommended in the extraction kit manual prior to elution of genomic DNA.
- For 5 PRIME PerfectPure DNA Blood Kit: Use only RNase-treated purification columns for extraction of genomic DNA.

The purified genomic DNA extracted using the two methods above should meet the DNA quality specifications per the manufacturer’s kit extraction manual.
Note: Both blood and cell line sample sources have been tested for the CytoScan HD assay.

The CytoScan HD Array includes 750,000 "genotype-able" SNPs and 1.9 million non-polymorphic probes. The CytoScan HD Array covers both constitutional and cancer genes with: - ISCA constitutional coverage at 1 marker /384 bases
- Complete cancer gene coverage at 1 marker / 553 bases
- 12,000 OMIM genes at 1 marker / 659 bases
- >36,000 RefSeq genes with 1 marker / 880 bases
- Backbone (non-gene) coverage of 1 marker / 1,737 bases across genome for breakpoints

Array format: 49
Feature size: 5 µM
Time to scan per array: ~32 mins
Hyb temperature: 50 degrees C

The CytoScan HD Array uses the following QC metrics:
- SNPQC ≥15
- MAPD ≤0.25
- Waviness SD ≤0.12

The Purification Wash Buffer and Elution Buffer are not affected by being frozen at -20 degrees C and are safe to use for purification.

A subset of Y probes is used and run through a Hidden Markov Model to determine gender. Copy number of 0 or 1. 0 indicates female and 1 indicates male.

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The Minor Allele Frequency (MAF) refers to the frequency at which the least common allele occurs in a given population.

For example: A SNP with an allele (G) frequency of 0.40 implies that 40% of the population has G allele versus the most common allele (major allele), which is found in 60% of the population. If an SNP allele frequency is 0.05 in a population of 100 people, the number of alleles for each SNP is: 100 * 2 = 200 (each individual has two alleles).
Total number alleles for your SNP with a MAF of 0.05: 200 *0.05 = 10.

The minor allele is present ten times in your population. However, you don't know how many are heterozygotes vs homozygotes.

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No, we do not offer probes in the Cytoscan HD array that cover the mitochondrial genome.

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We recommend using the following thermal cyclers with Cytoscan HD:

For pre-PCR, we recommend 2720 Thermal Cycler (Cat. No. 4359659)

For pre- and post-PCR , we recommend the following thermal cyclers:
- GeneAmp PCR System 9700 Gold (Cat. No. 4314878) (Note: This instrument has been discontinued and therefore cannot be ordered)
- GeneAmp PCR System 9700 Silver (Cat. No. N8050001) (Note: This instrument has been discontinued and therefore cannot be ordered)
- Applied Biosystems Veriti Dx 96-Well Thermal Cycler (Cat. No. 4452300)
- Applied Biosystems Veriti 96-Well Thermal Cycler (Cat. No. 4375786)
- Bio-Rad® DNA Engine® PTC-200 Thermal Cycler
- Eppendorf® Mastercycler® Pro S Thermal Cycler


Note: You must use thermal cyclers with silver or gold-plated silver blocks. Do not use thermal cyclers with aluminum blocks.

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Tough-Spots® labels are small adhesive stickers used to temporarily seal the backs of cartridge arrays during the overnight hybridization step. They are required to prevent loss of volume due to evaporation through the septa. We recommend using Tough-Spots® labels on Rolls from USA Scientific (Item No. 9185-0000)

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Proper hybridization volume is critical to obtaining an even signal across a given array. Too little volume can lead to black circles in the middle of the array. Too much volume can leak out of the back of the array. The correct hybridization volume leaves enough room for a small air bubble to circulate around the array surface during the overnight hybridization. Here are the recommended hybridization and fill volumes based on the array format:
Array Format; Hybridization Volume; Fill Volume
- 49 Format (Standard); 200 µL; 250 µL
- 64 Format; 200 µL; 250 µL
- 100 Format (Midi); 130 µL; 160 µL
- 169 Format (Mini); 80 µL; 100 µL
- 400 Format (Micro); 80 µL; 100µL

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The tested sources of human gDNA used in the CytoScan Assay are blood and cell line samples.
The genomic DNA (gDNA) sample must be double-stranded, not degraded and free of any contaminants (e.g., PCR inhibitors and other human/non-human gDNA). The recommended starting amount is 250 ng (5 µL with 50 ng/µL) dissolved in low EDTA TE buffer. High EDTA concentration may negatively impact the downstream enzymatic reactions. We recommend running 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 the CytoScan Assay User Guide, page 9, Genomic DNA general requirements and recommendations section .

