Mapping 250K/500K SNP assay

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206 GeneChip ® Mapping 500K Assay Manual DOWNSTREAM ANALYSIS CONSIDERATIONS Data Filtering For many genotyping applications, loss of accuracy can result in a significant decrease in genetic power. This loss of accuracy can be caused by systematic or sporadic errors that occur due to stochastic, sample or experimental factors. To filter out errors and exclude these SNPs in downstream analysis, a two-tiered filtering process is recommended. In the first filter, samples are included only if the call rate is greater than 93% at a 0.33 confidence score when using high quality DNA (see Chapter 3). Those filtered samples are then evaluated in a secondary SNP filter where any SNP with less than an 85% call rate across samples is eliminated from the analysis. These two filters together should result in a high quality set of samples and SNPs for use in downstream analysis. Studies on multiple 500K data sets have shown that SNPs with a lower per SNP call rate tend to have a higher error rate, and disproportionately contribute to the overall error rate in the experiment. Consider taking additional steps to identify and eliminate sporadic genotype errors. Steps may include eliminating SNPs out of Hardy Weinberg equilibrium in control samples from the population, or eliminating genotypes showing Mendel inconsistency or unlikely genotypes. GTYPE has functionality to identify SNPs showing Mendelian or Hardy Weinberg errors in user-defined samples. These genotyping errors can be filtered out upon data export. Please consult the GeneChip ® Genotyping Analysis Software User’s Guide for more information. Adjust the Default Value to Increase or Decrease Accuracy and Call Rate GTYPE 4.0 software provides flexible options to enable a trade off between Call Rate and genotyping accuracy. The Dynamic Model Mapping Algorithm uses the Wilcoxon Signed Rank Test to assign a confidence score measuring the reliability of a genotype call (see Appendix D in the GeneChip ® Genotyping Analysis Software User’s Guide) 1,2 .
chapter 7 | Analysis Workflow 207 We have chosen a confidence score value of 0.33 as the default because it gives a good compromise between accuracy and Call Rate, especially when the 85% per sample filter is applied. In cases where you see a lower Call Rate, you may wish to consider reanalyzing the data at the more stringent confidence score (e.g., 0.26), which will decrease the Call Rate for the sample, but may increase the accuracy of the genotypes. Data Clean Up For many genotyping applications, loss of accuracy can result in a significant decrease in genetic power. Genotyping errors can be caused by SNPs that give systematic errors, or by sporadic errors that occur due to stochastic, sample or experimental factors. SNPs that systematically give errors (e.g., out of Hardy Weinberg equilibrium) have been removed through a SNP selection process 1 . Overall the accuracy has been shown to be approximately 99.5% on the Mapping 500K Array Set based upon internal data. Prior to downstream analysis, the user should consider taking steps to identify and eliminate sporadic genotype errors. Steps may include eliminating SNPs out of Hardy Weinberg equilibrium in control samples from the population, or eliminating genotypes showing Mendel inconsistency or unlikely genotypes. GTYPE has functionality to identify SNPs showing Mendelian or Hardy Weinberg errors in user defined samples. These genotyping errors can be filtered out upon data export. Please consult the GeneChip ® Genotyping Analysis Software User’s Guide for more information. 1 Matsuzaki H., Dong S., Loi H., Di X., Liu G., Hubbell E., Law J., Berntsen T., Chadha M., Hui H., Yang G., Kennedy G., Webster T., Cawley S., Walsh P., Jones K., Fodor S., Mei R. Genotyping over 100,000 SNPs on a pair of oligonucleotide arrays. Nat Methods 1:109-111 (2004). 2 Di X., Matsuzaki H., Webster T.A., Hubbell E., Liu G., Dong S., Bartell D., Huang J., Chiles R., Yang G., Shen M.M., Kulp D., Kennedy G.C., Mei R., Jones K.W., Cawley S. Dynamic model based algorithms for screening and genotyping over 100K SNPs on oligonucleotide microarrays. Bioinformatics 21:1958-63 (2005). 1 See SNP Selection Criteria for the GeneChip Human Mapping 10K Array Xba 131 Technical Note. A similar process was used for the Mapping 500K SNP selection.
