Question 1

Why does the CytoScan Assay User Guide state on page 35 to always prepare Fragmentation Master Mix according to Table 9 even when processing less than 24 samples?

Accepted Answer

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

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 2

What are the recommended requirements for genomic DNA samples used in the CytoScan assay?

Accepted Answer

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 .

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 3

Which type of freezer should be used to store CytoScan Reagent kit components that require -20 degrees C?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 4

What is the maximum number of freeze/thaw cycles recommended for the CytoScan Reagent kit?

Accepted Answer

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

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 5

Why does the protocol state to prepare a fragmentation master mix enough for 24 samples even if a smaller number of samples is processed?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 6

What are the fill volumes of the reagents in the CytoScan Reagent Kit?

Accepted Answer

We can only guarantee the volume that is indicated on the label. Since our assurance guarantee is only provided for the label volume we advise not planning experiments or standard operating procedures based on potential overage volumes.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 7

When using ChAS, is it important for someone to back up their data?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 8

What data files are produced during assay and analysis process when using ChAS?

Accepted Answer

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).

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 9

What is the mosaic call determination in ChAS?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 10

What is the library file location for ChAS?

Accepted Answer

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

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 11

How do I unzip the array library file if I have to install it manually?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 12

How does someone export LogRatios for the whole genome?

Accepted Answer

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

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 13

Are there additional databases to refer to that will help in data analysis for the CytoScan HD Array?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 14

What samples are in the Reference Model file in the CytoScan HD Array and CytoScan 750K Array set?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 15

Which genotype is presented in each allele difference track?

Accepted Answer

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

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 16

What are the recommended graph settings for data interpretation in ChAS?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 17

Are individual genotypes generated from the CytoScan arrays?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 18

For CytoScan arrays, what is Waviness-SD?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 19

What are the definitions of the array QC metrics terms "MAPD" and "SNPQC"; what does each metric measure and how are they used in assessing array performance?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 20

What software is available for analysis for genoytping and copy number analysis?

Accepted Answer

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

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 21

Are there any shutdowns run after bleaching the fluidics station when working with the CytoScan arrays?

Accepted Answer

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

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 22

How long can CytoScan arrays filled with holding buffer be stored before scanning?

Accepted Answer

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

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 23

After hybridization, how long can CytoScan arrays be stored before washing and staining?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 24

What is the hybridization time for CytoScan arrays?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 25

How long can the labeled products be stored at -20 degrees C?

Accepted Answer

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

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 26

What can cause under-fragmentation for the CytoScan assay?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 27

What can cause over-fragmentation for the CytoScan assay?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 28

Do the samples for the CytoScan Assay need to be run on a gel and the Bioanalyzer during fragmentation QC?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 29

What should be done if a refrigerated centrifuge is not available for the fragmentation step in the CytoScan Assay?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 30

What is the purpose of the fragmentation step of the CytoScan Assay?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 31

How long can the purified PCR products be stored at -20 degrees C?

Accepted Answer

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

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 32

Can I stop after the PCR process and continue the next day with the purification step for the CytoScan Assay?

Accepted Answer

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

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 33

For the CytoScan Assay, what is the role of purified PCR product?

Accepted Answer

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

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 34

What is the typical yield range from PCR product purification?

Accepted Answer

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).

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 35

For the CytoScan assay, can a different magnetic rack be used instead of the recommended MagnaRack magnetic stand?

Accepted Answer

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

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 36

For the CytoScan assay, is there a plate purification option for the PCR product purification step?

Accepted Answer

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

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 37

What should I do if I see a smear in their negative control on their PCR gel when performing the CytoScan assay?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 38

For the CytoScan assay, does the PCR have to be set up in a separate pre-PCR clean room?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 39

In a hypothetical CytoScan assay failure in which PCR fragment size distribution observed on the PCR gel is significantly greater than 2,000 bp DNA smear, what processes in the assay could play a role in the failure?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 40

When performing the CytoScan Assay, what can cause faint or absent bands on the PCR gel?

Accepted Answer

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

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 41

How long can the ligated products for the CytoScan assay be stored at -20 degrees C?

Accepted Answer

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

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 42

How long can the ligated products for the CytoScan assay be stored on the thermal cycler?

Accepted Answer

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

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 43

What happens during the digestion and ligation steps of the CytoScan assay?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 44

What are the 10 CytoScan assay processing steps listed in order?

Accepted Answer

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

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 45

What are the stopping points in the CytoScan Reagent Kit assay?

Accepted Answer

After any stage in the assay (except digestion), samples can be stored at -20 degrees C if one is not proceeding directly to the next step. However, once a stage has been initiated, it must be completed before storage of the samples.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 46

How many freeze/thaw cycles are validated for CytoScan Reagent Kit (24 samples)?

