GeneChip™ Fluidics Station 450 - FAQs

The part number for Tubing, Peristatic Pump, 8.5" Long is 400110, which contains one tube per pack.

For most expression microarrays, the amount of wash buffer used during a single run is:
Wash A: approximately 150 mL
Wash B: approximately 50 mL

We recommend checking the user guide for your specific microarray for further details.

The array format and recommended fluidics protocols can be found in the packet insert of the array. This information can also be found in the the following document: Compatible Fluidics Script Table (https://assets.thermofisher.com/TFS-Assets/LSG/brochures/compatible_fluidics_script_table.pdf)
Note: Fluidics recommendations are based on the assumption that Affymetrix labeling kits and Hyb Wash Stain kits are being used.

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The components include: Affymetrix Mouse Diversity Genotyping Array
Affymetrix Genome-Wide Human SNP Nsp/Sty Assay kit 5.0/6.0
Affymetrix Power Tools (sed to run a modified BRLMM-P algorithm for genotype calling, GeneChip Fluidics Station 450
GeneChip Hybridization Oven 645
and GeneChip Scanner 3000 7G.

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Customers must purchase a minimum of 60 arrays to begin a study, because this number of samples is required for BLRMM-P to cluster the data properly. For subsequent orders, the minimum order is approximately 30 arrays.

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A number of service providers have experience running the SNP 5.0/6.0 assay and the genotyping call algorithm that is used with the Mouse Diversity Genotyping Array. The list includes The Jackson Laboratory, the microarray and computational analysis group that participated in the design and validation of the Mouse Diversity Genotyping Array. The Jackson Laboratory has experience assaying more than 1,000 samples, has access to the most up-to-date methods, and has achieved excellent genotyping call accuracy. Their service spans basic delivery of raw data and genotype calls to custom bioinformatics analysis. For more information, please visit their website or email jaxservices@jax.org.

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Affymetrix GTC software does not perform copy number analysis. However, the developers of this array (The Jackson Labs) have developed an R package that can be used for CNV analysis which can be found here: http://cgd.jax.org/tools/MouseDivGeno/.
Alternatively, The Jackson Labs Services group mentions CNV analysis: http://jaxservices.jax.org/mdarray/index.html.

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The average sample file size is 66 MB.

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Library files contain information about probe array design layout, probe use and content, scanning and analysis parameters, and other characteristics. These files are unique for each probe array type.

Library files for the Mouse Diversity Genotyping Array are called MouseDIV and are located on the website.

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Please visit the website for sample data, including 98 classic mouse strains and 50 F1 crosses. The mouse breeds that the samples originated from have been anonymized since they are proprietary mouse strains.

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The output of the GTC is a text file that is compatible with multiple third-party software tools. Affymetrix works closely a number of GeneChip-compatible software providers that offer genome-wide linkage or association analysis solutions for Affymetrix genotyping arrays. GeneChip-compatible software providers that have been shown to support the genotype format for the Mouse Diversity Genotyping Array include:

- Golden Helix SNP & Variation Suite (SVS) - Golden Helix has tested data from the Mouse Diversity Genotyping Array and demonstrated compatibility. SVS can import SNP calls from the output text files through an automated import wizard. Once imported, data can be easily manipulated, augmented, and prepared with a full complement of QC tools, and then analyzed with powerful association methods, in-depth statistical analyses, and robust visualization tools. SVS can also import intensity data directly from the array's CEL files, detecting changes in copy number and enabling CNV association studies. For more information, please visit www.goldenhelix.com or email info@goldenhelix.com.

- JMP Genomics from SAS - JMP Genomics can import text genotypes and annotation for the new array using the import individual text files process. Imported genotype text files should be transposed into standard JMP Genomics SNP format for downstream analysis (individuals in rows, SNPs in columns) using the transpose rectangular process. For more information, please visit www.jmp.com/software/genomics/ or email genomics@jmp.com.

- Partek Genomics Suite - Partek has tested sample data from the Mouse Diversity Genotyping Array and can import SNP calls from the output text files. Partek Genomics Suite supports single-marker association workflows as well as inheritance tests. For more information, please visit www.partek.com or email support@partek.com. Additional information about these and other GeneChip-compatible software providers can be found on our website.

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Genotype calls are made using Affymetrix Genotyping Console Software (GTC). However there are no QC methods, no signature SNPs, no copy number data analysis functionality and the output is not compatible with PLINK (http://pngu.mgh.harvard.edu/~purcell/plink/ ). A modified version of the BRLMM-P algorithm is used for the analysis. Birdseed is not available for this array. Detailed instructions for downloading and using GTC, along with sample CEL files, can be found on the website.

We strongly recommends against clustering data that have been run in different facilities, as this will introduce systematic errors.

