Pantone #2685 (Blue) 4/Color Equivalent (C:100.0 M:94.0 Y:0.0 K:0.0) Pantone #3288 (Green) 4/Color Equivalent (C:100.0 M:0.0 Y:56.0 K:18.5) Pantone #032 (Red) ® 4/Color Equivalent (C:0.0 M:91.0 Y:87.0 K:0.0) Affymetrix Genome-Wide Human SNP Array 5.0 Black AFFYMETRIX® PRODUCT FAMILY > DNA ARRAYS AND REAGENTS > Data Sheet II ® II Affymetrix Genome-Wide Human SNP Array 5.0 The new single-chip Affymetrix Introduction The Whole-genome Sampling Genome-Wide Human SNP Array The new single-chip Affymetrix Genome- Assay 5.0 features single nucleotide Wide Human SNP Array 5.0 contains all The Affymetrix Genome-Wide Human polymorphisms (SNPs) from the 500,568 single nucleotide polymorphisms SNP Nsp/Sty Assay Kit 5.0/6.0 (P/N original two-chip Mapping 500K (SNPs) from the two-array Mapping 500K 901152, 901015) was developed and vali- Array Set, as well as additional Array Set as well as an additional 420,000 dated for use in conjunction with the non-polymorphic probes that can non-polymorphic probes that can measure Genome-Wide Human SNP Array 5.0 measure other genetic differences, other genetic differences, such as copy num- (P/N 901167, 901071). Briefly, total such as copy number variation. The ber variation. SNPs on the array are present SNP 5.0 Array gives researchers a genomic DNA (500 ng) is digested with on 200 to 1,100 base pair (bp) Nsp I or Sty significant increase in information Nsp I and Sty I restriction enzymes and lig- I digested fragments in the human genome, above the original 500K Array Set, ated to adaptors that recognize the cohesive while reducing the array and are amplified using the fifth generation 4 bp overhangs. All fragments resulting processing time. of the Whole-genome Sampling Assay from restriction enzyme digestion, regard- (WGSA). This assay now combines the Nsp less of size, are substrates for adaptor liga- and Sty fractions previously assayed on two tion. A generic primer that recognizes the separate arrays. Using the current version of adaptor sequence is used to amplify adap- the Affymetrix® Genotyping Console, a set tor-ligated DNA fragments. PCR condi- of 440,794 SNPs on the array exhibit the tions have been optimized to preferentially performance capabilities detailed in this amplify fragments in the 200 to 1,100 bp data sheet. size range. PCR amplification products for One hundred thousand non-polymorphic each restriction enzyme digest are com- probes were chosen to cover 2,000 germline bined and purified using polystyrene beads. copy number variants (CNV) identified in The amplified DNA is then fragmented, the UCSC Genome Browser database with labeled and hybridized to a Genome-Wide 50 probes each. The other 320,000 were Human SNP Array 5.0. chosen to give even spacing across the The Affymetrix Genome-Wide Human genome, concentrating on areas that were SNP Nsp/Sty Assay Kit 5.0/6.0 contains not already represented by SNPs. Current validated and qualified reagents for the Affymetrix software does not support analy- most critical steps in the assay. This sis of the non-polymorphic probes. includes the PCR primer and adaptors, Please check the Affymetrix web site for reagents to fragment and label the PCR additional updates to the genotyping call- products and several control reagents. Kits ing algorithm and updates to support are available for either 50 or 100 reactions analysis of non-polymorphic probes. (refer to Ordering Information). the Affymetrix® Genotyping Console Manual. Figure 1: The fifth-generation Whole-genome Sampling Assay. Further details can be found in the snp5_probeset_genotypemanual (www.affymetrix.com/products/software/spe- Nsp INsp I Nsp I Sty I Sty I Sty I cific/genotyping_console_software.affx). RE Digestion RE Digestion Performance Data To test the performance of the SNP Array 5.0, Affymetrix and the Broad Institute Nsp Adaptor Ligation Sty Adaptor Ligation jointly ran the 270 samples from the International HapMap Project. In addi- tion, two external sites and one internal validation group ran a plate of 44 PCR: One Primer PCR: One Primer HapMap DNAs, which includes 30 Amplification Amplification unique samples, 10 trios and five samples with multiple replicates. Complexity The arrays that passed the QC call rate Reduction Clean Up threshold were analyzed using the BRLMM-P algorithm at the default set- Fragmentation and ting of 0.05. The average call rate for End-labeling each set was greater than 99 percent, and the concordance with HapMap geno- Hybridization types was observed to be greater than or and Wash equal to 99.5 percent. For the 10 trios, AA BB AB the Mendelian inheritance consistency was found to be greater than 99.9 per- Whole-genome-amplified material pre- sample should be repeated or used for cent. Reproducibility was measured at pared by the Qiagen REPLI-g® kits may downstream analysis. If the sample passes 99.9 percent. also be used as the starting material for the QC call rate, it is expected to have a the Genome-Wide Human SNP Assay minimum BRLMM-P call rate of 97 per- Data analyzed with BRLMM-P (0.05). Kit 5.0/6.0. cent. The user may adjust the default con- 270 fidence score of BRLMM-P to allow geno- HapMap Site 1 Site 2 Internal Genotype Calls Using typing with either greater accuracy or Call Rate 99.71 99.55 99.37 99.63 Affymetrix® Genotyping higher call rates, depending on what is 3 HapMap Console needed for the application of interest . 