Accepted Answer

CytoScan Reagent Kit (Cat. No. 901808) is validated for for 4 in-use freeze/thaw events but have to use the kit within a month (30 days) once opened even if they have not gone through 4 freeze-thaws.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 47

What is the recommended genomic DNA extraction methods used for the CytoScan assay?

Accepted Answer

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 at ThermoFisher Scientific:

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.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 48

What are the general genomic DNA requirements for the CytoScan assay?

Accepted Answer

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).

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 49

What sample types are validated on the CytoScan assay?

Accepted Answer

The CytoScan assay has been validated with blood and cell line samples.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 50

Which magnetic racks are supported for the CytoScan assay?

Accepted Answer

The use of the following magnetic racks is supported:
-DynaMag-2 Magnet (Cat. No. 123-21D)
-MagnaRack (Cat. No. CS15000)

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 51

Can electronic pipettes be used in the CytoScan assay instead of multichannel manual pipettes?

Accepted Answer

Pipetting viscous solutions using electronic pipettes may be very challenging and result in ineffective pipetting that could contribute to overall poor QC values. Therefore, the use of electronic pipettes is not recommended.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 52

Am I required to have all equipment current regarding calibration and maintenance?

Accepted Answer

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 ThermoFisher Scientific equipment, including the 645 hybridization oven, fluidics station(s), and scanner, have had their recommended maintenance prior to running the assay. For lab equipment, it is typically a yearly calibration. For ThermoFisher Scientific equipment, there is typically a yearly preventative maintenance.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 53

What is required for processing and viewing CytoScan array CEL files?

Accepted Answer

CytoScan array CEL files are processed and analyzed in ChAS. This is a free download from the ThermoFisher Scientific web site. For processing and viewing the CEL files, a 64-bit computer with a minimum of 8 GB of RAM, is required. The recommended system specifications are included in the Chromosome Analysis Suite User Manual (Cat. No. 702943; http://assets.thermofisher.com/TFS-Assets/LSG/manuals/ChAS_Manual.pdf).

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 54

How can I tell the difference between a silver and aluminum thermal cycler block?

Accepted Answer

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. In addition, an aluminum block has a honeycomb appearance between the wells, whereas a silver block is smooth.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 55

What thermal cycler can be used in for the CytoScan assay?

Accepted Answer

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)

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 56

What Thermo Fisher Scientific equipment is required for the CytoScan assay?

Accepted Answer

The CytoScan assay requires either GeneChip Scanner 3000 7G System with Workstation and AutoLoader or GeneChip System 3000Dx v.2 and a GeneChip Hybridization Oven 645.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 57

What is CytoScan Cytogenetics Suite?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 58

For the CytoScan 750K Array, is it important to back up my data in ChAS?

Accepted Answer

Out of the six data files (ARR, AUDIT, DAT, CEL, CYCHP and CHPCAR), it is important to backup and archive the ARR, CEL, CYCHP and CHPCAR file at a minimum. This will allow you to maintain the ability to either reanalyze from the CEL file, or re-visualize the results using the CYCHP/CHPCAR files.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 59

For the CytoScan 750K Array, what data files are produced during assay and analysis process?

Accepted Answer

A total of 6 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
CHPCAR file - This file stores user-annotated calls, interpretations, and modifications made to CHP file segment data
CYCHP file - This file is the output of ChAS and contains all of the analysis data

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 60

Are individual genotypes generated on the CytoScan 750K Array?

Accepted Answer

Yes, genotypes can be generated in the Chromosome Analysis Suite (ChAS 3.3) Software. 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.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 61

What can cause waviness SD failures when using the CytoScan Assay?

Accepted Answer

Waviness SD failures can be caused by degraded DNA, an incompatible sample type, or a sample-specific effect. See the CytoScan Assay User Manual (Cat. No. 703038, https://tools.thermofisher.com/content/sfs/manuals/cytoscan_assay_user_manual.pdf) and the Chromosome Analysis Suite User Manual (Cat. No. 702943) for more information.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 62

What can cause MAPD failures when using the CyotScan Assay?

Accepted Answer

MAPD failures can be caused by assay drift due to variation in assay execution or 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.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 63

What can cause SNPQC failures when using the CytoScan Assay?

Accepted Answer

SNPQC failures can be caused by contamination of samples, equipment, or reagents. Other possible causes include over or under fragmentation of samples or a hybridization oven that is out of calibration. See the troubleshooting section of the CytoScan Assay User Manual (Cat. No. 703038) for more information (https://tools.thermofisher.com/content/sfs/manuals/cytoscan_assay_user_manual.pdf).

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

Question 64

What are the array QC metrics for the CytoScan 750K Array?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

CytoScan™ 750K Suite - FAQs