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GeneChip ® Mapping 500K Assay Manu
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Contents CHAPTER 1 Overview 1 ABOUT
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contents v Equipment and Consumable
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contents vii Reagents Required 118
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contents ix CHAPTER 8 Troubleshooti
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contents xi STEP 4: PCR 257 Reagent
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Chapter 1 Overview
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About This Manual This manual is a
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chapter 1 | Overview 5 LD patterns
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Figure 1.1 GeneChip ® Mapping Assa
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chapter 1 | Overview 9 Gilman, B.,
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chapter 1 | Overview 11 23. Schaid,
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chapter 1 | Overview 13 35. Hu, N.,
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chapter 1 | Overview 15 50. Bignell
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chapter 1 | Overview 17 64. Gabriel
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Chapter 2 Laboratory Setup
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Introduction to Laboratory Setup Pl
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Main Lab Pre-PCR Clean Room chapter
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Chapter 3 Genomic DNA General Requi
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Introduction This chapter describes
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Sources of Human Genomic DNA chapte
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chapter 3 | Genomic DNA General Req
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Chapter 4 96-Well Plate Protocol
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ABOUT THIS PROTOCOL 96-Well Plate P
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chapter 4 | 96-Well Plate Protocol
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PREPARING THE WORK AREA FOR EACH ST
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PROGRAM YOUR THERMAL CYCLERS chapte
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EQUIPMENT AND CONSUMABLES REQUIRED
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ALIQUOTING PREPARED GENOMIC DNA To
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Negative Controls chapter 4 | 96-We
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Table 4.7 Nsp I Digestion Master Mi
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Stage 3: Ligation ABOUT THIS STAGE
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REAGENTS REQUIRED chapter 4 | 96-We
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DILUTE THE SAMPLES To dilute the sa
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Stage 4: PCR ABOUT THIS STAGE chapt
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About Controls chapter 4 | 96-Well
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B P1 P2 P3 Figure 4.2 Transferring
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Table 4.16 PCR Master Mix ADD PCR M
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WHAT YOU CAN DO NEXT chapter 4 | 96
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POOL THE PCR PRODUCTS chapter 4 | 9
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Figure 4.5 Loading Optical Plates w
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Process Control Metrics Evaluate th
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Table 4.24 PROBLEM: Average Sample
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IMPORTANT INFORMATION ABOUT THIS ST
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Figure 4.6 Work Area Set Up for Sta
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Stage 8: Labeling ABOUT THIS STAGE
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LOCATION AND DURATION • Main Lab
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Hybridizing Samples Using Heat Bloc
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Table 4.37 Hybridization Master Mix
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Load the Samples onto Arrays chapte
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Figure 4.8 Applying Tough-Spots ®
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Chapter 5 Washing, Staining, and Sc
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Introduction This chapter contains
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Reagent Preparation Wash A: Non-Str
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Experiment and Fluidics Station Set
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chapter 5 | Washing, Staining, and
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HANDLING THE GENECHIP ® PROBE ARRA
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Chapter 6 Fluidics Station Care and
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Page 201 and 202: FILE SETS chapter 7 | Analysis Work
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Page 205 and 206: NETAFFX SNP ANNOTATION chapter 7 |
Page 207 and 208: CALL RATE chapter 7 | Analysis Work
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Page 215 and 216: B2 OLIGO PERFORMANCE chapter 7 | An
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Page 223 and 224: Assay Recommendations 211 Genotypin
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Page 227 and 228: Problem Likely Cause Solution Faint
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Page 231 and 232: Table 8.2 PROBLEM: Average Sample O
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Page 249 and 250: Appendix B Thermal Cycler Programs
Page 251 and 252: ABOUT THIS APPENDIX 500K DIGEST 500
Page 253 and 254: 500K FRAGMENT 500K LABEL 500K HYB a
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Page 263 and 264: DIGESTION PROCEDURE PRE-PCR CLEAN A
Page 265 and 266: STEP 3: Ligation REAGENTS AND EQUIP
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STEP 4: PCR REAGENTS AND EQUIPMENT
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PCR PROCEDURE PRE-PCR CLEAN ROOM ap
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MAIN LAB appendix C | Low Throughpu
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STEP 5: PCR Purification and Elutio
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appendix C | Low Throughput Protoco
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STEP 7: Fragmentation REAGENTS AND
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FRAGMENTATION PROCEDURE 1. Pre-heat
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LABELING PROCEDURE MAIN LAB appendi
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STEP 9: Target Hybridization REAGEN
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HYBRIDIZATION PROCEDURE appendix C
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Appendix D Reagents, Instruments, a
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Appendix D
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appendix D | Reagents, Instruments,
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* Adaptor, Nsp 5' ATTATGAGCACGACAGA
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Table D.4 Reagents Supplied by Affy
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Fragmentation and Labeling appendix
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Mapping 250K/500K SNP assay

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