Mapping 250K/500K SNP assay

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198 GeneChip ® Mapping 500K Assay Manual detection. The MPAM calling algorithm, used to make genotype calls for the Mapping 10K array, is employed in this analysis to make genotype calls on a subset of ~8,000 SNPs chosen for each of the GeneChip ® Mapping 250K Nsp and 250K Sty Arrays. These SNPs were chosen based upon the ability to be a suitable detector of sample contamination across large data sets. Using this subset also allows the software to quickly derive contamination metrics without the additional computational burden of having to make an extra set of calls on the entire array. In a pure sample, the proportion of labeled DNA target for two alleles of any SNP will typically be present at one of three allelic ratios, 100:0, 50:50 or 0:100. The models used to make genotype calls from probe hybridization data apply this information to determine the genotype call for each SNP, and assign a no call if the observed data fall too far from these predicted ratios. If a mixed or contaminated DNA sample is genotyped, the assumption of only three possible allelic ratios will be violated for many SNPs, resulting in lower Call Rates. However, whether or not the sample is contaminated, the MPAM Detection Rate (MDR) will remain high as it is based only on the extent to which PM probes are brighter than MM probes. Therefore, a reduction in Call Rate (MCR < 0.94), accompanied by no decrease in MDR (MDR > 0.99), is a characteristic indicator of sample contamination (Figure 7.7 and Figure 7.8). Note that this contamination metric does not apply if the MDR is < 99%.
chapter 7 | Analysis Workflow 199 Figure 7.7 The effect of genomic DNA contamination on MPAM Detection Rate (MDR), Concordance Rate, and MPAM Call Rate (MCR) (Nsp). Figure 7.8 The effect of genomic DNA contamination on MPAM Detection Rate (MDR), Concordance Rate, and MPAM Call Rate (MCR) (Sty). If you suspect a sample has DNA contamination, you may want to consider testing it with microsatellite markers.
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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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Page 159 and 160: chapter 5 | Washing, Staining, and
Page 161 and 162: HANDLING THE GENECHIP ® PROBE ARRA
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Page 165 and 166: Chapter 6 Fluidics Station Care and
Page 167 and 168: Introduction This chapter provides
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Page 179 and 180: White Clamp Figure 6.7 Releasing pe
Page 181 and 182: Figure 6.8 Troubleshooting decision
Page 183 and 184: PROBLEMS AND SOLUTIONS Table 6.3 Co
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Page 193 and 194: Introduction Software Requirements
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Page 197 and 198: Figure 7.3 Dynamic Model Mapping Al
Page 199 and 200: Table 7.1 Dynamic Model Mapping Alg
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 237 and 238: Reagents, Equipment, and Consumable
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Page 245 and 246: Consumables Required GENECHIP ® AR
Page 247 and 248: Supplier Contact List Table A.10 Su
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
Page 255 and 256: Appendix C Low Throughput Protocol
Page 257 and 258: Introduction The Affymetrix GeneChi
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STEP 2: Restriction Enzyme Digestio
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DIGESTION PROCEDURE PRE-PCR CLEAN A
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STEP 3: Ligation REAGENTS AND EQUIP
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PCR STAGING AREA appendix C | Low T
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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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