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The reagents that require storage at -20 degrees C must not be stored in a frost-free freezer. The activity of the enzymes will be decreased.

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The CytoScan Reagent kit has been validated for less than or equal to 5 freeze/thaw cycles.

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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. The recommendation is to always prepare a master mix for 24 samples even if a smaller number of samples are processed.

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Thermal cyclers with the following specifications are compatible with the CytoScan Assay protocol:

• Capable of holding 200 µL volume and 96-well plate
• Heat block capable of holding temperature of 4-99.9°C
• Temperature accuracy of +/- 0.25°C (at 35-99.9°C)
• Average heating and cooling rate of 2.6°C /second
• Thermal uniformity of +/- 0.5°C

The following PCR instruments have been verified for use in the CytoScan Assay (newer thermal cyclers may use simulation mode to mimic these settings):

• GeneAmp PCR System 9700 Gold/Silver
• Applied Biosystems Veriti 96-Well Thermal Cycler (Cat. No. 4375786)
• Applied Biosystems Veriti Dx 96-Well Thermal Cycler (Cat. No. 4452300)
• Bio-Rad DNA Engine PTC-200 Thermal Cycler (Calculated Temperature Mode)
• Eppendorf Mastercycler Pro S Thermal Cycler (Safe Temperature Mode) (Cat. No. 950030020 and Cat. No. 950040025).

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The plate can be tightly sealed and stored at -20 degrees C after all steps of the assay. The plate can be left over night at 4 degrees C after the PCR reaction and labeling reaction have been completed.

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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.

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A total of six 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.
CHPCAR file: this file stores user-annotated calls, interpretations, and modifications made to CHP file segment data (ChAS 2.0 and higher).

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The algorithm is designed to detect only mosaicism between ~30-70% for CNs between 1 and 3 for regions on the order of 5,000 markers in size or larger. The endpoint location of mosaic segments is less precise than the CN segmentation, with endpoint variation within 500 markers being typical for segments of 5,000 markers or larger. Some regions of full integer CN 1 or 3 below 5,000 markers in length may be incorrectly called as mosaic segments. Some regions of CN below 1 or above 3, mosaic or otherwise, less than 5,000 markers in length may be incorrectly called as mosaic segments.

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The library file location for ChAS 2.1 and above is C:\Affymetrix\ChAS\Library.

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Right click the zip folder and click extract all. They can also use an already installed software that unzips files (e.g., Winzip, 7-zip, or any other equivalent software). Browse to the location to save the folder and click Extract. Go to the folder to find the files.

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

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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.

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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.

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

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We recommend changing the weighted log2 ratio graph from the default to a minimum at -1.5 to a maximum at 1.5, using data type as points. We recommend changing the allele peaks graph from the default to a minimum at -2 to a maximum at 2, using data type as points. The other graphs can stay with the default values.

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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.

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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.

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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.

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

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

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The correct fluidics script is CytoScanHD_Array_450.

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Each array takes ~32 minutes to scan.

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

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CytoScan HD Array covers both constitutional and cancer genes with:
Overall intragenic coverage at 1 marker/880 bases
ClinGen (previously CCG 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

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

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

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After the washing and staining step, arrays can be stored for up to 24 hrs at 4 degrees C before scanning.

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Arrays must be put onto the fluidics station immediately after removal from the hybridization oven. Do not remove arrays from the oven you are ready to wash them.

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The hybridization time is 16-18 hrs. If there are a limited number of fluidics stations, the hybridization process can be staggered by two hrs. 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 hrs later. After 16 hrs, the first four arrays can be placed on the fluidics station, and then the next four arrays placed on the fluidics station two hrs later. Thus, all eight samples will have hybridized for 16 hrs. Another option is to wash the first set of samples at 16 hrs and the second set at 18 hrs. It is not recommended to stagger more than three wash/stain cycles in an eight-hour workday. Please consult technical support at techsupport@thermofisher.com for more details on the hybridization process.

