Mapping 250K/500K SNP assay

More documents

Recommendations

Info

204 GeneChip ® Mapping 500K Assay Manual CONCORDANCE WITH REFERENCE GENOTYPES ON REFERENCE GENOMIC DNA 103 A simple test that can be used to determine if the assay is performing to specifications is to measure the concordance between two samples. The Reference Genomic DNA 103, included in the GeneChip ® Mapping 250K Assay Kits, can be used for this purpose. The calls generated from the reference DNA should be > 99% concordant with the reference calls. The following method allows users to quickly assess the concordance of their data with the reference calls. Microsoft Excel is limited to a maximum of 65,536 rows. Because your data will exceed this number, Microsoft Excel will give you a warning when you paste in your data. 1. Open the reference and experimental Genotype calls in GTYPE. A. Sort the calls in ascending order on the SNP ID. B. Check that the SNP IDs match. 2. Copy and paste the calls from GTYPE and paste into Microsoft Excel. Create a header row. Table 7.3 Columns used to calculate concordance Excel Column Header A Reference DNA SNP ID B Reference DNA call C Test DNA call D Concordance check E No call F Valid concordant SNP
chapter 7 | Analysis Workflow 205 3. Create column called “concordance check”. This column will contain a 1 if the two genotype calls match and a 0 if they do not. A. Enter the following formula “=IF(B2=C2,1,0)”. B. Auto fill down the column. 4. Create a second Column Called “No Call”. This column will contain “TRUE” if either call column contains a “NC”. A. Enter the following formula “=OR(B2="NoCall",C2="NC")”. B. Auto fill down the column. 5. Create a third column called “Valid Concordant SNP”. This column will contain a “TRUE” if the SNP calls match and neither is a “NC”. A. Enter the following formula “=AND(D2=1,E2=FALSE)”. B. Auto fill down the column. 6. Count the number of SNPs that had a genotype call in both experiments. A. Within the spreadsheet, enter the following formula into a cell: “=COUNTIF(E2:65536,"FALSE")”. B. Press return. 7. Count the number of SNPs that have a genotype call in both experiments and are concordant. A. Elsewhere within the spreadsheet, enter the following formula into a cell: “=COUNTIF(F2:65536,"TRUE")”. B. Press return. 8. The Concordance rate can be calculated by dividing the value from step 7 by the value from step 6. The concordance rate should always be greater than 99%. Reasons for a decline in the concordance rate include: Selecting the wrong data set to compare to the reference DNA. Contamination at some step in the assay. Poor sample preparation.
Page 1 and 2:
GeneChip ® Mapping 500K Assay Manu
Page 3 and 4:
Contents CHAPTER 1 Overview 1 ABOUT
Page 5 and 6:
contents v Equipment and Consumable
Page 7 and 8:
contents vii Reagents Required 118
Page 9 and 10:
contents ix CHAPTER 8 Troubleshooti
Page 11 and 12:
contents xi STEP 4: PCR 257 Reagent
Page 13 and 14:
Chapter 1 Overview
Page 15 and 16:
About This Manual This manual is a
Page 17 and 18:
chapter 1 | Overview 5 LD patterns
Page 19 and 20:
Figure 1.1 GeneChip ® Mapping Assa
Page 21 and 22:
chapter 1 | Overview 9 Gilman, B.,
Page 23 and 24:
chapter 1 | Overview 11 23. Schaid,
Page 25 and 26:
chapter 1 | Overview 13 35. Hu, N.,
Page 27 and 28:
chapter 1 | Overview 15 50. Bignell
Page 29 and 30:
chapter 1 | Overview 17 64. Gabriel
Page 31 and 32:
Chapter 2 Laboratory Setup
Page 33 and 34:
Introduction to Laboratory Setup Pl
Page 35 and 36:
Main Lab Pre-PCR Clean Room chapter
Page 37 and 38:
Chapter 3 Genomic DNA General Requi
Page 39 and 40:
Introduction This chapter describes
Page 41 and 42:
Sources of Human Genomic DNA chapte
Page 43 and 44:
chapter 3 | Genomic DNA General Req
Page 45 and 46:
Chapter 4 96-Well Plate Protocol
Page 47 and 48:
ABOUT THIS PROTOCOL 96-Well Plate P
Page 49 and 50:
chapter 4 | 96-Well Plate Protocol
Page 51 and 52:
PREPARING THE WORK AREA FOR EACH ST
Page 53 and 54:
PROGRAM YOUR THERMAL CYCLERS chapte
Page 55 and 56:
EQUIPMENT AND CONSUMABLES REQUIRED
Page 57 and 58:
ALIQUOTING PREPARED GENOMIC DNA To
Page 59 and 60:
Page 61 and 62:
Negative Controls chapter 4 | 96-We
Page 63 and 64:
Table 4.7 Nsp I Digestion Master Mi
Page 65 and 66:
Stage 3: Ligation ABOUT THIS STAGE
Page 67 and 68:
REAGENTS REQUIRED chapter 4 | 96-We
Page 69 and 70:
Page 71 and 72:
DILUTE THE SAMPLES To dilute the sa
Page 73 and 74:
Stage 4: PCR ABOUT THIS STAGE chapt
Page 75 and 76:
Page 77 and 78:
About Controls chapter 4 | 96-Well
Page 79 and 80:
B P1 P2 P3 Figure 4.2 Transferring
Page 81 and 82:
Table 4.16 PCR Master Mix ADD PCR M
Page 83 and 84:
Page 85 and 86:
WHAT YOU CAN DO NEXT chapter 4 | 96
Page 87 and 88:
Page 89 and 90:
Page 91 and 92:
POOL THE PCR PRODUCTS chapter 4 | 9
Page 93 and 94:
Page 95 and 96:
Page 97 and 98:
Page 99 and 100:
Page 101 and 102:
Figure 4.5 Loading Optical Plates w
Page 103 and 104:
Process Control Metrics Evaluate th
Page 105 and 106:
Table 4.24 PROBLEM: Average Sample
Page 107 and 108:
Page 109 and 110:
Page 111 and 112:
IMPORTANT INFORMATION ABOUT THIS ST
Page 113 and 114:
Figure 4.6 Work Area Set Up for Sta
Page 115 and 116:
Page 117 and 118:
Page 119 and 120:
Stage 8: Labeling ABOUT THIS STAGE
Page 121 and 122:
Page 123 and 124:
Page 125 and 126:
Page 127 and 128:
LOCATION AND DURATION • Main Lab
Page 129 and 130:
Hybridizing Samples Using Heat Bloc
Page 131 and 132:
Page 133 and 134:
Page 135 and 136:
Table 4.37 Hybridization Master Mix
Page 137 and 138:
Load the Samples onto Arrays chapte
Page 139 and 140:
Figure 4.8 Applying Tough-Spots ®
Page 141 and 142:
Page 143 and 144:
Page 145 and 146:
Chapter 5 Washing, Staining, and Sc
Page 147 and 148:
Introduction This chapter contains
Page 149 and 150:
Reagent Preparation Wash A: Non-Str
Page 151 and 152:
Experiment and Fluidics Station Set
Page 153 and 154:
chapter 5 | Washing, Staining, and
Page 155 and 156:
Page 157 and 158:
Page 159 and 160:
Page 161 and 162:
HANDLING THE GENECHIP ® PROBE ARRA
Page 163 and 164:
Page 165 and 166: Chapter 6 Fluidics Station Care and
Page 167 and 168: Introduction This chapter provides
Page 169 and 170: chapter 6 | Fluidics Station Care a
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
Page 185 and 186: Table 6.3 (Continued) Common error
Page 187 and 188: Table 6.4 (Continued) Common Error
Page 189 and 190: Table 6.5 (Continued) Other Problem
Page 191 and 192: Chapter 7 Analysis Workflow
Page 193 and 194: Introduction Software Requirements
Page 195 and 196: Figure 7.2 GTYPE main window with t
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
Page 203 and 204: chapter 7 | Analysis Workflow 191 3
Page 205 and 206: NETAFFX SNP ANNOTATION chapter 7 |
Page 207 and 208: CALL RATE chapter 7 | Analysis Work
Page 209 and 210: chapter 7 | Analysis Workflow 197 G
Page 211 and 212: chapter 7 | Analysis Workflow 199 F
Page 213 and 214: chapter 7 | Analysis Workflow 201 S
Page 215: B2 OLIGO PERFORMANCE chapter 7 | An
Page 219 and 220: chapter 7 | Analysis Workflow 207 W
Page 221 and 222: Chapter 8 Troubleshooting
Page 223 and 224: Assay Recommendations 211 Genotypin
Page 225 and 226: chapter 8 | Troubleshooting 213 10.
Page 227 and 228: Problem Likely Cause Solution Faint
Page 229 and 230: Problem Likely Cause Solution Posit
Page 231 and 232: Table 8.2 PROBLEM: Average Sample O
Page 233 and 234: When to Contact Technical Support c
Page 235 and 236: Appendix A Reagents, Equipment, and
Page 237 and 238: Reagents, Equipment, and Consumable
Page 239 and 240: appendix A | Reagents, Equipment, a
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
Page 259 and 260: BEFORE YOU BEGIN appendix C | Low T
Page 261 and 262: STEP 2: Restriction Enzyme Digestio
Page 263 and 264: DIGESTION PROCEDURE PRE-PCR CLEAN A
Page 265 and 266: STEP 3: Ligation REAGENTS AND EQUIP
Page 267 and 268:
PCR STAGING AREA appendix C | Low T
Page 269 and 270:
STEP 4: PCR REAGENTS AND EQUIPMENT
Page 271 and 272:
PCR PROCEDURE PRE-PCR CLEAN ROOM ap
Page 273 and 274:
MAIN LAB appendix C | Low Throughpu
Page 275 and 276:
STEP 5: PCR Purification and Elutio
Page 277 and 278:
appendix C | Low Throughput Protoco
Page 279 and 280:
STEP 7: Fragmentation REAGENTS AND
Page 281 and 282:
FRAGMENTATION PROCEDURE 1. Pre-heat
Page 283 and 284:
Page 285 and 286:
Page 287 and 288:
LABELING PROCEDURE MAIN LAB appendi
Page 289 and 290:
STEP 9: Target Hybridization REAGEN
Page 291 and 292:
HYBRIDIZATION PROCEDURE appendix C
Page 293 and 294:
Page 295 and 296:
Appendix D Reagents, Instruments, a
Page 297 and 298:
Appendix D
Page 299 and 300:
appendix D | Reagents, Instruments,
Page 301 and 302:
* Adaptor, Nsp 5' ATTATGAGCACGACAGA
Page 303 and 304:
Table D.4 Reagents Supplied by Affy
Page 305 and 306:
Page 307 and 308:
Page 309 and 310:
Fragmentation and Labeling appendix
Page 311 and 312:
show all

Mapping 250K/500K SNP assay

Create successful ePaper yourself

Delete template?

Save as template?