99.69 99.67 99.56 99.69 Concordance The Genome-Wide Human SNP Array 5.0 Using the current version of the algo- ® Mendelian is used in conjunction with the Affymetrix rithm BRLMM-P, a set of 440,794 SNPs 99.96 99.95 99.94 99.96 Consistency Genotyping Console. Genotyping Console exhibit the performance capabilities detailed implements two algorithms; the Dynamic below. Future versions of the algorithm have Reproducibility NA 99.9 99.9 99.9 Model algorithm (DM) generates a quality the potential to add more SNPs to this control (QC) call rate for each array by test- default set of 440,794 SNPs. In addition, ® ing 3,022 SNPs specifically chosen for eval- advanced users can analyze the full set of Refer to the Affymetrix Genome-Wide uating data quality, and the BRLMM-P 500,568 SNPs using an alternative library Human SNP Nsp/Sty Assay 5.0 Manual (P/N algorithm uses data from multiple arrays to file (CDF) that reveals all of the SNP content 702419) for details on the QC call rate thresholds, as well as procedures on DNA make genotype calls1, 2. BRLMM-P is an on the array. This advanced workflow cannot updated version of the previous BRLMM be conducted using Genotyping Console; it target preparation, target hybridization, flu- (Bayesian Robust Linear Model with can only be performed with the command- idics setup, array scanning and data analysis. Mahalanobis distance classifier) algorithm line “snp5-probeset-genotype” software. FLUIDICS PROTOCOL REQUIRED that only analyzes perfect match probes. The details of the Genotyping Console data GenomeWideSNP5v1_450 The QC call rate for an individual array analysis workflow using BRLMM-P, includ- (at a DM confidence threshold of 0.33) ing step-by-step instructions to install and LIBRARY FILES REQUIRED should be used to determine whether a run the Genotyping Console are described in GenomeWideSNP_5 II II 2 Library files contain information about REFERENCES probe array design layout and other charac- 1. Rabbee N., et al. A genotype calling algorithm for Affymetrix SNP arrays. Bioinformatics 22:7-12 teristics, probe use and content, and scan- (2006). ning and analysis parameters. 2. Affymetrix White Paper, BRLMM: An Improved These files are unique for each probe Genotype Calling Method for the Mapping 500K Array Set. array type. Library files are available 3. Matsuzaki H., et al. Genotyping over 100,000 from the Affymetrix web site at: SNPs on a Pair of Oligonucleotide Arrays. Nature Methods 1:109-111 (2004). www.affymetrix.com/support/technical/lib 4. Papassotiropoulos A., et al. Common Kibra Alleles raryfilesmain.affx. Are Associated with Human Memory Two alternate SNP List (CDF) files are Performance. Science 314(5798):475-8 (2006). available. These files identify which SNPs are available for downstream analysis. GenomeWideSNP_5.cdf is the default set of 440,794 SNPs that are accessible via the Genotyping Console (BRLMM-P algorithm). GenomeWideSNP_5.Full.cdf is for advanced users who wish to look at all SNPs from the previous-generation Mapping 500K Array Set. This CDF file can only be accessed from the command- line tool, snp5-probeset-genotype, which is part of the Affymetrix Power Tools distri- bution. The full CDF file includes SNPs that may have lower per-SNP accuracy or call rates. It is expected that the perform- ance of some of these SNPs will improve with different or future algorithms. Affymetrix products can be pur- chased directly from Affymetrix in the United States and many European countries. For all other territories, refer to our list of distribution partners located at: www.affymetrix.com/site/con- tact/index.affx. II II 3 Product Information Number of SNPs on the array 500,568 Ordering Information Number of SNPs accessible using BAT 2.0 440,794 Number of Arrays 1 DNA Required 500ng Affymetrix® Genome-Wide Human SNP Expected minimum BRLMM-P Array 5.0 Call Rate (0.05) 97 percent ≥ 901167 Contains 50 arrays Average Minor Allele Frequency (MAF) 0.22 901071 Contains 100 arrays Average Heterozygosity 0.31 PCR Primers 1 per sample Affymetrix® Genome-Wide Human SNP Instrumentation GeneChip® Scanner 3000 7G with AutoLoader Nsp/Sty Assay Kit 5.0/6.0 Throughput >21 million