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The labeled products can be stored at -20 degrees C for no more than 10 days.

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Common causes of under-fragmentation include improper storage or handling of the enzyme, an incorrect volume of enzyme used based on the unit activity, and improper mixing of fragmentation master mix. See the troubleshooting section of the CytoScan Assay User Manual (Cat. No. 703038, https://tools.thermofisher.com/content/sfs/manuals/cytoscan_assay_user_manual.pdf) for more information about under-fragmentation.

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Common causes of over-fragmentation include excess enzyme due to pipetting errors or an incorrect volume of enzyme used based on the unit activity. In addition, warming up of the assembled reaction prior to initiating the 37 degrees C incubation can lead to over-fragmentation. See the troubleshooting section of the CytoScan Assay User Manual (Cat. No. 703038, https://tools.thermofisher.com/content/sfs/manuals/cytoscan_assay_user_manual.pdf) for more information about over-fragmentation.

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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.

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A refrigerated centrifuge is highly recommended. If one is not available, a plastic plate rack that has been stored at -20 degrees C may be used. It is recommended to keep the samples chilled and work quickly prior to initiating the incubation at 37 degrees C.

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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.

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The purified PCR products can be stored at -15 to -25 degrees C for 10 days.

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Yes, the purified PCR products can be stored at -15 to -25 degrees C, and one can continue with the purification step later.

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

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The minimum recommended yield is 2.5 µg/µL for a sample. 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 Manual (Cat. No. 703038, https://tools.thermofisher.com/content/sfs/manuals/cytoscan_assay_user_manual.pdf).

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Yes, the assay is currently validated for the following magnetic racks:
DynaMag-2 Magnet (Cat. No. 123-21D)
MagnaRack (Cat. No. CS15000)

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No, only the single-tube purification process is validated with the assay.

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A smear in the negative control indicates there has been a contamination. If the negative control band is a light smear, it could be a low-level environmental bacterial contaminant introduced through plastic surfaces. This has shown not to have a material impact on the CytoScan HD data. If a bright band or a smaller smear is seen, then that could be a real cross-contaminant from a sample, and the samples would need to be checked for contamination. If a smear is seen in the negative control well, you should re-run the negative control to verify this was not a result of sample bleed-over from gel loading.

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Using two separate rooms greatly reduces the risk of sample contamination 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 a one-room configuration is being used, it is highly recommended to use a laminar flow cabinet for the pre-PCR clean area. See the CytoScan Assay User Manual (Cat. No. 703038, https://tools.thermofisher.com/content/sfs/manuals/cytoscan_assay_user_manual.pdf) for more details about the recommended laboratory setup.

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The digestion step could be root cause. If the reaction did not go to completion, there may be, on average, longer fragments at the end of the reaction. These longer fragments then pass on to ligation and PCR reactions.

Insufficient amounts of PCR primer were added to facilitate suppression PCR. The primer concentration shifts the PCR reaction equilibrium toward larger fragment distributions, in this case, by increasingly unfolding (linearizing) the stem-loop structure at increasing primer concentration when the same adaptor ends hybridize.

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Several things can cause this problem. To troubleshoot this problem, first determine if the positive control worked properly. Common reasons for this failure include incomplete digestion of genomic DNA or inefficient ligation of adaptors, ligation samples that are not properly diluted or mixed, and degraded DNA (if only the positive control worked). See the troubleshooting section of the CytoScan Assay User Manual (Cat. No. 703038) for more information

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A plate can be frozen at -15 to -25 degrees C for up to one week.

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A plate can be held in thermal cycler at 4 degrees C for up to 60 hrs.

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

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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

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Always use deionized (DI) water on the fluidics stations for all protocols, including the shut down and bleach protocols.

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Common causes of under-fragmentation include improper storage or handling of the enzyme, an incorrect volume of enzyme used based on the unit activity, and improper mixing of fragmentation master mix. See the troubleshooting section of the CytoScan Assay User Manual (Cat. No. 703038, https://tools.thermofisher.com/content/sfs/manuals/cytoscan_assay_user_manual.pdf) for more information about under-fragmentation.