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The protocol for processing the SNP Affymetrix Genome-Wide Human SNP 5.0/6.0 Assay is manual. Some customers have automated assay processing using their own liquid-handling equipment. At this time, we do not recommend a particular automated protocol.

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Use the GenomeWideSNP6_450 protocol with the GeneChip Fluidics Station 450.

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Scanning an individual Mouse Diversity Genotyping Array takes about 35 mins.

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The assay requires 500 ng of high-quality, double-stranded genomic DNA that is not highly degraded. Genomic DNA must be free of PCR and other enzymatic inhibitors such as high salt, heme, and EDTA. For details about general assay requirements for genomic DNA, please refer to Chapter 3, page 19 of Affymetrix Genome-Wide Human SNP Nsp/Sty 6.0 User Guide (Cat. No. 702504). We have tested a variety of extraction and purification methods as well as cleanup procedures that are also listed in this user guide.

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Total genomic DNA (500 ng) is digested with Nsp I and Sty I restriction enzymes and ligated to adaptors that recognize the cohesive 4 base pair (bp) overhangs. All fragments resulting from restriction enzyme digestion, regardless of size, are substrates for adaptor ligation. A generic primer that recognizes the adaptor sequence is used to amplify adaptor-ligated DNA fragments. PCR conditions have been optimized to preferentially amplify fragments in the 200 to 1,100 bp size range. PCR amplification products for each restriction enzyme digest are combined and purified using polystyrene beads. The amplified DNA is then fragmented, labeled, and hybridized to a Mouse Diversity Genotyping Array.

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The Mouse Diversity Genotyping Array is designed for use with the Affymetrix Genome-Wide Human SNP 6.0 Core Kit. It is available in 100 reactions.

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Content found on the Mouse Diversity Genotyping Array is so comprehensive that it will not fit on the format required for array plates.

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In a study by The Jackson Laboratory comparing two inbred strains, 99.7% of the called genotypes were correct. Among the 114,625 polymorphisms detected, 112,759 SNPs had genotypes for these strains in the Single Nucleotide Polymorphism database (dbSNP). 99.7% (112,452) of the SNPs had the same genotypes between the Mouse Diversity Genotyping Array analysis results and the dbSNP. Among the 307 discordant SNPs, 301 were genotyped by Perlegen and the other five were computed genotypes from the Sanger Institute.

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We have performance metrics for using the Mouse Diversity Genotyping Array with classic inbred laboratory strains and F1 crosses between these inbred strains, but we do not currently offer comprehensive metrics for wild-derived strains.

Table 2 of the supplement to the publication “A customized and versatile high-density genotyping array for the mouse” (Yang H., et al., Nature Methods, 2009), shows performance (call, heterozygosity, and concordance rates) of the array for six wild-derived strains: WSB/EiJ, PWD/EiJ, MOLF/EiJ, CAST/EiJ, SPRET/EiJ, and PANCEVO/EiJ, representing M. m. domesticus, M. M. musculus, M. m. castaneus, M. m. molosinnus subspecies, and M. spretus and M. spicilegus species, respectively. The array performance was highest for wild-derived strains from M. m. domesticus subspecies, followed by strains from other Mus subspecies, which reflects the degree of genetic divergence from the reference strain used C57BL/6J.

The least divergent strain, WSB/EiJ, had the highest call and concordance rates (98.041, 0.992) and lowest heterozygosity rate (2.445), while the most divergent strain, SPRET/EiJ, had the lowest call and concordance rates (92.386, 0.986) and highest heterozygosity rate (14.797).

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SNPs were selected to represent polymorphisms from a comprehensive set of common inbred laboratory and wild-derived mice. For additional information, please see “A customized and versatile high-density genotyping array for the mouse” (Yang H., et al., Nature Methods, 2009).

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It is extremely important to change the vials each time a sample is removed or loaded onto a probe array. This prevents cross-contamination as well as sample loss. RNase contamination is not an issue with gene expression applications due to the fact that the cRNA sample is fragmented prior to hybridization and is removed prior to array processing on the fluidics station.

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With normal use (e.g., 20 arrays/module/week), we recommend the following schedule: Every week, the needle bleaching protocol (i.e., "Bleach" fluidics protocol) should be performed; on a monthly basis, the full-fluidics bleaching protocol (i.e., "Monthly Decontamination" protocol) should be performed and the peristaltic-pump tubing replaced. Please refer to Section 4, Fluidics Station Maintenance Procedures, for more detail.

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If any of the subgrids fail to align, the GeneChip Command Console (GCC) will fail to generate a .cel file. The failed subgrids will be visualized with red borders and an "x" in the center, rather than white borders for successfully aligned subgrids.

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