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Common causes of over-fragmentation include excess enzyme due to pipetting errors or an incorrect volume of enzyme used based on the unit activity. In addition, warming up of the assembled reaction prior to initiating the 37 degrees C incubation can lead to over fragmentation. See the troubleshooting section of the CytoScan Assay User Manual (Cat. No. 703038, https://tools.thermofisher.com/content/sfs/manuals/cytoscan_assay_user_manual.pdf) for more information about over-fragmentation.

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No, the routine QC includes running samples only on a gel. You will save some samples for analysis in case you need them for additional analysis on the Bioanalyzer.

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A refrigerated centrifuge is highly recommended, but if one is not available, you may use a plastic plate rack that has been stored at -20 degrees C. It is recommended that you keep the samples chilled and work quickly prior to initiating the incubation at 37 degrees C. See the CytoScan Assay and Data Analysis Training Video (www.thermofisher.com/us/en/home/life-science/microarray-analysis/microarray-analysis-learning-center/microarray-analysis-training.html) for more details.

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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 Manual (Cat. No. 703038, https://tools.thermofisher.com/content/sfs/manuals/cytoscan_assay_user_manual.pdf).

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No, the Magna Rack is the only magnetic rack that is validated with the assay.

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Several things can cause this problem. The first step in troubleshooting this problem is to determine if the positive control worked. Common reasons for this failure include incomplete digestion of genomic DNA or inefficient ligation of adaptors, ligation samples that are not properly diluted or mixed, and degraded DNA (if only the positive control worked). See the troubleshooting section of the CytoScan Assay User Manual (Cat. No. 703038; https://tools.thermofisher.com/content/sfs/manuals/cytoscan_assay_user_manual.pdf) for more information.

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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.

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The CytoScan Assay has significantly fewer pipetting steps and requires less hands-on time than the Affymetrix Genome-Wide Human SNP 6.0 Assay. The CytoScan Assay uses only the Nsp I restriction enzyme and has been optimized for cytogenetics applications. The CytoScan Assay is not intended for genome-wide association studies.

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After any stage in the assay (including fragmentation) you can store the samples at -20 degrees 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.

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The expiration date applies to the date of manufacturing. For the CytoScan Reagent Kit, it can be used for up to one year from the date of manufacturing.

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No. The CytoScan Assay has been optimized for performance with the CytoScan Reagent Kit, which is therefore required.

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We recommend running 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

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The CytoScan Reagent Kit (Cat. No. 901808) is validated for five freeze/thaw cycles.

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For successful execution of the CytoScan Assay, it is important that your equipment is properly maintained and calibrated per manufacturers' specifications. Please assure that your centrifuges, pipettes, thermal cyclers, and Affymetrix equipment, including the 645 Hybridization Oven, Fluidics Station(s), and Scanner, have had their last recommended maintenance prior to running the assay. For lab equipment, it is typically a yearly calibration. For the Affymetrix equipment, there is typically a yearly preventative maintenance.

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Look at the serial number on the block. An “A” in the serial number indicates the block is aluminum; an “S” indicates it is silver. Also, an aluminum block has a honeycomb appearance between the wells, whereas a silver block is smooth.

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The ABI 9700 (silver or gold-plated silver block only; do not use an aluminum block) and the ABI 2720 (pre-PCR room only) thermal cyclers have been validated for the CytoScan Assay.

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The CytoScan Assay, along with the CytoScan 750K Arrays, the GeneChip Command Console Software (AGCC), and the Affymetrix Chromosome Analysis Suite (ChAS) Software, enable you to perform high-resolution genome-wide DNA copy number analysis. This Affymetrix solution for cytogenetics also provides genotyping information, enabling detection of copy neutral loss of heterozygosity (LOH), which can be used to detect uniparental isodisomy (isodisomic UPD). The combined high-resolution DNA copy number data and the ability to detect gains, losses, and UPDs on a single array makes the CytoScan 750K Solution ideal for next-generation cytogenetics studies.

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